iGEM 2018 — Team TUDelft

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# Team:TUDelft/Notebook

ADOPE

[![](https://static.igem.org/mediawiki/2018/0/09/T--TUDelft--2018_adope_logo_white.png)](https://2018.igem.org/Team%3ATUDelft)

* Project
  + [Description](https://2018.igem.org/Team%3ATUDelft/Description)
  + [Design](https://2018.igem.org/Team%3ATUDelft/Design)
  + [Results](https://2018.igem.org/Team%3ATUDelft/Results)
  + [Demonstration](https://2018.igem.org/Team%3ATUDelft/Demonstrate)
* Wetlab
  + [Experiments (Protocols)](https://2018.igem.org/Team%3ATUDelft/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ATUDelft/Notebook)
  + [InterLab](https://2018.igem.org/Team%3ATUDelft/InterLab)
  + [Safety](https://2018.igem.org/Team%3ATUDelft/Safety)
  + [Measurement](https://2018.igem.org/Team%3ATUDelft/Measurement)
* Parts
  + [Parts Overview](https://2018.igem.org/Team%3ATUDelft/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3ATUDelft/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ATUDelft/Composite_Parts)
  + [Part Collection](https://2018.igem.org/Team%3ATUDelft/Part_Collection)
  + [Sequences](https://2018.igem.org/Team%3ATUDelft/Primers)
* Modeling
  + [Model](https://2018.igem.org/Team%3ATUDelft/Model)
  + [Software Tool](https://2018.igem.org/Team%3ATUDelft/Software)
  + [Improvement](https://2018.igem.org/Team%3ATUDelft/Improve)
* Human Practices
  + [Integrated Human Practices](https://2018.igem.org/Team%3ATUDelft/Human_Practices)
  + [Education and Engagement](https://2018.igem.org/Team%3ATUDelft/Public_Engagement)
  + [Entrepreneurship](https://2018.igem.org/Team%3ATUDelft/Entrepreneurship)
  + [Applied Design](https://2018.igem.org/Team%3ATUDelft/Applied_Design)
* [EurAsian Meetup](https://2018.igem.org/Team%3ATUDelft/EurAsianMeetup)
* Team
  + [Team Members](https://2018.igem.org/Team%3ATUDelft/Team)
  + [Collaborations](https://2018.igem.org/Team%3ATUDelft/Collaborations)
  + [Contributions](https://2018.igem.org/Team%3ATUDelft/Contributions)
  + [Team Building](https://2018.igem.org/Team%3ATUDelft/Team_Building)
* Acknowledgements
  + [Attributions](https://2018.igem.org/Team%3ATUDelft/Attributions)
  + [Sponsors](https://2018.igem.org/Team%3ATUDelft/Sponsors)
* + [Judging form](https://igem.org/2018_Judging_Form?team=TUDelft)
  + [Medal Requirements](https://2018.igem.org/Team%3ATUDelft/Medals)
  + [Awards](https://2018.igem.org/Team%3ATUDelft/Awards)

![Wetlab Notebook](https://static.igem.org/mediawiki/2018/b/b7/T--TUDelft--2018_Wetlabnotebookhead.png)

Please select the the model of which you would like to view the notebooks.

Sample Preparation
Pre-screen
Strain Construction
*In Vitro*
*In Vivo*
Purification
Sequencing

* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberSamplePrep1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberSamplePrep2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberSamplePrep3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberSamplePrep4)
* October
  + [Week: 01/10/2018 - 07/10/2018](#OctoberSamplePrep1)
  + [Week: 08/10/2018 - 14/10/2018](#OctoberSamplePrep2)

### September

[ ]

[ ]

[ ]

### October

[ ]

[ ]

* July
  + [Week: 16/07/2018 - 22/07/2018](#JulyPreScreen3)
* August
  + [Week: 27/08/2018 - 02/09/2018](#AugustPreScreen5)
* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberPreScreen1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberPreScreen2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberPreScreen3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberPreScreen4)
* October
  + [Week: 01/10/2018 - 07/10/2018](#OctoberPreScreen1)
  + [Week: 08/10/2018 - 14/10/2018](#OctoberPreScreen2)

### July

[ ]

### August

[ ]

### September

[ ]
Week 1 03/09/2018 - 09/09/2018

Wednesday, 05/09/2018

Date
:   05/09/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the EPO (582 bp) coding sequence, high fidelity PCR was done using the EPO Gblock ordered from IDT as template DNA and 038 and 039 as forward and reverse primers. Two 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- |
| 038 | 039 | EPO | 582 |

The thermocycler programme was installed to have an extension time of 0 seconds and an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4 °C.

Close

[ ]
Week 2 10/09/2018 - 16/09/2018

Monday, 10/09/2018

Date
:   10/09/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The obtained PCR products with EPO coding sequence were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

The EPO fragment that should be amplified by the PCR is 582 bp in size. As can be seen on the gel, EPO was successfully amplified.

Date
:   10/09/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 20 μL of each PCR reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   10/09/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| EPO | 56.0 | 1.90 | 1.58 |

Close

Friday, 14/09/2018

Date
:   14/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt

The dAuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/3/32/T--TUDelft--20180904_saltaggregation.png)

The aggregation of d-AuNPs occured at a final NaCl concentration of 1M\*⅓ = 333 mM, but increased when adding more salt. At a final concentration of 2M\*⅓=666 mM NaCl, the aggregates were the most intensively changed of color. One mistake was made by pipetting the complete d-AuNPs tube into the wells without mixing the tube constantly. The d-AuNPs were sedimentating to the bottom of the tube and this was pipetted into wells E1 and E2. These results should be left out of the data analysis.

From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted. Due to pipetting errors and mixing errors, the standard deviations at lower salt concentrations were high. Additionally, the from all the samples with different salt concentrations, the ratio 620/520 was calculated and plotted over the salt concentration.

Close

[ ]
Week 3 17/09/2018 - 23/09/2018

Monday, 17/09/2018

Date
:   17/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt

The dAuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/6/68/T--TUDelft--20180917_saltaggregation.png)

The aggregation of d-AuNPs occured at a final NaCl concentration of 1M\*⅓ = 333 mM, but increased when adding more salt. At a final concentration of 2M\*⅓=666 mM NaCl, the aggregates were the most intensively changed of color.

From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted. In comparison with the previous measurements, the error bars are lower. Additionally, the from all the samples with different salt concentrations, the ratio 620/520nm was calculated and plotted over the salt concentration.

Date
:   17/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt with ssDNAp

The dAuNPs are tested for their stability under different salt concentrations and the influence of ssDNAp is determined. 20 µL of the d-AuNPs solution, 16 µL MilliQ, [4 µL ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 96 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/b/be/T--TUDelft--20180917_ssDNAp.png)

The aggregation of d-AuNPs occured at a final NaCl concentration of 1500 mM \* ⅓ = 500 mM, but increased when adding more salt. At a final concentration of 2M\*⅓=666 mM NaCl, the aggregates were the most intensively changed of color.
From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted. Additionally, the from all the samples with different salt concentrations, the ratio 620/520 nm was calculated and plotted over the salt concentration.

Close

Wednesday, 19/09/2018

Date
:   19/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt with(out) ssDNAp

The dAuNPs are tested for their stability under different salt concentrations and the influence of ssDNAp is determined. 20 µL of the d-AuNPs solution, 16 µL MilliQ, [4 µL ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. As negative controls, 0 L ssDNAp 1 was added. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/0/01/T--TUDelft--20180919_ssDNAp.png)

The d-AuNPs aggregated when salt was added from a final concentration of 1M\*⅓=333 mM to 2M\*⅓=666 mM NaCl. When ssDNAp (1 uM) were added, the concentrations at which the d-AuNPs aggregated increased. However, at 2 M NaCl, the d-AuNPs still aggregated although the aggregation was less intense compared to the salt aggregation without ssDNAp’s.

From the data, the spectrum of the mixtures with 0 mM, 1000 mM and 2000 mM NaCl were plotted from both samples: with and without ssDNAp. Additionally, the from all the samples either with or without ssDNAp addition, the ratio 620/520 was calculated and plotted over the salt concentration.

Date
:   19/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Generation

In order to generate dextrin-capped gold nanoparticles, first a gold chloride (HAuCl4.H2O) stock solution of 50 mL was prepared by dissolving 393.8 mg of HAuCl4.H2O in 50 mL of distilled sterile water. After preparation, this solution was stored at 4 °C covered in aluminium foil.
Secondly, a 10% sterile sodium carbonate (Na2CO3) solution was prepared by dissolving 4.985 g Na2CO3 in 50 mL MilliQ water, after which the solution was filter sterilized.
Then, 200 mL distilled sterile water was be adjusted to pH 9 by carefully adding 1 M sodium hydroxide (NaOH) drops of 20 uL at a time, while keeping the solution sterile under a flame.
Subsequently, 25 mL of dextrin solution was prepared by dissolving 625 mg of dextrin powder in 25 mL of sterile MilliQ water to obtain a concentration of 25 g/L. This solution was then added to a sterile 250 mL flask.

Then, 5 mL of the gold chloride stock solution was added to the 250 mL flask and the pH was adjusted to 9 with the sterile 10% sodium carbonate solution, while working sterile.
Lastly, the reaction volume was completed to 50 mL by adding 20 mL distilled water (pH = 9).

For the actual generation of the d-AuNPs the flask was then incubated at 50 °C, covered in aluminium foil with continuous shaking for 3 to 8 hours. After 3 to 8 hours shaking, a reddish colour was observed on the solution that is characteristic for the correct size nanoparticle formation. The solution was observed every 30 minutes to check for red color formation.

Transmission Electron Microscope analysis was performed to check the size and size distribution of the d-AuNPs. the suspension of d-AuNPs in distilled water (ratio 1:4) and sonicate it for 5 min. Then, after plasma treatment this solution was be transferred to a copper grid for imaging. The correct size of the d-AuNPs be in the order of 10 nm.

![TEM](https://static.igem.org/mediawiki/2018/b/b6/T--TUDelft--180919_B2_Unag_igem_001.tif)

We observed d-AuNPs in a relatively low concentration. The size distribution is ranging from 8 nm to 13 nm, which is sufficient for d-AuNPs analysis for prescreening.

Date
:   19/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt

The newly generated d-AuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 2000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The plate view is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/e/e2/T--TUDelft--20180919_Saltaggregation3.jpg)

The aggregation of d-AuNPs occured at a final NaCl concentration of 100 mM. In comparison with the old d-AuNPs batch, they aggregate at a lower salt concentration indicating that they are less stable. This observation can be explained by the time that the gold nanoparticles were in solution. During the generation, dextrin solution was added. The incubation time was approximately 8 hours and in this time the gold nanoparticles were capped by dextrin. The dextin makes the gold nanoparticles more stable. However, when the gold nanoparticles stay in the solution for a longer time, the remaining dextrin can adhere to the gold nanoparticles, making them more stable.

Close

Thursday, 20/09/2018

Date
:   20/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt

The newly generated dAuNPs are tested for their stability under different salt concentrations. 20 µL of the d-AuNPs solution, 20 µL MilliQ and 20 µL NaCl solutions (range from 0 mM to 1000 mM) were added in one of the 96 wells on the 92 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/c/c7/T--TUDelft--d-AuNPs_saltaggregation.png)

With the new batch of d-AuNPs, the salt concentration variation was performed again. The range of salt concentration is adapted from 0 to 1000 mM NaCl added concentration. From the plate view it can be seen that the d-AuNPs are aggregating at a lower salt concentration than the previous (older) batch on 14/09/2018, 17/09/2019 and 19/09/2018.

Date
:   20/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt with(out) ssDNAp

The dAuNPs are tested for their stability under different salt concentrations and the influence of ssDNAp is determined. 20 µL of the d-AuNPs solution, 16 µL MilliQ, [4 µL ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) and 20 µL NaCl solutions (range from 0 mM to 1000 mM) were added in one of the 96 wells on the 96 wells plate. As negative controls, 0 L ssDNAp 1 was added. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/0/0d/T--TUDelft--d-AuNPs_ssDNAp.png)

With the new batch of d-AuNPs, the salt concentration variation was performed again. The range of salt concentration is adapted from 0 to 1000 mM NaCl added concentration. Additionally, ssDNAp cause a stabilizing effect as the color change occurs at a higher salt concentration compared to the solutions without ssDNAp.

Date
:   20/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: d-AuNPs stability upon salt with(out) ssDNAp

The dAuNPs are tested for their stability under different salt concentrations and the influence of ssDNAp is determined. 20 µL of the d-AuNPs solution and 20 µL NaCl solutions (250, 500 and 1000 mM respectively) were added in the 96 wells plate. To test the influence of the ssDNAp concentration on d-AuNPs stabilization, different amounts of [0 to 10 µL ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was added in subsequent wells. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/a/ae/T--TUDelft--20180920_ssDNAp_variation.png)

With a higher ssDNAp concentration, the d-AuNPs are being stabilized. The color change to purple, which indicates destabilization, occurs at a higher salt concentration.

Date
:   20/09/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the EPO (582 bp) coding sequence, high fidelity PCR was done using the EPO Gblock ordered from IDT as template DNA and 038 and 039 as forward and reverse primers. Two 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- |
| 038 | 039 | EPO | 582 |

The thermocycler programme was installed to have an extension time of 0 seconds and an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   20/09/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 20 μL of each PCR reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   20/09/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| EPO (1) | 144.2 | 1.89 | 1.94 |
| EPO (2) | 150.9 | 1.88 | 2.06 |
| EPO (3) | 128.2 | 1.88 | 1.99 |

Close

Friday, 21/09/2018

Date
:   21/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

The aggregation of d-AuNPs due to increasing salt concentration starts at 500 mM of NaCl. When a ssDNAp is added, the aggregation is extended to a NaCl concentration of 600 mM. However, presence of dsDNA target with a ssDNAp that can specifically bind to the target dsDNA does not influence the aggregation behavior drastically compared to the aggregation when dsDNA target is absent. Presence of only dsDNA target does decrease the stabilization of the d-AuNPs as they already aggregate at a NaCl added of 250 mM.

Date
:   21/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. The ssDNAp concentration is decreased to avoid to much stabilization of d-AuNPs by the ssDNAp. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1nM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/4/4d/T--TUDelft--20180921_ssDNAp_EPO.png)

Date
:   21/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. The ssDNAp concentration is decreased to avoid to much stabilization of d-AuNPs by the ssDNAp. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (100nM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/9/97/T--TUDelft--20180921_ssDNAp_lowC_EPO.png)

Date
:   21/09/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. The ssDNAp concentration is increased to check the stabilization effect on d-AuNPs by the ssDNAp. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA, 6 µL hybridization buffer, 4 µL [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (100nM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/7/70/T--TUDelft--20180921_ssDNAp_highC_EPO.png)

Close

### October

[ ]
Week 1 01/10/2018 - 07/10/2018

Tuesday, 02/10/2018

Date
:   02/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the sensitivity of the prescreening, the EPO target DNA is lowered to 100 pM. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The ssDNAp, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 nM and 100 pM), 6 µL hybridization buffer, 4 µL [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/8/8d/T--TUDelft--20181002_ssDNAp_EPO_lowC_1nM_100pM.png)

Close

[ ]
Week 2 08/10/2018 - 14/10/2018

Monday, 08/10/2018

Date
:   08/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 2 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The [ssDNAp 2](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was used in this experiment to target the EPO DNA. The ssDNAp 2, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL [ssDNAp 2](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/7/70/T--TUDelft--20181008_ssDNAp2_EPO.png)

Date
:   08/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 3 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The [ssDNAp 3](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was used in this experiment to target the EPO DNA. The ssDNAp 3, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL [ssDNAp 3](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/0/03/T--TUDelft--20181008_ssDNAp3_EPO.png)

Date
:   08/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 4 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The [ssDNAp 4](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was used in this experiment to target the EPO DNA. The ssDNAp 4, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL [ssDNAp 4](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/3/3f/T--TUDelft--20181008_ssDNAp4_EPO.png)

Date
:   08/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the versatility of the prescreening, multiple variants of the ssDNAp were designed to target the EPO DNA. The ssDNAp 5 targets the EPO coding sequence. Prior, the ssDNAp is heated up to let the ssDNAp anneal to EPO target DNA and create the secondary structure. The [ssDNAp 5](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was used in this experiment to target the EPO DNA. The ssDNAp 5, EPO target DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL [ssDNAp 5](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/2/2b/T--TUDelft--20181008_ssDNAp5_EPO.png)

Close

Tuesday, 09/10/2018

Date
:   09/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the effect of background DNA on the ability to screen for target DNA, linear DNA was added. The [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was used in this experiment to target the EPO DNA while background DNA is present. The ssDNAp 1, EPO target DNA, background DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

![d-AuNPs salt stability](https://static.igem.org/mediawiki/2018/a/a8/T--TUDelft--20181009_ssDNAp1_EPO_background.png)

Date
:   09/10/2018

Operator
:   Nicole Bennis

#### d-AuNPs Testing: ssDNAp interaction with EPO target DNA

The prescreening with d-AuNPs is tested with EPO DNA as target. To test the effect of background DNA on the ability to screen for target DNA, linear DNA was added. The [ssDNAp 1](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) was used in this experiment to target the EPO DNA while background DNA is present. The ssDNAp 1, EPO target DNA, background DNA and hybridization buffer were incubated:

+ 5 minutes at 95 ºC (denature dsDNA target)
+ 1 minute at 57 ºC (anneal ssDNAp to dsDNA target)
+ 10 minutes at 21 ºC

20 µL d-AuNPs, 10 µL target dsDNA (1 µM), 6 µL hybridization buffer, 4 µL [ssDNAp 2](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp) (1 µM) and 20 µL of a varying salt concentration (range of 0 mM to 1000 mM) were added to the 96 wells plate. Again, the primer 051 was used as off-target ssDNAp. The plate was incubated at 21 ºC during 10 minutes. The visible absorption spectrum from 450 nm to 650 nm of the solutions was measure and the ratio 620/520 nm was calculated for analysis. The experiment was performed in triplicate. The plate view, relevant absorption spectra and ratio 620/520 nm is shown.

In order to check the versatility of the prescreening, the effect of background DNA present in the sample, the d-AuNPs test is also performed with [ssDNAp 3, 4 and 5](https://2018.igem.org/Team%3ATUDelft/Primers#ssDNAp).

Close

* May
  + [Week: 28/05/2018 - 03/06/2018](#MayStrainCon)
* June
  + [Week: 04/06/2018 - 10/06/2018](#JuneStrainCon1)
  + [Week: 11/06/2018 - 17/06/2018](#JuneStrainCon2)
  + [Week: 18/06/2018 - 24/06/2018](#JuneStrainCon3)
  + [Week: 25/06/2018 - 01/07/2018](#JuneStrainCon4)
* July
  + [Week: 02/07/2018 - 08/07/2018](#JulyStrainCon1)
  + [Week: 09/07/2018 - 15/07/2018](#JulyStrainCon2)
  + [Week: 16/07/2018 - 22/07/2018](#JulyStrainCon3)
* August
  + [Week: 30/07/2018 - 05/08/2018](#AugustStrainCon1)
  + [Week: 06/08/2018 - 12/08/2018](#AugustStrainCon2)
  + [Week: 13/08/2018 - 19/08/2018](#AugustStrainCon3)
  + [Week: 20/08/2018 - 26/08/2018](#AugustStrainCon4)
  + [Week: 27/08/2018 - 02/09/2018](#AugustStrainCon5)
* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberStrainCon1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberStrainCon2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberStrainCon3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberStrainCon4)
* October
  + [Week: 01/10/2018 - 07/10/2018](#OctoberStrainCon1)
  + [Week: 08/10/2018 - 14/10/2018](#OctoberStrainCon2)

### May

[ ]
Week 5 28/05/2018 - 03/06/2018

Thursday, 30/05/2018

Date
:   30/5/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

An overnight starter culture of *E. coli* DH5α harboring plasmid pTXB1-Tn5 was subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   30/5/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pTXB1-Tn5 | 36.3 | 1.92 | 2.06 |
| pTXB1-Tn5 | 40.4 | 1.88 | 2.17 |
| pTXB1-Tn5 | 47 | 1.82 | 1.75 |

Close

Friday, 01/06/2018

Date
:   1/6/2018

Operator
:   Kavish Kohabir

#### Primer working stock preparation

The following primers were resuspended in sterile Milli-Q water, in order to make 100µM stock solutions.

| Primer name | Amount (ng) | Volume (uL) |
| --- | --- | --- |
| 001 | 290 | 277 |
| 002 | 380 | 304 |
| 003 | 280 | 377 |
| 004 | 280 | 344 |
| 005 | 4nmol | 40\* |
| 006 | 260 | 255 |
| 007 | 290 | 284 |
| 008 | 4nmol | 40\* |
| 009 | 250 | 290 |
| 010 | 270 | 320 |
| 011 | 220 | 308 |
| 012 | 200 | 301 |
| 013 | 220 | 357 |
| 014 | 180 | 301 |
| 015 | 200 | 334 |
| 016 | 200 | 330 |
| 017 | 190 | 350 |
| 018 | 170 | 348 |
| 019 | 290 | 345 |
| 020 | 220 | 365 |
| 021 | 240 | 343 |
| 022 | 230 | 334 |
| 023 | 200 | 319 |
| 024 | 230 | 336 |
| 025 | 220 | 339 |
| 026 | 160 | 355 |
| 027 | 150 | 301 |
| 028 | 170 | 343 |

The primers in these 100 µM stocks were heated to 60 °C for 20 minutes and centrifuged at maximum speed (~17,000 x g) for 2 minutes. 10µM primer working stock solutions were prepared by making 10x dilution aliquots with sterile Milli-Q, each in a total volume of 100µl.

Close

### June

[ ]
Week 1 04/06/2018 - 10/06/2018

Monday, 04/06/2018

Date
:   04/06/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 005 | 006 | Linker-Tn5 | 1508 |
| 2 | 007 | 006 | Tn5 | 1452 |
| 3 | 007 | 008 | Tn5-Linker | 1505 |

The thermocycler programme was installed to have an extension time of 1 min 30 seconds and an annealing temperature of 55 °C for one of the tubes and 60 °C for the second tubes. After the programme was finished, samples were kept at 4 °C.

Date
:   04-06-2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The obtained PCR products Linker-Tn5, Tn5, Tn5-Linker were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/19/T--TUDelft--2018_20180604_PCR_linkers_Tn5.png)

The fragments that should be amplified by the PCR are respectively 1508, 1452 and 1505 bp in size. As can be seen on the gel, only the Tn5 amplification was successfully amplified. The Linker-Tn5 and Tn5-Linker fragments were not successfully amplified.

Close

Tuesday, 05/06/2018

Date
:   05/06/2018

Operator
:   Nicole Bennis

#### PCR clean-up

Two times 50 μL of PCR product Tn5 was subjected to PCR Clean-Up, according to the [DNA clean up protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL prewarmed Milli-Q.

Date
:   05-06-2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5 (annealed at 55 °C) | 59.6 | 1.88 | 1.65 |
| Tn5 (annealed at 60 °C) | 47.4 | 1.82 | 0.59 |

Date
:   05/06/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

*E.coli* strain harbouring the plasmid pTXB1-Tn5 was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with ampicillin) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Close

Wednesday, 06/06/2018

Date
:   06/06/2018

Operator
:   Nicole Bennis

#### Restriction

Restriction cloning was done for assembling Tn5 into pACYC-Duet. The following table shows an overview of what fragment(s) were cut with which restriction enzyme(s).

| Component | Volume (uL) |
| --- | --- |
| 10x CutSmart buffer | 2.5 |
| Fragment (pACYC-Duet) | 10 (1000 ng) |
| Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 |
| MilliQ | 10.5 |

| Component | Volume (uL) |
| --- | --- |
| 10x CutSmart buffer | 2.5 |
| Fragment (Tn5) | 20.5 (1000 ng) |
| Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 |
| MilliQ | 0 |

The samples were incubated for 2 hours at 37 °C.

Close

Friday, 08/06/2018

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### PCR Clean up

25 μL of restriction mixture with Tn5 or pACYCDuet-1 was subjected to DNA Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL prewarmed Milli-Q.

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5 55 °C (EcoRI, KpnI restricted) | 8.8 | 1.97 | 0.79 |
| Tn5 60 °C (EcoRI, KpnI restricted) | 6.8 | 1.68 | 0.52 |
| Duet-1 (EcoRI, KpnI restricted) | 5.6 | 2.05 | 0.40 |

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments Tn5 55 °C/60 °C and pACYCDuet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3768 | EcoRI, KnpI |
| Fragment Tn5 | 1442 | EcoRI, KpnI |

A 20 μL ligation reaction mixture was prepared with the following composition:

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 |
| T4 DNA Ligase | 1 || DNA vector (~50 ng) | 9 |
| DNA fragment (~60 ng) | 8 |
| MilliQ | 0 |

The samples were incubated for 72 hours (over the weekend) at 4°C for ligation.

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. As the PCR reaction was not successful before, different conditions were tested to optimize the PCR. Multiple 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol.](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll)

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 005 | 006 | Linker-Tn5 | 1508 |
| 2 | 007 | 006 | Tn5 | 1452 |
| 3 | 007 | 008 | Tn5-Linker | 1505 |

The sets of conditions tested were:

+ Touch Down PCR with HF buffer
+ Touch Down PCR with GC buffer
+ 2 step PCR with HF buffer
+ 2 step PCR with GC buffer

Touch Down PCR program

| Step | Time (s) | Temperature (°C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| Denaturation | 10 | 98 |
| Annealing | 15 | 60 down to 50 |
| Extension | 1 min 30 sec | 72 |
| Final extension | 300 | 72 |
| Hold | ∞ | 4 |

2 Step PCR program

| Step | Time (s) | Temperature (°C) |
| --- | --- | --- |
| Initial denaturation | 30 | 98 |
| Denaturation | 10 | 98 |
| Annealing | 15 | 72 |
| Extension | 1 min 30 sec | 72 |
| Final extension | 300 | 72 |
| Hold | ∞ | 4 |

After the programme was finished, samples were kept at 4 °C.

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The obtained PCR products Linker-Tn5, Tn5, Tn5-Linker were run on a 0.8% agarose gel in TAE buffer. 4 μL DNA was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/4/4c/T--TUDelft--2018_20180608_PCR_linkers_Tn5.png)

The fragments that should be amplified by the PCR are respectively 1508, 1452 and 1505 bp in size. As can be seen on the gel, only the Linker-Tn5 amplification was successfully amplified in the 2 Step PCR program with GC buffer. The Linker-Tn5, Tn5 and Tn5-Linker fragments were not successfully amplified when a touch down PCR program was performed.

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using the amplified Tn5 (in previous PCR reactions) as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two times eight 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 005 | 006 | Linker-Tn5 | 1508 |
| 2 | 007 | 008 | Tn5-Linker | 1505 |

To test the effect of DMSO on the PCR performance, one extra tube per amplicon was made that was supplemented with 1.5 μL DMSO (3% final) and 34 μL MilliQ.
Additionally, instead of the amplified Tn5 PCR product as template, the original pTXB1-Tn5 plasmid was used as template.
Finally, a negative control sample was taken into account that contained no primers and 37.5 μL MilliQ instead.
The thermocycler programme was installed to have an extension time of 1 min 30 seconds and an annealing temperature of 60 °C. In order to check the difference between two PCR machines (older one and new one), the tubes were divided over the two thermocyclers. After the programme was finished, samples were kept at 4 °C.

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The obtained PCR products Linker-Tn5 and Tn5-Linker were run on a 0.8% agarose gel in TAE buffer. 4 μL DNA was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/65/T--TUDelft--2018_20180608_PCR_linkers_Tn52.png)

As can be seen, the PCR amplification with primers 005 and 006 resulting in Linker-Tn5 were successful. The primers 007 and 008 did not result in any PCR amplicons. Also, the HF buffer supplemented with 3% DMSO resulted in the required amplicon. Remarkably, the old PCR machine did result in thicker and brighter bands DNA. This might be due to the differences in speed when changing temperature.

Date
:   08/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Linker-Tn5, 7 tubes (new machine) | 117.9 | 1.82 | 1.35 |
| Linker-Tn5, 1 tube (old machine) | 67.7 | 1.82 | 1.17 |

Date
:   08-06-2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two times three 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 005 | 006 | Linker-Tn5 | 1508 |
| 2 | 007 | 006 | Tn5 | 1452 |
| 3 | 007 | 008 | Tn5-Linker | 1505 |

The thermocycler programme was installed to have an extension time of 2 min 30 seconds (1 minute longer than PCR reactions before) and an annealing temperature of 60 °C. Again, in order to check the difference between two PCR machines (older one and new one), the tubes were divided over the two thermocyclers. After the programme was finished, samples were kept at 4 °C.

Date
:   08-06-2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

All the PCR amplicons were run on a 0.8% agarose gel in TAE buffer. 4 μL DNA was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/13/T--TUDelft--2018_20180608_PCR_linkers_Tn53.png)

As can be seen, the PCR amplification with primers 005, 006, 007 and 008 resulting in Linker-Tn5, Tn5, Tn5-Linker (respective sizes: 1508, 1452 and 1505 bp) were successful. In contradiction with the previous results, the new PCR machine did result in thicker and brighter bands DNA and is therefore considered more efficient for this PCR reaction. From this experiment it can be concluded that the standard PCR conditions work sufficient for this purpose.

Close

[ ]
Week 2 11/06/2018 - 17/06/2018

Monday, 11/06/2018

Date
:   11/06/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pACYC1Duet-1\_Tn5 (5210 bp) | Restriction/Ligation | 20 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_Tn5(5210 bp) | Restriction/Ligation | 20 | LB Cam |
| *E.coli* DH5α | pTXB1-Tn5 | - | 2 | LB Amp |
| *E.coli* DH5α | pACYCDuet-1 (pos.control) | - | 1 | LB Cam |
| *E.coli* DH5α | none (neg. control) | Restriction/Ligation | 0 | LB Cam |

200 μl LB medium was added as recovery medium.

Close

Tuesday, 12/06/2018

Date
:   12/06/2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1\_Tn5 after transformation from 11-06-2018. This plasmid contains the Tn5 insert, which can be checked with primers 029 and 030. As negative control, the colonies containing pACYCDuet-1 were checked according to the same protocol.

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Tn5 |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1686 bp |

Six tubes were prepared for colony PCR, containing pACYCDuet-1\_Tn5 as template DNA. Additionally, one tube with pACYCDuet-1 was taken along as positive control. The thermocycler programme was installed to have an extension time of 2 min and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Close

Wednesday, 13/06/2018

Date
:   13/06/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons of the colony PCR performed on 12-06-2018 were run on a 0.8% agarose gel in TAE buffer. 4μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/7/7a/T--TUDelft--2018_20180613_ColonyPCR.png)

Unfortunately, the PCR reaction did not result in the required PCR amplicons. However, the positive control colony (pACYCDuet-1) did also not results in any PCR amplicon. Therefore, it is likely that the PCR reaction itself was not successful and needs to be repeated.

Date
:   13-06-2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

The PCR amplicons made on 08-06-2018 were subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   13-06-2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The PCR amplicons were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Linker-Tn5 | 494.1 | 1.88 | 1.95 |
| Tn5 | 463.1 | 1.86 | 1.90 |
| Tn5-Linker | 398.3 | 1.88 | 1.87 |

Close

Thursday, 14/06/2018

Date
:   14/06/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

The six colonies that have grown on LB Cam after transformation on 11-06-2018 and probably harbour our desired plasmid pACYCDuet-1\_Tn5 were used for inoculation of 50 mL liquid starter culture (Luria Broth, complemented with Cam) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll). Additionally, as positive control one colony of pACYCDuet-1 that has also grown was inoculated in the same way.

Close

Friday, 15/06/2018

Date
:   15/06/2018

Operator
:   Nicole Bennis

#### Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 |
| Fragment (Tn5) | 4.5 (1000 ng) | Fragment (Linker-Tn5) | 4 (1000 ng) | Fragment (Tn5-Linker) | 5 (1000 ng) |
| Enzyme (EcoRI) | 1 | Enzyme (FseI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 | Enzyme (KpnI) | 1 | Enzyme (NotI) | 1 |
| MilliQ | 38.5 | MilliQ | 39 | MilliQ | 38 |

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute at 65 °C heat inactivation of the restriction enzyme(s).

Date
:   15/06/2018

Operator
:   Timothy Páez

#### Restriction

Restriction cloning was done for assembling Tn5-lin and lin-Tn5 into pACYCduet-1. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (Tn5-lin) | 9 |
| Enzyme (ECORI HF) | 1 |
| Enzyme (NotI HF) | 1 |
| MilliQ | 7 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC) | 16 |
| Enzyme (EcoRI HF) | 1 |
| Enzyme (NotI HF) | 1 |
| MilliQ | 0 |

The samples were incubated for 1 hour at 37 °C.

Date
:   15/06/2018

Operator
:   Timothy Páez

#### PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- Tn5-lin: 30.4 ng/µL
- pACYC: 8.5 ng/µL

Date
:   15/06/2018

Operator
:   Timothy Páez

#### Ligation

In order to assemble cut fragments Tn5-lin into pACYCduet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet | 1.5 kbp | EcoRI and NotI |
| Tn5-lin | 1.5 kbp | EcoRI and NotI |

The ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 µL || T4 DNA Ligase | 1 µL || pACYC (~100 ng) | 13.3 µL || Tn5 (300 ng) | 3.7 µL || MilliQ | 0 µL |

The samples were incubated overnight at 4 ºC for ligation.

Close

Saturday, 16/06/2018

Date
:   16/06/2018

Operator
:   Timothy Páez

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pACYC Tn5 (3000 bp) | Restriction Ligation | 10 µL | LB with Chloroamphenicol |

400 µL LB medium was added as recovery medium.

Close

Sunday, 17/06/2018

Date
:   17/06/2018

Operator
:   Timothy Páez

#### Transformation results from 16/06/2018

No colonies grew on LB plates with chloroamphenicol. Repeat protocol with higher DNA concentrations starting from restriction.

Close

[ ]
Week 3 18/06/2018 - 24/06/2018

Monday, 18/06/2018

Date
:   18-06-2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of Tn5 (two times) and pACYCDuet-1 (two times) were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   18-06-2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5 (1) | 119.1 | 2.02 | 2.08 |
| Tn5 (2) | 127.3 | 2.00 | 2.32 |
| pACYCDuet-1 (1) | 47.4 | 1.96 | 2.03 |
| pACYCDuet-1 (2) | 238.9 | 2.12 | 2.44 |

Date
:   18-06-2018

Operator
:   Nicole Bennis

#### Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 |
| Fragment (Tn5) | 4.5 (2000 ng) | Fragment (Linker-Tn5) | 4 (2000 ng) | Fragment (Tn5-Linker) | 5 (2000 ng) | Fragment (pACYCDuet-1) | 5 (2000 ng) |
| Enzyme (EcoRI) | 1 | Enzyme (FseI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 | Enzyme (KpnI) | 1 | Enzyme (NotI) | 1 | Enzyme (NotI) | 1 |
| MilliQ | 38.5 | MilliQ | 39 | MilliQ | 38 | MilliQ | 38 |

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute at 65 °C heat inactivation of the restriction enzyme(s).

Date
:   18/06/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

The restriction reactions Tn5, Tn5-Linker, Linker-Tn5 and pACYCDuet-1 were subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   18-06-2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pACYCDuet-1\_Tn5 (5210 bp) | Restriction/Ligation | 5 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_Linker-Tn5 (5477 bp) | Restriction/Ligation | 5 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 restricted (neg. control) | Restriction | 5 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 (pos. control) | - | 0.5 | LB Cam |

200 μl LB medium was added as recovery medium.

Date
:   08-06-2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

The colonies that have grown on the plates of the transformation performed on 11-06-2018 were used for inoculation of 5 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll). This was done to be able to test the colonies for correct insert.

Close

Tuesday, 19/06/2018

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Two times three 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 005 | 006 | Linker-Tn5 | 1508 |
| 2 | 007 | 006 | Tn5 | 1452 |
| 3 | 007 | 008 | Tn5-Linker | 1505 |

The thermocycler programme was installed to have an extension time of 2 min 30 seconds (1 minute longer than PCR reactions before) and an annealing temperature of 60 °C. Again, in order to check the difference between two PCR machines (older one and new one), the tubes were divided over the two thermocyclers. After the programme was finished, samples were kept at 4 °C.

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of the colonies from the transformation performed on 11-06-2018 were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 20 μL of pre-warmed MilliQ.

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Colony 1: pACYCDuet-1\_Tn5 | 19.7 | 1.75 | 0.86 |
| Colony 2: pACYCDuet-1\_Tn5 | 38.6 | 1.66 | 0.79 |
| Colony 3: pACYCDuet-1\_Tn5 | 93.4 | 1.56 | 0.62 || Colony 4: pACYCDuet-1\_Tn5 | 34.6 | 1.75 | 0.98 |
| Colony 5: pACYCDuet-1\_Tn5 | 57.4 | 1.62 | 0.74 || Colony 6: pACYCDuet-1\_Tn5 | 39.8 | 1.73 | 0.99 |
| Colony 7: pACYCDuet-1\_Tn5 | 32.5 | 1.77 | 1.10 || Colony pACYCDuet-1 | 54.3 | 1.73 | 1.05 |

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYCDuet-1\_Tn5 after transformation from 11-06-2018. This plasmid contains Tn5, which can be checked with primers 006 and 007. Additionally, the primers binding at the T7 promoter and termination were used to check the Tn5 insert.

|  |  |  |
| --- | --- | --- |
| Template DNA | pACYCDuet-1\_Tn5 | pACYCDuet-1\_Tn5 |
| Fw primer | 007 | 029 |
| Rv primer | 006 | 030 |
| expected amplicon size (bp) | 1452 bp | 1576 bp |

Eight tubes were prepared for colony PCR, containing pACYCDuet-1-Tn5 as template DNA. Additionally, MgCl2 was added to increase the efficiency of the PCR reaction. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The colony PCR’s of the transformation of 11-06-2018 were run on a 0.8% agarose gel in TAE buffer. 5 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/ac/T--TUDelft--2018_20180619_ColonyPCR.png)

Colonies 1,2,3,4,6 and 7 show the presence of Tn5 and T7p-Tn5-T7t, indicating that these colonies were positive for Tn5 integration.

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify Linker-Tn5 (1508 bp), Tn5 (1452 bp) and Tn5-Linker (1505 bp) coding sequence, high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP005, FP006, FP007 and FP008 as forward and reverse primers. Multiple 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol.](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll)

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 005 | 006 | Linker-Tn5 | 1508 |
| 2 | 007 | 006 | Tn5 | 1452 |
| 3 | 007 | 008 | Tn5-Linker | 1505 |

The thermocycler programme was installed to have an extension time of 2 min 30 seconds (1 minute longer than PCR reactions before) and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of the strain with pACYCDuet-1 were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   19/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 (1) | 15.5 | 1.85 | 1.36 |
| pACYCDuet-1 (2) | 14.0 | 1.60 | 0.86 |

Date
:   19-06-2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

Colonies 1,2,3,4,6 and 7 harbouring the pACYCDuet-1\_Tn5 plasmid and strains containing the plasmids pTXB1-Tn5 and pACYCDuet-1 were used for inoculation of 50 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Close

Wednesday, 20/06/2018

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of pACYCDuet-1 and pTXB1-Tn5 were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 (1) | 120.9 | 1.67 | 0.85 |
| pACYCDuet-1 (2) | 108.1 | 1.70 | 1.03 |
| pACYCDuet-1 (3) | 56.8 | 1.86 | 1.52 || pACYCDuet-1 (4) | 113.8 | 1.68 | 0.91 |
| pTXB1-Tn5 (1) | 90.6 | 1.81 | 1.45 || pTXB1-Tn5 (2) | 96.6 | 1.82 | 1.65 |
| pTXB1-Tn5 (3) | 106.4 | 1.79 | 1.36 || pTXB1-Tn5 (4) | 121.7 | 1.73 | 1.18 |

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

Two sets of 50 μL of PCR product of Tn5, Linker-Tn5 and Tn5-linker performed on and 19-06-2018 was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL prewarmed Milli-Q.

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Linker (1) | 190.3 | 1.90 | 1.38 |
| Tn5-linker (2) | 206.0 | 1.90 | 1.98 |
| Tn5 (1) | 258.4 | 1.88 | 2.04 || Tn5 (2) | 260.7 | 1.88 | 2.06 |
| Linker-Tn5 (1) | 199.7 | 1.88 | 1.86 || Linker-Tn5 (2) | 234.6 | 1.89 | 1.99 |

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 |
| Fragment (Tn5-Linker) | 10 (2000 ng) | Fragment (Tn5) | 8 (2000 ng) | Fragment (Linker-Tn5) | 9 (2000 ng) |
| Enzyme (FseI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 | Enzyme (KpnI) | 1 | Enzyme (NotI) | 1 |
| MilliQ | 6 | MilliQ | 8 | MilliQ | 7 |

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 |
| Fragment (pACYCDuet-1) | 16 (2000 ng) | Fragment (pACYCDuet-1) | 16 (2000 ng) | Fragment (pACYCDuet-1) | 16 (2000 ng) |
| Enzyme (FseI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 | Enzyme (KpnI) | 1 | Enzyme (NotI) | 1 |
| MilliQ | 0 | MilliQ | 0 | MilliQ | 0 |

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute heat inactivation (65 °C) of the restriction enzyme(s).

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 20 µL Milli-Q.

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 cut with FseI and KpnI | 18.7 | 1.71 | 1.02 |
| pACYCDuet-1 cut with EcoRI and KpnI | 41.2 | 1.80 | 1.44 |
| pACYCDuet-1 cut with EcoRI and NotI | 22.6 | 1.77 | 0.56 |
| Tn5-Linker cut with FseI and KpnI | 56.7 | 1.85 | 1.63 |
| Tn5 cut with EcoRI and KpnI | 59.2 | 1.84 | 1.20 |
| Linker-Tn5 cut with EcoRI and NotI | 70.0 | 1.87 | 1.73 |

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pACYCDuet-1 and Tn-Linker, Tn5 and Linker-Tn5 respectively, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3970 | EcoRI and NotI |
| Fragment Tn5-Linker | 1490 | EcoRI and NotI |

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3768 | KpnI and EcoRI |
| Fragment Tn5 | 1442 | KpnI and EcoRI |

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3984 | FseI and KpnI |
| Fragment Linker-Tn5 | 1493 | FseI and KpnI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector (~200 ng) | 10 | DNA vector (~200 ng) | 6 | DNA vector (~200 ng) | 11 |
| DNA Tn5-Lin (~80 ng) | 1.5 | DNA Tn5 (~80 ng) | 2 | DNA Lin-Tn5 (~80 ng) | 2 |
| MilliQ | 6.5 | MilliQ | 9 | MilliQ | 4 |

The samples were incubated for 4 hours at 20°C for ligation.

Date
:   20/06/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pACYCDuet-1\_Tn5-Lin (5460 bp) | Restriction/Ligation | 4 | LB-Cam || *E.coli* DH5α | pACYCDuet-1\_Tn5 (5210 bp) | Restriction/Ligation | 4 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_Linker-Tn5 (5477 bp) | Restriction/Ligation | 4 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 restricted (neg. control) | Restriction | 1 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 (pos. control) | - | 1 | LB Cam |

200 μl LB medium was added as recovery medium.

Close

Thursday, 21/06/2018

Date
:   21-06-2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1\_Lin-Tn5 after transformation from 20-06-2018. This plasmid contains the Lin-Tn5 insert, which can be checked with primers 029 and 030.

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Lin-Tn5 |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1722 bp |

23 tubes were prepared for colony PCR, containing pACYCDuet-1\_Lin-Tn5 as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   21-06-2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

Strain E.coli DH5a harbouring the pACYCDuet-1 plasmid was used for inoculation of 50 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll)

Close

Friday, 22/06/2018

Date
:   22-06-2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1\_Lin-Tn5 after transformation from 20-06-2018. This plasmid contains the Lin-Tn5 insert, which can be checked with primers 029 and 030.

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Lin-Tn5 |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1722 bp |

23 tubes were prepared for colony PCR, containing pACYCDuet-1\_Lin-Tn5 as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   21-06-2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The colony PCR performed on 21-06-2018 checking for pACYCDuet-1\_Linker-Tn5 was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/ca/T--TUDelft--2018_20180622_ColonyPCR.png)

Unfortunately, no band was observed that corresponds to the Linker-Tn5 fragment in pACYCDuet-1. The bands are at the same height as the positive control pACYCDuet-1, meaning that no insertion has taken place

Close

[ ]
Week 4 25/06/2018 - 01/07/2018

Wednesday, 27/06/2018

Date
:   27/06/2018

Operator
:   Nicole Bennis

#### Restriction

Restriction cloning was done for assembling Tn5, Tn5-Linker and Linker-Tn5 separately into pACYCDuet-1. The following table shows an overview of what fragment(s) were cut with what restriction enzymes.

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 |
| Fragment (Tn5-Linker) | 10 (2000 ng) | Fragment (Tn5) | 8 (2000 ng) | Fragment (Linker-Tn5) | 9 (2000 ng) |
| Enzyme (FseI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 | Enzyme (KpnI) | 1 | Enzyme (NotI) | 1 |
| MilliQ | 6 | MilliQ | 8 | MilliQ | 7 |

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 |
| Fragment (pACYCDuet-1) | 16 (2000 ng) | Fragment (pACYCDuet-1) | 16 (2000 ng) | Fragment (pACYCDuet-1) | 16 (2000 ng) |
| Enzyme (FseI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (KpnI) | 1 | Enzyme (KpnI) | 1 | Enzyme (NotI) | 1 |
| MilliQ | 0 | MilliQ | 0 | MilliQ | 0 |

The samples were incubated for 1 hour at 37 °C, and followed by 20 minute heat inactivation (65 °C) of the restriction enzyme(s).

Date
:   20-06-2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   27/06/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 cut with FseI and KpnI | 24.9 | 1.89 | 0.66 |
| pACYCDuet-1 cut with EcoRI and NotI | 20.8 | 1.81 | 1.08 |
| Tn5-Linker cut with NotI and EcoRI | 56.0 | 1.85 | 1.61 |
| Linker-Tn5 cut with FseI and KpnI | 53.4 | 1.88 | 1.57 |

Date
:   27/06/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pACYCDuet-1 and Tn-Linker and Linker-Tn5 respectively, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3970 | EcoRI and NotI |
| Fragment Tn5-Linker | 1490 | EcoRI and NotI |

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3984 | FseI and KpnI |
| Fragment Linker-Tn5 | 1493 | FseI and KpnI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector (~200 ng) | 14.5 | DNA vector (~200 ng) | 14.5 |
| DNA Tn5-Lin (~80 ng) | 2.5 | DNA Lin-Tn5 (~80 ng) | 2.5 |
| MilliQ | 0 | MilliQ | 0 |

The samples were incubated overnight at 20°C for ligation.

Close

Thursday, 28/06/2018

Date
:   28/06/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pACYCDuet-1\_Tn5-Lin (5460 bp) | Restriction/Ligation | 1.5 (~20 ng) | LB-Cam || *E.coli* DH5α | pACYCDuet-1\_Tn5-Lin (5460 bp) | Restriction/Ligation | 4 (~50 ng) | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_Linker-Tn5 (5477 bp) | Restriction/Ligation | 1.5 (~20 ng) | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_Linker-Tn5 (5477 bp) | Restriction | 4 (~50 ng) | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 (pos. control) | - | 0.1 (~10 ng) | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 restricted (neg. control) | Restriction | 2.5 | LB Cam |

200 μl LB medium was added as recovery medium.

Date
:   28/06/2018

Operator
:   Timothy Páez

#### Restriction

Restriction cloning was done for assembling Tn5-lin and lin-Tn5 into pACYCduet-1. The following table shows an overview of what fragments were cut with what restriction enzymes.
First Reaction:

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (Tn5-lin) | 9 |
| Enzyme (ECORI HF) | 1 |
| Enzyme (NotI HF) | 1 |
| MilliQ | 7 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC) | 16 |
| Enzyme (EcoRI HF) | 1 |
| Enzyme (NotI HF) | 1 |
| MilliQ | 0 |

Second reaction:

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (lin-Tn5) | 10 |
| Enzyme (FseI HF) | 1 |
| Enzyme (KpnI HF) | 1 |
| MilliQ | 6 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC) | 16 |
| Enzyme (FseI HF) | 1 |
| Enzyme (KpnI HF) | 1 |
| MilliQ | 0 |

The samples were incubated for 1 hour at 37 °C.

Date
:   28/06/2018

Operator
:   Timothy Páez

#### PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- Tn5-lin: 32.3 ng/µL
- pACYC: 12.2 ng/µL
- lin-Tn5: 33.6 ng/µL
- pACYC: 11.2 ng/µL

Date
:   28/06/2018

Operator
:   Timothy Páez

#### Ligation

In order to assemble cut fragments Tn5-lin into pACYCduet-1 and lin-Tn5 into pACYCduet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

First reaction:

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet | 1.5 kbp | EcoRI and NotI |
| Tn5-lin | 1.5 kbp | EcoRI and NotI |

The ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 µL || T4 DNA Ligase | 1 µL || pACYC (~100 ng) | 7.35 µL || Tn5 (300 ng) | 3.39 µL || MilliQ | 6.26 µL |

Second reaction:

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet | 1.5 kbp | FseI and KpnI |
| lin-Tn5 | 1.5 kbp | FseI and KpnI |

The ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 µL || T4 DNA Ligase | 1 µL || pACYC (~100 ng) | 8.77 µL || lin-Tn5 (300 ng) | 3.4 µL || MilliQ | 4.8 µL |

The samples were incubated overnight at 4 ºC for ligation.

Date
:   28/06/2018

Operator
:   Timothy Páez

#### Liquid Starter Culture Preparation

*Escherichia coli* DH5 alpha harbouring pACYC1-duet plasmid was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

Close

Friday, 29/06/2018

Date
:   29/06/2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1\_Lin-Tn5 after transformation from 28-06-2018. This plasmid contains the Lin-Tn5 or Tn5-Lin insert, which can be checked with primers 029 and 030. There are two ways for colony PCR. The first one is picking a colony and directly resuspending the cells in PCR mixture. The second one is resuspending the colony in MilliQ, heating the suspension, centrifugation the broken cell pieces and taking the supernantant for PCR. In orde to test the efficiency of both methods, I tested both simultaneously. 22 colony PCR were performed by picking the colony and direct resuspension in the PCR mixture, while 20 colonies were picked and heated prior to pipetting the DNA into the PCR mixture.

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Lin-Tn5 |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1722 bp |

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Tn5-Lin |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1935 bp |

The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   29/06/2018

Operator
:   Timothy Páez

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pACYC lin-Tn5 (3000 bp) | Restriction Ligation | 10 µL | LB with Chloroamphenicol |

400 µL LB medium was added as recovery medium.

Date
:   29/06/2018

Operator
:   Timothy Páez

#### Liquid Starter Culture Preparation

*Escherichia coli* DH5 alpha harbouring plasmid with dxCas9 from Addgene was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with ampicillin) following the Liquid Starter Culture Protocol.

Close

Saturday, 30/06/2018

Date
:   30/06/2018

Operator
:   Timothy Páez

#### Colony PCR

Colony PCR was done to verify propagation of *Escherichia coli* DH5 alpha cells after transformation with pACYC containing lin-Tn5 (reaction 1) or Tn5-lin (reaction 2) as insert. This insertion can be checked with primers 029 and 030 (on T7 promoter and terminator).

| Component |  |
| --- | --- |
| Template DNA | Colony solution |
| Forward primer | 029 |
| Reverse primer | 030 |
| Expected amplicon size (bp) | 1500 bp |

The number of colonies screened were:
- Tn5-lin: Colonies 1-6
- lin-Tn6: Colonies 8-10
- Control self ligation: Colony 11
The thermocycler programme was installed to have an extension time of 1 min 30 sec and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4°C.

Date
:   30/06/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

Colony PCR products were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane (no loading buffer required as PCR buffer contains loading buffer).
- 4 µL of 100 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/7/71/T--TUDelft--Lin-Tn5.png)

Figure 1. Gele electrophoresis from colony PCR. Lanes are (L) ladder 100 bp, (1) Colony 1,
(2) Colony 2, (3) colony 3, (4) colony 4, (5) colony 5, (6) colony 6, (7) colony 7, (8) colony 8, (9) colony 9, (10) colony 10, (11) colony 11

Date
:   30/06/2018

Operator
:   Timothy Páez

#### Liquid Starter Culture Preparation

*Escherichia coli* DH5 alpha cells that possibly harbour positive pACYC1-duet plasmids with lin-Tn5 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

Close

### July

[ ]
Week 1 02/07/2018 - 08/07/2018

Monday, 02/07/2018

Date
:   2/7/2018

Operator
:   Kavish Kohabir

#### Sequence Verification

##### Option 1: Sequence from plasmid

For verification of pTXB1-Tn5, primer FP030 was used for sequencing. The sequencing starts from the T7 terminator, in the reverse orientation respective to the Tn5 CDS. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | Volume (uL) | Final concentration |
| --- | --- | --- |
| Plasmid DNA (500ng) | 5.3 | 50 ng/uL |
| 10µM primer 030 | 2.5 | 25 pmol |
| MilliQ | 2.1 | - |

Within 7 days, the sequence was retrievable online and alignment with the *in silico* designed molecule showed 100% match of the 1050 bp downstream of the sequencing primer binding site.

Date
:   02/07/2018

Operator
:   Timothy Páez

#### Sequence Verification

##### Sequence from plasmid

For verification of Tn5-lin insertion in pACYC, primers 029 and 030 was used for sequencing. The sequencing starts from the -20 bp position of fragment Tn5-lin, flanking the promoter of T7 and terminator from T7 of the vector. Sequencing sample was prepared in 1.5 mL tubes for each primer as indicated my Macrogen for specific concentration of plasmid DNA.

Within 2 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the insert lin-Tn5, by aligning both primer sequences obtained.

Date
:   02/07/2018

Operator
:   Timothy Páez

#### High Fidelity PCR

To amplify dxCas9 (4 kbp), high fidelity PCR was done using Addgene plasmid harbouring dxCas9 as template DNA and 001 and 002 as forward and reverse primers respectively. Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 60 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   02/07/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

dxCas9 PCR product was run on a 0.8 % agarose gel in TAE buffer. 5μL was pipetted into each lane with 1 µL of loading dye. The gel was imaged using a gel documentation system with UV-light.

 No amplification was observed. It is recomended to do PCR amplification again with longer extention time.

Close

Tuesday, 03/07/2018

Date
:   3/7/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify dxCas9 (4172bp), high fidelity PCR was done using dxCas(3.7)-VPR as template DNA and FP001 and FP002 as forward and reverse primers respectively. 6 25µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.
The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4°C.

Date
:   3/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

dxCas9 PCR reaction products were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel shows that only samples 1, 2, 4 and 5 were successful attempts.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/0/0e/T--TUDelft--20180704KavishdxCas9PCR.jpg)

Date
:   02/07/2018

Operator
:   Timothy Páez

#### High Fidelity PCR

To amplify dxCas9 (4 kbp), high fidelity PCR was done using Addgene plasmid harbouring dxCas9 as template DNA and 001 and 002 as forward and reverse primers respectively. Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C and 65 ºC. After the programme was finished, samples were kept at 4 °C.

Date
:   03/07/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

PCR products of dxCas9 PCR were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane with 1 µL of loading buffer.
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/dd/T--TUDelft--03_07_dxCas9.png)

Figure 1. Gel electrophoresis from dxCas9 PCR. Lanes are (1-2) Amplification at 60 ºC anhelaing temperature, (3) negative control, (4-5) amplification at 65 ºC anhealing temperature and (6) negative control.

Close

Wednesday, 04/07/2018

Date
:   04/07/2018

Operator
:   Timothy Páez

#### Restriction

Restriction cloning was done for assembling Cas9 into pACYCduet-1 and pACYC-duet lin-Tn5. The following table shows an overview of what fragments were cut with what restriction enzymes.
First Reaction (dxCas9 into pACYC):

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (dxCas9) | 15 |
| Enzyme (NotI HF) | 0.5 |
| Enzyme (KpnI HF) | 2.5 |
| MilliQ | 0 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC) | 15 |
| Enzyme (NotI HF) | 0.5 |
| Enzyme (KpnI HF) | 2.5 |
| MilliQ | 0 |

Second reaction (dxCas9 into pACYC lin-Tn5):

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (dxCas9) | 15 |
| Enzyme (NotI HF) | 0.5 |
| Enzyme (FseI HF) | 2.5 |
| MilliQ | 0 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC lin-Tn5) | 15 |
| Enzyme (NotI HF) | 0.5 |
| Enzyme (FseI HF) | 2.5 |
| MilliQ | 0 |

The samples were incubated for 1 hour at 37 °C.

Date
:   04/07/2018

Operator
:   Timothy Páez

#### PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- pACYC: 12.7 ng/µL
- dxCas9: 40 ng/µL
- pACYC Tn5-lin: 9 ng/µL
- dxCas9: 31 ng/µL

Date
:   04/07/2018

Operator
:   Timothy Páez

#### Ligation

In order to assemble cut fragments Tn5-lin into pACYCduet-1 and lin-Tn5 into pACYCduet-1, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

First reaction:

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet | 1.5 kbp | NotI and KpnI |
| dxCas9 | 4 kbp | NotI and KpnI |

The ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 µL || T4 DNA Ligase | 1 µL || pACYC (~100 ng) | 5.13 µL || dxCas9 (300 ng) | 11.87 µL || MilliQ | 0 µL |

Second reaction:

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet lin Tn5 | 3 kbp | NotI and FseI |
| dxCas9 | 4 kbp | NotI and FseI |

The ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 µL || T4 DNA Ligase | 1 µL || pACYC linTn5 (~100 ng) | 8.55 µL || dxCas9 (300 ng) | 8.45 µL || MilliQ | 0 µL |

The samples were incubated overnight at 4 ºC for ligation.

Close

Thursday, 05/07/2018

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify Tn5-linker (1505bp), high fidelity PCR was done using pTXB1-Tn5 as template DNA and FP007 and FP008 as forward and reverse primers respectively. 3 100µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.
The thermocycler programme was installed to have an extension time of 1 minute and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Tn5-Linker PCR reaction products were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light. The gel was not saved properly, but the results showed correct amplification of a ~1.5kb amplicon in sample 1 and 3.

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

200 μL of PCR product was subjected to PCR Clean-Up, according to the protocol. Elution was done in 60 µL Milli-Q.

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Linker | 50 | 1.63 | 2.26 |

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 into pACYC-Duet & for assembling dxCas9 into pACYC-Duet. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (µL) | Volume (µL) | Volume (µL) | Volume (µL) | Volume (µL) || 10x CutSmart buffer | 2 | 2 | 2 | 2 | 2 |
| Fragment | 10 (~500ng) (Tn5-Linker) | 15 (~1.5µg)(dxCas9) | 15 (~1.5µg)(dxCas9) | 15.25 (~2µg) (pACYC-DUET) | 15.25 (~2µg) (pACYC-DUET) |
| Enzyme | 0.25 (EcoRI) | 2.5 (FseI) | 0.5 (NotI) | 2.5 (FseI) | 0.5 (NotI) |
| Enzyme | 0.5 (NotI) | 0.5 (NotI) | 0.5 (KpnI) | 0.25 (EcoRI) | 0.5 (KpnI) |
| MilliQ | 7.25 | 0 | 2 | 0 | 2 |

The samples were incubated for 1 hour(s) at 37 °C, and were not heat inactivated.

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

All restriction reaction mixtures were subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Linker (cut with EcoRI & NotI) | 6.5 | 1.74 | 2.26 |
| dxCas9 (cut with NotI & FseI) | 71 | 1.88 | 2.13 |
| pACYC-Duet (cut with EcoRI & FseI) | 24 | 1.76 | 2.19 |
| dxCas9 (cut with NotI & KpnI) | 84 | 1.77 | 2.22 |
| pACYC-Duet (Cut with Noti & KpnI) | 26 | 1.69 | 2.16 |

Date
:   5/7/2018

Operator
:   Kavish Kohabir

#### Ligation

In order to assemble cut Tn5-Linker and dxCas9 into pACYC-Duet, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector (pACYC-DUET) | 3792 | EcoRI & FseI |
| Fragment 1 (Tn5-Linker) | 1490 | EcoRI & NotI |
| Fragment 2 (dxCas9) | 4172 | FseI & NotI |

A 20µL ligation reaction mixture was prepared with the following composition\*

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (µL) || Ligase Buffer (10x) | 2 || T4 DNA Ligase | 1 || DNA vector (~100 ng) | 8 || DNA fragment 1 (26 ng) | 4 || DNA fragment 2 (150 ng) | 2.4 || MilliQ | 2.6 |

In order to assemble cut dxCas9 into pACYC-Duet, another ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector (pACYC-DUET) | 3806 | KpnI & NotI |
| Fragment 1 (dxCas9) | 4161 | KpnI & NotI |

A 20µL ligation reaction mixture was prepared with the following composition\*

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (µL) || Ligase Buffer (10x) | 2 || T4 DNA Ligase | 1 || DNA vector (~100 ng) | 2.2 || DNA fragment 1 (26 ng) | 2 || MilliQ | 12.8 |

*\*For both ligations, a negative control was included separately (in which the insert fragment was substituted with equivalent volume of milli-Q).*
All samples were incubated overnight on the bench (room temperature) for ligation.

Date
:   05/07/2018

Operator
:   Timothy Páez

#### Transformation of chemically competent cells

Using the ligate reactions from 04/07/2018, the following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5αlpha | pACYC dxCas9-lin-Tn5 (7500 bp) pACYC dxCas9 (5500 bp) | Restriction Ligation | 10 µL | LB with Chloroamphenicol |

400 µL LB medium was added as recovery medium.

Close

Friday, 06/07/2018

Date
:   6/7/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pACYC-Tn5Linker-dxCas9 (9419bp) | Restriction-ligation | 2.78 (~50ng) | LB+Cam || *E.coli*DH5α | pACYC-dxCas9 (7967bp) | Restriction-ligation | 4.6 (~50ng) | LB+Cam || *E.coli*DH5α | 3 negative controls (2x ligate without insert, 1x plain DH5α) | Restriction-ligation | 20 | LB+Cam || *E.coli*DH5α | pACYC-DUET (pos. ctrl) | - | 3 | LB+Cam |

500 μl LB medium was added as recovery medium. 1x and 10x dilutions were plated for each transformation.

Date
:   06/07/2018

Operator
:   Timothy Páez

#### Colony PCR

Colony PCR was done to verify propagation of *Escherichia coli* DH5 alpha cells after transformation with pACYC containing dxCas9 (reaction 1) or pACYC containing dxCas9-lin-Tn5 (reaction 2) as insert. This insertion can be checked with primers 029 and 030 (on T7 promoter and terminator). Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

| Component |  |
| --- | --- |
| Template DNA | Colony solution |
| Forward primer | 029 |
| Reverse primer | 030 |
| Expected amplicon size (bp) | (R1) 4000 bp  (R2) 6000 bp |

The number of colonies screened were:
- Cas9-lin-Tn5: Colonies 1-17
- Cas9: Colonies 18-45
- Control lin-Tn5: Colonies 46-68
- Control pACYC: Colonies 69-70
The thermocycler programme was installed to have an extension time of 6 min and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4°C.

Date
:   06/07/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

Colony PCR products were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane (no loading buffer required as PCR buffer contains loading buffer).
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/4/43/T--TUDelft--Colony_PCR1.png)

Figure 1. Gel electrophoresis from colony PCR. Lanes are (L) ladder 500 bp, (1-30) Colonies 1-39.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/50/T--TUDelft--07_07Colony_PCR2.png)

Figure 2. Gel electrophoresis from colony PCR. Lanes are (L) ladder 500 bp, (40-45) Colonies 40-45.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/6d/T--TUDelft--06_07_PCR3.png)

Figure 3. Gel electrophoresis from colony PCR. Lanes are (L) ladder 500 bp, (46-62) Colonies 46-62.

Date
:   06/07/2018

Operator
:   Timothy Páez

#### Liquid Starter Culture Preparation

*Escherichia coli* DH5 alpha cells that possibly harbour positive pACYC1-duet plasmids with dxCas9 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

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[ ]
Week 2 09/07/2018 - 15/07/2018

Monday, 09/07/2018

Date
:   09/07/2018

Operator
:   Nicole Bennis

#### Restriction

In our laboratory, we experienced some trouble with restriction ligation cloning. Therefore, I aimed for checking the efficiency of all restriction enzymes used in the cloning process. I used the pACYCDuet-1 plasmid and restricted this plasmid with the restriction enzyme XbaI and one of the restriction enzymes EcoRI, KpnI, NotI or FseI. To compare, I restricted the pACYCDuet-1 plasmid only with XbaI and as negative control I used no restriction enzyme to cut pACYCDuet-1.

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s).

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 | 10x CutSmart buffer | 2 |
| pACYCDuet-1 | 10 (~1000 ng) | pACYCDuet-1 | 10 (~1000 ng) | pACYCDuet-1 | 10 (~1000 ng) |
| Enzyme (XbaI) | 0.5 | Enzyme (XbaI) | 0.5 | Enzyme (none) | 0 |
| Enzyme (EcoRI, KpnI, FseI or NotI) | 0.5 | Enzyme (none) | 0 | Enzyme (none) | 0 |
| MilliQ | 7 | MilliQ | 7.5 | MilliQ | 8 |

The samples were incubated for 4 hour(s) at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date
:   09/07/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The restriction efficiency experiment performed on 09/07/2018 checking for activity of NotI, EcoRI, FseI and KnpI was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/ed/T--TUDelft--2018_20180709_Restriction_check.png)

From the DNA gel electrophoresis, it can be concluded that the restriction enzymes work efficiently for restriction ligation cloning purposes.

Date
:   9/7/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 6/7/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the plates were stored at 4°C until 9/7/2018 for observing the results

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli* DH5α | pACYC-Tn5Linker-dxCas9 (9419bp) | LB+Cam | 20 colonies (1x diluted)  4 colonies (10x diluted) |
| *E.coli* DH5α | pACYC-dxCas9 (7967bp) | LB+Cam | 3 colonies (1x diluted)  No colonies (10x diluted) |
| *E.coli* DH5α | None (all neg. controls) | LB+Cam | no colonies |
| *E.coli* DH5α | pACYC-Duet (pos. ctrl.) | LB+Cam | >500 colonies |

Date
:   9/7/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify propagation of pACYC-Tn5Linker-dxCas9 (9419bp) and pACYC-dxCas9 (7967bp) after transformation on 6/7/2018. These plasmids contain the T7 expression system, which allow primers FP029 and FP030 to anneal.

| **Template DNA** | pACYC-Tn5Linker-dxCas9 | pACYC-dxCas9 |
| --- | --- | --- |
| **FW primer** | 029 | 029 |
| **RV primer** | 030 | 030 |
| **Expected amplicon size (bp)** | 5894 | 4442 |

27 tubes were prepared for colony PCR, containing picked colony as template DNA. pACYC was used as positive control template. The thermocycler programme was installed to have an extension time of 6 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   9/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

All colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light. None of the reactions, not even the positive control showed presence of amplicon.

Date
:   09/07/2018

Operator
:   Timothy Páez

#### Sequence Verification

##### Sequence from plasmid

For verification of Tn5-lin insertion in pACYC, primers 003, 012, 029 and 030 were used for sequencing. The sequencing starts from the -20 bp position of fragment Tn5-lin, flanking the promoter of T7 and terminator from T7 of the vector. Sequencing sample was prepared in 1.5 mL tubes for each primer as indicated my Macrogen for specific concentration of plasmid DNA.

Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

Within 2 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the insert lin-Tn5, by aligning both primer sequences obtained.

Close

Tuesday, 10/07/2018

Date
:   10/07/2018

Operator
:   Nicole Bennis

#### Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 separately into pACYCDuet-1 or pACYCDuet-1\_Lin-Tn5, respectively. The following table shows an overview of what fragment(s) were cut with what restriction enzymes.

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 |
| pACYCDuet-1 | 41 (2000 ng) | Fragment (Tn5-Lin) | 41 (2000 ng) |
| Enzyme (NotI) | 2 | Enzyme (NotI) | 2 |
| Enzyme (EcoRI) | 2 | Enzyme (EcoRI) | 2 |
| MilliQ | 0 | MilliQ | 0 |

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| 10x CutSmart buffer | 5 | 10x CutSmart buffer | 5 |
| pACYCDuet-1\_Lin-Tn5 | 41 (2000 ng) | Fragment (dxCas9) | 41 (2000 ng) |
| Enzyme (NotI) | 2 | Enzyme (NotI) | 2 |
| Enzyme (FseI) | 2 | Enzyme (FseI) | 2 |
| MilliQ | 0 | MilliQ | 0 |

The samples were incubated overnight at 37 °C and followed by 20 minute heat inactivation (65 °C) of the restriction enzymes.

Date
:   10/07/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   10/07/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Lin cut with EcoRI and NotI | 36.3 | 1.80 | 0.09 |
| pACYCDuet-1 cut with EcoRI and NotI | 53.6 | 1.88 | 0.17 |
| dxCas9 cut with NotI and FseI | 59.5 | 1.87 | 0.49 |
| pACYCDuet-1\_Lin-Tn5 cut with FseI and NotI | 50.0 | 2.00 | 0.47 |

Date
:   10/07/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pACYCDuet-1 with Tn-Linker and pACYCDuet-1\_Lin-Tn5 with dxCas9 ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1 | 3970 | EcoRI and NotI |
| Fragment Tn5-Linker | 1490 | EcoRI and NotI |

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1\_Lin-Tn5 | 5299 | FseI and NotI |
| Fragment dxCas9 | 4150 | FseI and NotI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector (~200 ng) | 5 | DNA vector (~200 ng) | 5 |
| DNA Tn5-Lin (~100 ng) | 5 | DNA dxCas9 (~600 ng) | 12 |
| MilliQ | 7 | MilliQ | 0 |

The samples were incubated for 8 hours at 4°C for ligation.

Date
:   20-06-2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pACYCDuet-1\_Tn5-Lin (5460 bp) | Restriction/Ligation | 4 | LB-Cam || *E.coli* DH5α | pACYCDuet-1\_dxCas9-Linker-Tn5 (9449 bp) | Restriction/Ligation | 4 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 restricted (neg. control) | Restriction | 1 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1 (pos. control) | - | 1 | LB Cam |

200 μl LB medium was added as recovery medium.

Date
:   10/7/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify propagation of pACYC-Tn5Linker-dxCas9 (9419bp) and pACYC-dxCas9 (7967bp) after transformation on 6/7/2018. These plasmids contain the T7 expression system, which allow primers FP029 and FP030 to anneal.

| **Template DNA** | pACYC-Tn5Linker-dxCas9 | pACYC-dxCas9 |
| --- | --- | --- |
| **FW primer** | 029 | 029 |
| **RV primer** | 030 | 030 |
| **Expected amplicon size (bp)** | 5894 | 4442 |

27 tubes were prepared for colony PCR, containing boiled colony as template DNA (colonies were picked from the restreaked plate on 9/7/2018). Vector pACYC was used as positive control template. The thermocycler programme was installed to have an extension time of 6 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   10/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

All colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
Colonies 11, 13, 20, 21, 22, 23, 24 and 25 show vague smears around the expected height (Tn5LCas = 5894bp)

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/2c/T--TUDelft--20180710KAVISHboiledcolonypcr.jpg)

Date
:   10/7/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Colonies 11, 13, 20, 21, 22, 23, 24 and 25 (all potentially pACYC-Tn5Linker-dxCas9) were used for inoculation of 5mL liquid starter culture (LB+Cam) each, following the Liquid Starter Culture Protocol.

Close

Wednesday, 11/07/2018

Date
:   11/07/2018

Operator
:   Nicole Bennis

#### Restriction

In our laboratory, we experienced some trouble with restriction ligation cloning. Therefore, will use a different restriction buffer: the universal buffer of JenaBioscience. Also, I will try to ligate the Tn-Linker and Linker-Tn5 separately to the dxCas9 prior to cloning into pACYCDuet-1. The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s).

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| 10x Universal buffer | 2 | 10x Universal buffer | 2 |
| dxCas9 | 10 (~2000 ng) | Tn5-Linker | 10 (~2000 ng) |
| Enzyme (NotI) | 0.5 | Enzyme (NotI) | 0.5 |
| MilliQ | 7.5 | MilliQ | 7.5 |

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| 10x Universal buffer | 2 | 10x Universal buffer | 2 |
| dxCas9 | 10 (~2000 ng) | Linker-Tn5 | 10 (~2000 ng) |
| Enzyme (FseI) | 0.5 | Enzyme (FseI) | 0.5 |
| MilliQ | 7.5 | MilliQ | 7.5 |

The samples were incubated for 4 hour(s) at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date
:   11/07/2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1\_Tn5-Lin after transformation from 10/07/2018. This plasmid contains the Tn5-Lin insert, which can be checked with primers 029 and 030. Also, the pACYC1Duet-1\_dxCas9-Lin-Tn5 transformation resulted in colonies, which were also checked. However, on the negative control transformation plate with restricted pACYCDuet-1, colonies were observed.

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Tn5-Lin |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1935 bp |

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_dxCas9-Lin-Tn5 |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 5924 bp |

15 tubes were prepared for colony PCR, containing pACYCDuet-1\_dxCas9-LinTn5 as template DNA. Additionally, 14 tubes were prepared for colony PR containing pACYCDuet-1\_Tn5-Lin as template DNA. For positive control, 4 tubes for colonies with pACYCDuet-1 were used. The thermocycler programme was installed to have an extension time of 6 and 4 minutes respectively and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   11/7/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of colonies 11, 13, 19, 20, 21, 22, 23, and 24 (all potentially pACYC-Tn5Linker-dxCas9) were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   11/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

Plasmid isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentrations were measured and verified to be higher than 10ng/µl. The purity of the DNA (260/280 and 260/230 ratios) was also checked to be approximately 1.8~1.9.

Date
:   11/7/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Diagnostic PCR on isolated plasmids from inoculated colonies was done as attempt to verify propagation of pACYC-Tn5Linker-dxCas9 (9419bp) after transformation on 6/7/2018. This plasmids contains the T7 expression system, which allow primers FP029 and FP030 to anneal.

| **Template DNA** | pACYC-Tn5LinkerdxCas9 |
| --- | --- |
| **Fw primer** | 029 |
| **Rv primer** | 030 |
| **Expected amplicon size (bp)** | 5894 |

8 tubes were prepared for colony PCR, containing isolated plasmid as template DNA (isolated from colonies inoculated on 10/7/2018). Vector pACYC was used as positive control template. The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   11/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel showed all of the plasmid isolate templates result in double bands, which is corresponding to the pACYC-DUET control (lane 9). Compared to the Tn5 insert (lane 10), this indicates all of the plasmid isolates are pACYC-DUET and contain no insert.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/7/7d/T--TUDelft--20180711kavishPCRonplasmid.jpg)

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Thursday, 12/07/2018

Date
:   12/07/2018

Operator
:   Nicole Bennis

#### Colony PCR

Colony PCR was done to verify propagation of pACYC1Duet-1\_Lin-Tn5 after transformation from 11/07/2018. This plasmid contains the Lin-Tn5 insert, which can be checked with primers 029 and 030.

|  |  |
| --- | --- |
| Template DNA | pACYCDuet-1\_Lin-Tn5 |
| Fw primer | 029 |
| Rv primer | 030 |
| expected amplicon size (bp) | 1722 bp |

23 tubes were prepared for colony PCR, containing pACYCDuet-1\_Lin-Tn5 as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   12/06/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The colony PCR performed on 11-07-2018 checking for pACYCDuet-1\_Tn5-Linker and pACYCDuet-1\_dxCas9-Lin-Tn5 was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/12/T--TUDelft--2018_20180712_Colony_PCR.png)

Unfortunately, no band was observed that corresponds to the dxCas9 fragment in pACYCDuet-1\_Linker-Tn5. Also, no band was observed that indicates integration of Tn5-Lin into pACYCDuet. The bands are at the same height as the positive control pACYCDuet-1, meaning that no insertion has taken place.

Date
:   12/7/2018

Operator
:   Kavish Kohabir

#### Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 into pACYC-Duet & for assembling dxCas9 and Linker-Tn5 into pACYC-Duet. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (µL) | Volume (µL) | Volume (µL) | Volume (µL) | Volume (µL) || 10x CutSmart buffer | 5 | 5 | 5 | 5 | 5 |
| Fragment | 5 (~1µg) (Tn5-Linker) | 14 (~1µg)(dxCas9) | 25 (~1µg)(pACYC-DUET) | 5 (~2µg) (Linker-Tn5) | 25 (~1µg) (pACYC-DUET) |
| Enzyme | 0.5 (EcoRI) | 1 (FseI) | 1 (FseI) | 1 (FseI) | 0.5 (NotI) |
| Enzyme | 0.5 (NotI) | 0.5 (NotI) | 0.25 (EcoRI) | 0.5 (KpnI) | 0.5 (KpnI) |
| MilliQ | 39 | 29.5 | 18.5 | 38.5 | 19 |

The samples were incubated for 4 hour(s) at 37 °C, and were not heat inactivated.

Date
:   12/7/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

Restriction products from 12/7/2018 were subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date
:   12/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Cut with | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- | --- |
| Tn5-Linker | EcoRI&NotI | 48 | 1.9 | 1.1 |
| Linker-Tn5 | FseI&KpnI | 30 | 2 | 0.9 |
| dxCas9 | FseI & NotI | 8.2 | 2.2 | 0.3 |
| pACYC-DUET | FseI&EcoRI | 18 | 1.8 | 0.6 |
| pACYC-DUET | FotI&KpnI | 15 | 1.8 | 0.6 |

Date
:   12/7/2018

Operator
:   Kavish Kohabir

#### Ligation

In order to assemble cut Tn5-Linker and cut dxCas9 into cut pACYC-Duet, ligation was performed. This table gives an overview of which fragments were cut with which enzymes. the approach taken is a single-pot ligation, in which both inserts (dxCas9 and Tn5)

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector (pACYC-DUET) | 3792 | EcoRI & FseI |
| Fragment 1 (Tn5-Linker) | 1490 | EcoRI & NotI |
| Fragment 2 (dxCas9) | 4172 | NotI & FseI |

A 20 µl ligation reaction mixture was prepared with the following composition\*

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 3 || T4 DNA Ligase | 1.5 || DNA vector (~50 ng) | 2.5 || DNA fragment 1 (~50 ng) | 1 || DNA fragment 2 (~60 ng) | 20 || MilliQ | 2 |

In order to assemble cut dxCas9 and cut Linker-Tn5 into cut pACYC-Duet, another ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector (pACYC-DUET) | 3806 | NotI & KpnI |
| Fragment 1 (dxCas9) | 4161 | FseI & NotI |
| Fragment 2 (Linker-Tn5) | 1508 | FseI & KpnI |

A 20 µl ligation reaction mixture was prepared with the following composition\*

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 3 || T4 DNA Ligase | 1.5 || DNA vector (~50 ng) | 2.5 || DNA fragment 1 (~50 ng) | 20 || DNA fragment 2 (~50 ng) | 1.8 || MilliQ | 1.2 |

*\*For both ligations, a negative control was included separately (in which ONE of the insert fragments was substituted with equivalent volume of milli-Q).*
All samples were incubated overnight on the bench (room temperature) for ligation.

Close

Friday, 13/07/2018

Date
:   13/07/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture done on 11/07/2018 was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   13/07/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Lin cut with NotI | 58.1 | 1.92 | 0.70 |
| dxCas9 cut with NotI | 72.3 | 1.84 | 1.68 |
| Linker-Tn5 cut with FseI | 59.5 | 1.88 | 1.34 |
| dxCas9 cut with FseI | 48.7 | 1.84 | 0.76 |

Date
:   13/07/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments dxCas9 with Tn-Linker and dxCas9 with Linker-Tn5 respectively, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| dxCas9 | 4172 | NotI |
| Fragment Tn5-Linker | 1495 | NotI |

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| dxCas9 | 4157 | FseI |
| Linker-Tn5 | 1508 | FseI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA dxCas9 (~200 ng) | 4 | DNA dxCas9 (~200 ng) | 4 |
| DNA Tn5-Lin (~100 ng) | 2 | DNA Lin-Tn5 (~100 ng) | 2 |
| MilliQ | 11 | MilliQ | 11 |

The samples were incubated for 2.5 hours at 4°C for ligation.

Date
:   19-06-2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp) coding sequence from the ligation product, high fidelity PCR was done. The template DNA is the ligation mixture and the primers that are used are the combinations 001 & 006 and 002 & 007 respectively. Two 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 001 | 006 | dxCas9-Linker-Tn5 | 5655 |
| 2 | 002 | 007 | Tn5-Linker-dxCas9 | 5660 |

The thermocycler programme was installed to have an extension time of 6 min 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   13/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

The restriction products from 11/7/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel shows that one of the dxCas9 samples (lane 3) was not purified.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/62/T--TUDelft--20180713kavishrestrictionCheck.jpg)

Date
:   13/7/2018

Operator
:   Kavish Kohabir

#### Diagnostic PCR

Diagnostic PCR on ligation samples from 12/7/2018 were done to check for presence of correct constructs. Primer sets were chosen based on unique binding opportunities either to pACYC-Duet, dxCas9 and/or Tn5. The expected bands were estimated based on *in silico* design.

| Template DNA | pACYC-Tn5Linker-dxCas9 | | | | pACYC-dxCas9-LinkerTn5 | | | |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Fw primer | 029 | 007 | 001 | 029 | 029 | 001 | 007 | 029 |
| Rv primer | 030 | 002 | 030 | 006 | 030 | 006 | 030 | 002 |
| Expected amplicon size (bp) | 5907 | 5660 | 4295 | 1576 | 5924 | 5655 | 1561 | 4332 |

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a9/T--TUDelft--20180713kavishprimersetupOnePotLigation.png)

The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C

Date
:   13/7/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (µL) | Medium |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pACYC-Tn5Linker-dxCas9 (~10kb) | Restriction-ligation | 20µL | LB+Cam || *E.coli*DH5α | pACYC-dxCas9-LinkerTn5 (~10kb) | Restriction-ligation | 20µL | LB+Cam || *E.coli*DH5α | pACYC-dxCas9-LinkerTn5 (~10kb) | Restriction-ligation | - | LB+Cam |

500 μl LB medium was added as recovery medium. 1x and 10x dilutions were plated for each transformation.

Close

[ ]
Week 3 16/07/2018 - 22/07/2018

Monday, 16/07/2018

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The ligation performed on 13/07/2018 for construction of Tn5-Linker-dxCas9 and dxCas9-Lin-Tn5 was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/1e/T--TUDelft--2018_20180716_Ligation_fusion.png)

The ligation reaction resulted in the expected sizes of the dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp) coding sequence.

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp), high fidelity PCR was done using the ligation reactions as template DNA ausing the forward and reverse primers sets 001 & 007 and 002 & 006 respectively. 2 times 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 001 | 006 | dxCas9-Linker-Tn5 | 5655 |
| 2 | 002 | 007 | Tn5-Linker-dxCas9 | 5660 |

The thermocycler programme was installed to have an extension time of 60 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

To amplify the ligation products Tn5-Linker-dxCas9 and dxCas9-Lin-Tn5 a High Fidelity PCR was performed and the result was run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/3/3b/T--TUDelft--20180716_PCR_Ligation_fusion.png)

The PCR reaction on the ligation products resulted in the expected sizes of the dxCas9-Linker-Tn5 (5655 bp) Tn5-Linker-dxCas9 (5660 bp) coding sequence in large amounts of DNA. However, there is also one other band of DNA present, indicating an off-target amplicon generated during the PCR.

Date
:   16-07-2018

Operator
:   Nicole Bennis

#### DNA Gel Excision and Purification

Due to presence of off-target amplicons, the PCR mixture is again loaded on a gel. Subsequently, the required fragments containing Tn5-Linker-dxCas9 and dxCas9-Lin-Tn5 were excised. The agarose slice containing the desired DNA were purified by a DNA Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL prewarmed Milli-Q.

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Linker-dxCas9 | 37.0 | 1.32 | 0.77 |
| dxCas9-Lin-Tn5 | 35.9 | 1.87 | 0.52 |

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify dxCas9, Linker-Tn5 (1508 bp) and Tn5-Linkr (1505 bp), high fidelity PCR was done. The forward and reverse primers sets that were used are 001 & 002 and 005 & 006 and 007 & 008 respectively. Two times 3 times 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll). However, one of the sets of 3 times 50µL PCR mixtures uses HF buffer and Phusion Polymerase, while the other set of 3 times 50µL PCR mixtures uses Q5 buffer and Q5 Polymerase.

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 001 | 002 | dxCas9 | 4172 |
| 2 | 005 | 006 | Linker-Tn5 | 1508 |
| 3 | 007 | 008 | Tn5-Linker | 1505 |

The thermocycler programme was installed to have an extension time of 6 minutes and 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The amplicons generated during the High Fidelity PCR were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/6c/T--TUDelft--20180716_PCR_dxCas9_Tn5_linkers.png)

The PCR reaction o resulted in the expected sizes of the dxCas9 (4172 bp) Tn5-Linker (1505 bp) and Linker-Tn5 (1508 bp) coding sequence in large amounts of DNA.

Date
:   16/07/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

*E.coli* strains harbouring the plasmid pACYCDuet-1 and pACYCDuet-1\_Lin-Tn5 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with ampicillin) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Date
:   16/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Diagnostic PCR reaction products from 13/7/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
This gel shows that partially, the constructs have been ligated (e.g. lane 8 & 9), but the complete plasmid does not seem to be constructed (lanes 2 & 6).

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/87/T--TUDelft--20180716kavishOnePotPCR.jpg)

Close

Tuesday, 17/07/2018

Date
:   17/07/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of pACYCDuet-1\_Lin-Tn5 and pACYCDuet-1 were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   17/07/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 μL of each PCR reaction done on 16/07/2018 was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   17-07-2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 | 7.1 | 2.19 | 1.23 |
| pACYCDuet-1 | 7.9 | 1.81 | 1.19 |
| pACYCDuet-1 | 10.7 | 1.52 | 0.77 |
| pACYCDuet-1\_Lin-Tn5 | 15.5 | 1.69 | 0.89 |
| pACYCDuet-1\_Lin-Tn5 | 10.1 | 1.59 | 0.99 |
| pACYCDuet-1\_Lin-Tn5 | 10.2 | 1.64 | 0.97 |
| Q5: Lin-Tn5 | 180.9 | 1.80 | 1.52 |
| Q5: Tn5-Lin | 148.3 | 1.80 | 1.92 |
| Q5: dxCas9 | 259.6 | 1.84 | 1.82 |
| Phusion: Lin-Tn5 | 183.8 | 1.85 | 1.86 |
| Phusion: Tn5-Lin | 164.1 | 1.85 | 1.86 |
| Phusion: dxCas9 | 233.9 | 1.83 | 1.77 |

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

The following high fidelity PCR reactions were carried out:

| Amplicon | Size (bp) | Fw primer | Rv primer | template | Elongation time (sec) | Annealing temperature (°C) |
| --- | --- | --- | --- | --- | --- | --- |
| LacZ-gRNA | 314 | IV003 | IV004 | Gblock | 60 | 60 |
| KanR CDS | 862 | IV005 | IV006 | pSB1K3 | 60 | 60 |
| dxCas9 | ~4200 | 001 | 002 | dxCas9(3.7)-VPR | 240 | 60 |
| Tn5-Linker | ~1500 | 007 | 008 | pACYC-Tn5Linker | 240 | 60 |
| Linker-Tn5 | ~1500 | 005 | 006 | pACYC-LinkerTn5 | 240 | 60 |
| dxCas9-LinkerTn5 | ~6000 | 001 | 006 | dxCas9 & Tn5 | 240 | 60 |
| Tn5Linker-dxCas9 | ~6000 | 007 | 002 | dxCas9 & Tn5 | 240 | 60 |

PCR mixtures were prepared, according to the High Fidelity PCR protocol. After the programme was finished, samples were kept at 4°C.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

PCR products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 50 µL Milli-Q.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | 260/280 ratio | 260/230 ratio | Concentration (ng/uL) |
| --- | --- | --- | --- |
| LacZ-gRNA |  |  | 85 |
| KanR cassette |  |  | 209 |
| dxCas9 |  |  | 128 |
| Tn5Linker |  |  | 162 |
| LinkerTn5 | 1.87 | 2.02 | 181 |
| dxCas9Linker-Tn5 | 1.82 | 1.88 | 74.8 |
| Tn5Linker-dxCas9 | 1.81 | 1.91 | 78.7 |
| dxCas9Tn5+upstr EcoRI | 1.79 | 1.81 | 56.8 |
| Tn5dxCas9 + upstr EcoRI | 1.8 | 1.99 | 89.4 |
| dxCas9Tn5 + downstr EcoRI | 1.85 | 1.93 | 109 |
| Tn5dxCas9 + downstr EcoRI | 1.82 | 1.99 | 101.4 |
| dxCas9 | 1.85 | 2.11 | 138 |

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Restriction

Restriction cloning was done for assembling Tn5-Linker and dxCas9 into pACYC-Duet & for assembling dxCas9 into pACYC-Duet. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) | Volume (uL) | Volume (uL) | Volume (uL) | Volume (uL) || 10x CutSmart buffer | 5 | 5 | 5 | 5 | 5 |
| Fragment | 43 (pACYC-LinkerTn5) | 10 (dxCas9) | 43 (pACYC-LinkerTn5) | 15 (Tn5-Linker) | 10 (dxCas9) |
| Enzyme | 1 (NotI) | 1 (FseI) | 1 (EcoRI) | 1 (EcoRI) | 1 (NotI) |
| Enzyme | 1 (FseI) | 1 (NotI) | 1 (KpnI) | 1 (NotI) | 1 (KpnI) |
| MilliQ | 0 | 0 | 0 | 28 | 33 |

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) | Volume (uL) | Volume (uL) | Volume (uL) | Volume (uL) || 10x CutSmart buffer | 5 | 5 | 5 | 5 | 5 |
| Fragment | 43 (pACYC-DUET) | 25 (Tn5Linker-dxCas9) | 15 (Tn5-Linker) | 43 (pACYC-DUET) | 10 (dxCas9-LinkerTn5) |
| Enzyme | 1 (NotI) | 1 (FseI) | 1 (EcoRI) | 1 (EcoRI) | 1 (NotI) |
| Enzyme | 1 (FseI) | 1 (NotI) | 1 (KpnI) | 1 (NotI) | 1 (KpnI) |
| MilliQ | 0 | 0 | 0 | 28 | 33 |

The samples were incubated for 4 hour(s) at 37 °C, and were not heat inactivated.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

Restriction products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 50 µL Milli-Q.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Fragment | Cut with | 260/280 ratio | 260/230 ratio | Concentration (ng/uL) |
| --- | --- | --- | --- | --- |
| pACYC-LinkerTn5 | NotI & FseI | 1.75 | 0.69 | 8 |
| dxCas9 | NotI & FseI | 1.85 | 1.51 | 2.37 |
| pACYC | EcoRI & KpnI | 1.88 | 1.05 | 10.6 |
| Tn5Linker | NotI & EcoRI | 1.82 | 1.33 | 26.1 |
| dxCas9 | NotI & KpnI | 1.86 | 1.51 | 29.6 |
| pACYC-DUET | NotI & KpnI | 1.81 | 1.03 | 13.8 |
| dxCas9 | NotI & FseI | 1.84 | 1.19 | 20.8 |
| Linker-Tn5 | FseI & KpnI | 1.84 | 1.54 | 42 |
| pACYC-DUET | NotI & KnpI | 1.79 | 1.06 | 13.1 |
| dxCas9-LinkerTn5 | NotI & KnpI | 1.79 | 0.54 | 5.5 |
| pACYC-DUET | EcoRI & KpnI | 1.67 | 0.97 | 13 |
| Tn5Linker-dxCas9 | EcoRI & KpnI | 2 | 0.78 | 6.1 |

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Ligation

In order to assemble cut fragments, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

A 50µl ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | L1\* Volume (uL) | L2\* Volume (uL) | L3\* Volume (uL) | L4\* Volume (uL) | L5\* Volume (uL) || Ligase Buffer (10x) | 5 | 5 | 5 | 5 | 5 || T4 DNA Ligase | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 || DNA vector | 24 (pACYC-LinkerTn5, ~200ng) | 17 (pACYC, ~200ng) | 12.5 (pACYC, ~150ng) | 4 (pACYC, ~50ng) | 4.3 (pACYC, ~100ng) || DNA insert 1 | 18 (dxCas9, ~200ng) | 17.5 (Tn5Linker, ~200ng) | 25 (dxCas9, ~500ng) | 39(dxCas9LinkerTn5, ~200ng) | 17.5 (Tn5LinkerdxCas9, ~200ng) |  || DNA nsert 2 (optional) |  | 18 (dxCas9, ~200ng) | 5 (LinkerTn5, ~200ng) |  |  |  || MilliQ | 0.5 | 0 | 0 | 0 | 0.2 |

*\*L1, L2, L3, L4 and L5 are the names of the ligation reactions. L2 and L3 are single-pot ligation reactions, which aim to ligate two inserts into 1 vector simultaneously.*
The samples were incubated for 6 hours at 37°C for ligation. Samples were not heat inactivated.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

Solid Luria Broth (LB) medium was prepared, complemented with chloramphenicol (Cam)

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | L1 (Ligate) | Restriction&Ligation | 20 | LB+Cam || *E.coli*DH5α | L2 (Ligate) | Restriction&Ligation | 20 | LB+Cam || *E.coli*DH5α | L3 (Ligate) | Restriction&Ligation | 20 | LB+Cam || *E.coli*DH5α | L4 (Ligate) | Restriction&Ligation | 20 | LB+Cam || *E.coli*DH5α | L5 (Ligate) | Restriction&Ligation | 20 | LB+Cam |

200 uL LB medium was added as recovery medium.

Date
:   17/07/2018

Operator
:   Timothy Páez

#### Restriction

Restriction cloning was done for assembling Cas9 into pACYCduet lin-Tn5 or lin-Tn5 into pACYCduet dxCas9. Both approaches will render the same insert. The following table shows an overview of what fragments were cut with what restriction enzymes.
First Reaction (lin-Tn5 into pACYC dxCas9):

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (lin-Tn5) | 16 |
| Enzyme (Fse HF) | 1 |
| Enzyme (KpnI HF) | 1 |
| MilliQ | 0 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC dxCas9) | 16 |
| Enzyme (Fse HF) | 1 |
| Enzyme (KpnI HF) | 1 |
| MilliQ | 0 |

Second reaction (dxCas9 into pACYC lin-Tn5):

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Fragment (dxCas9) | 16 |
| Enzyme (NotI HF) | 1 |
| Enzyme (FseI HF) | 1 |
| MilliQ | 0 |

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || 10x CutSmart buffer | 2 |
| Vector (pACYC lin-Tn5) | 16 |
| Enzyme (NotI HF) | 1 |
| Enzyme (FseI HF) | 1 |
| MilliQ | 0 |

The samples were incubated for 4 hours at 37 °C.

Date
:   17/07/2018

Operator
:   Timothy Páez

#### PCR clean-up

20 μL of restriction product were subjected to PCR Clean-Up, according to the protocol. Elution was done in 20 µL Milli-Q.

Products were quantified with Nanodrop, yielding concentration of:
- pACYC dxCas9: 42.7 ng/µL
- lin-Tn5: 34 ng/µL
- pACYC lin-Tn5: 31 ng/µL
- dxCas9: 46 ng/µL

Date
:   17/07/2018

Operator
:   Timothy Páez

#### Ligation

In order to assemble cut fragments lin-Tn5 into pACYC dxCas9 (Reaction 1) and dxCas9 into pACYC lin-Tn5, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

First reaction:

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet dxCas9 | 5.5 kbp | Fse and KpnI |
| lin-Tn5 | 1.5 kbp | Fse and KpnI |

Two different molar ratios between insert and plasmid were tried to attempt higher integration efficiency. Ratios of 1:3 and 1:10 (vector:plasmid) were incubated with T4 ligase (1 µL) and ligase buffer (2 µL). Total volume of reaction was 20 µL.

Second reaction:

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pACYCDuet lin Tn5 | 3 kbp | NotI and FseI |
| dxCas9 | 4 kbp | NotI and FseI |

Two different molar ratios between insert and plasmid were tried to attempt higher integration efficiency. Ratios of 1:3 and 1:10 (vector:plasmid) were incubated with T4 ligase (1 µL) and ligase buffer (2 µL). Total volume of reaction was 20 µL.

The samples were incubated overnight at 4 ºC for ligation.

Close

Wednesday, 18/07/2018

Date
:   18/7/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 17/7/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli* DH5α | L1 | LB+Cam | 2 colonies || *E.coli* DH5α | L2 | LB+Cam | 30 colonies || *E.coli* DH5α | L3 | LB+Cam | 2 colonies || *E.coli* DH5α | L4 | LB+Cam | 4 colonies || *E.coli* DH5α | L5 | LB+Cam | 19 colonies |

Date
:   18/7/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify propagation of ligate constructs after transformation (17/7/2018). This plasmid contains T7 expression system, which can be checked with primers 029 and 030.

| Component | L1 | L2 | L3 | L4 | L5 |
| --- | --- | --- | --- | --- | --- |
| Fw primer | 029 | 029 | 029 | 029 | 029 |
| Rv primer | 030 | 030 | 030 | 030 | 030 |
| Expected amplicon size (kbp) | ~5.7 | ~5.7 | ~5.7 | ~5.7 | ~5.7 |

48 tubes were prepared for colony PCR, containing boiled colony as template DNA. The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   18/7/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f8/T--TUDelft--20180718kavishColonyPCRjpg.png)

It is apparent that no amplicons of interest are found, other than template/uncut/self-closing plasmids (lane 3 & 25). Judging from the double bands around 200 and 500, most of the clones are identified to contain plain pACYC-DUET, which contains double T7 promoter, resulting in double amplicons around that height.

Date
:   18/07/2018

Operator
:   Timothy Páez

#### Transformation of chemically competent cells

Using the ligate reactions from 17/07/2018, the following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5αlpha | pACYC dxCas9-lin-Tn5 (7500 bp) | Restriction Ligation | 10 µL | LB with Chloroamphenicol |

400 µL LB medium was added as recovery medium.

Close

Thursday, 19/07/2018

Date
:   19/07/2018

Operator
:   Timothy Páez

#### Colony PCR

Colony PCR was done to verify propagation of *Escherichia coli* DH5 alpha cells after transformation with pACYC dxCas9 containing lin-Tn5 (Reaction 1) or pACYC linTn5 containing dxCas9 (Reaction 2) as insert. This insertion can be checked with primers 029 and 030 (on T7 promoter and terminator), but were also tested with primers 001 with 002 for dxCas9 amplification and 005 with 006 for Tn5 amplification. Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

| Component |  |
| --- | --- |
| Template DNA | Colony solution |
| Forward primer | 029, 001, 005 |
| Reverse primer | 030, 002, 006 |
| Expected amplicon size (bp) | 6 kbp, 4 kbp, 1.5 kbp |

The number of colonies screened were:
- Tn5 control : Colony 1
- Cas9-lin-Tn5 (Reaction 1): Colonies 2-10
- Cas9-lin-Tn5 (Reaction 2): Colonies 11-14
The thermocycler programme was installed to have an extension time of 6 min and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4°C.

Date
:   19/07/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

Colony PCR products were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane (no loading buffer required as PCR buffer contains loading buffer).
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/0/02/T--TUDelft--19_07_PCRfusion.png)

Figure 1. Gel electrophoresis from colony PCR for dxCas9-lin-Tn5 amplification. Expected size of integration is 6000 bp. Lanes are (L) ladder 500 bp, (1-14) Colonies 1-14.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/1b/T--TUDelft--19_07_PCR_fusionCas9.png)

Figure 2. Gel electrophoresis from colony PCR for dxCas9 amplification. Expected size of amplification is 4000 bp. Lanes are (L) ladder 500 bp, (1-14) Colonies 1-14.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a4/T--TUDelft--19_07_PCR_fusionTn5.png)

Figure 3. Gel electrophoresis from colony PCR for Tn5 amplification. Expected size of integration is 1500 bp. Lanes are (L) ladder 500 bp, (1-14) Colonies 1-14.

Close

### August

[ ]
Week 1 30/07/2018 - 05/08/2018

Friday, 03/07/2018

Date
:   03/08/2018

Operator
:   Timothy Páez

#### Liquid Starter Culture Preparation

Strains *Escherichia coli DH5 alpha* harbouring plasmid with possible fusion protein dxCas9-lin-Tn5 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloroamphenicol) following the Liquid Starter Culture Protocol.

Close

Saturday, 04/07/2018

Date
:   04/08/2018

Operator
:   Timothy Páez

#### Plasmid Isolation

Overnight starter cultures of *Escherichia coli DH5 alpha* harbouring plasmid with possible fusion protein dxCas9-lin-Tn5 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   04/08/2018

Operator
:   Timothy Páez

#### High Fidelity PCR

To amplify dxCas9-lin-Tn5 (6 kbp), high fidelity PCR was done using colonies' plasmid harbouring the fusion protein as template DNA and 029 and 030 as forward and reverse primers respectively. Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6 minutes and an annealing temperature of 55 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   03/07/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

PCR products of dxCas9 PCR were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane with 1 µL of loading buffer.
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/66/T--TUDelft--04_08_PCR_Fusion.png)

Figure 1. Gel electrophoresis from fusion protein dxCas9-lin-Tn5. Lanes are (L) Ladder 500 bp, (1) Blank, (2-3) Positive integration.

Date
:   04/08/2018

Operator
:   Timothy Páez

#### Sequence Verification

##### Sequence from plasmid

For verification of dxCas9-Tn5-lin insertion in pACYC, primers 003, 012, 029 and 030 were used for sequencing. Sequencing sample was prepared in 1.5 mL tubes for each primer as indicated my Macrogen for specific concentration of plasmid DNA.

Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

Within 2 days, the sequence was retrievable online and alignment with the in silico designed molecule showed 100% match of the insert lin-Tn5, by aligning both primer sequences obtained.

Close

[ ]
Week 2 06/08/2018 - 12/08/2018

Wednesday, 08/08/2018

Date
:   08/08/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

*E.coli* strain harbouring the plasmid pACYCDuet-1\_Tn5-Lin was used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Close

Thursday, 09/08/2018

Date
:   09/08/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter culture of pACYCDuet-1\_Tn5-Lin were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   09/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1\_Tn5-Lin | 66.5 | 1.88 | 1.83 |
| pACYCDuet-1\_Tn5-Lin | 70.1 | 1.83 | 1.77 |
| pACYCDuet-1\_Tn5-Lin | 90.2 | 1.72 | 1.06 |
| pACYCDuet-1\_Tn5-Lin | 108.8 | 1.73 | 1.09 |

Date
:   09/08/2018

Operator
:   Nicole Bennis

#### Restriction

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s) to obtain fragments Tn5-Lin-dxCas9 and pACYCDuet-1 for ligation.

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| 10x CutSmart | 2 | 10x CutSmart | 2 |
| dxCas9 | 8.7 (~2000 ng) | pACYCDuet-1\_Tn5-Linker | 17.2 (~2000 ng) |
| Enzyme (NotI) | 0.4 | Enzyme (NotI) | 0.4 |
| Enzyme (KpnI) | 0.4 | Enzyme (KpnI) | 0.4 |
| MilliQ | 8.5 | MilliQ | 0 |

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| 10x CutSmart | 2 | 10x CutSmart | 2 |
| Tn5-Lin | 12.5 (~2000 ng) | pACYCDuet-1\_dxCas9 | 17.2 (~2000 ng) |
| Enzyme (NotI) | 0.4 | Enzyme (NotI) | 0.4 |
| Enzyme (EcoRI) | 0.4 | Enzyme (EcoRI) | 0.4 |
| MilliQ | 4.7 | MilliQ | 0 |

The samples were incubated for 4 hour(s) at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Close

Friday, 10/08/2018

Date
:   10/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   10/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5-Lin cut with NotI and EcoRI | 88.1 | 1.83 | 1.69 |
| pACYCDuet-1\_dxCas9 cut with NotI and EcoRI | 11.8 | 1.44 | 0.69 |
| dxCas9 cut with NotI and EcoRI | 34.5 | 1.76 | 1.31 |
| pACYC\_Tn5-Linker cut with NotI and EcoRI | 41.9 | 1.73 | 1.36 |

Date
:   10/08/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pACYCDuet-1\_Tn5-Linker with dxCas9 Tn-Linker with pACYCDuet-1\_dxCas9 respectively, ligation was performed. to optimize the protocol, I used both vector to insert ratio 1:3 and 1:10. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1\_dxCas9 | 7929 | EcoRI and NotI |
| Fragment Tn5-Linker | 1490 | EcoRI and NotI |

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pACYCDuet-1\_Tn5-Lin | 5258 | NotI and KpnI |
| Fragment dxCas9 | 4161 | NotI and KpnI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector pACYCDuet-1\_dxCas9 (~100 ng) | 8.5 | DNA vector pACYCDuet-1\_dxCas9 (~100 ng) | 8.5 | DNA vector pACYCDuet-1\_dxCas9 (~100 ng) | 8.5 |
| none |  | DNA Lin-Tn5 (~60 ng) | 0.7 | DNA Lin-Tn5 (~190 ng) | 2.2 |
| MilliQ | 12 | MilliQ | 11.3 | MilliQ | 9.8 |

| Component | Volume (μL) | Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector pACYCDuet-1\_Tn5-Linker (~100 ng) | 2.5 | DNA vector pACYCDuet-1\_Tn5-Linker (~100 ng) | 2.5 | DNA vector pACYCDuet-1\_Tn5-Linker (~100 ng) | 2.5 |
| none |  | DNA dxCas9 (~220 ng) | 5.4 | DNA dxCas9 (~730 ng) | 14.5 |
| MilliQ | 14.5 | MilliQ | 7.1 | MilliQ | 0 |

The samples were incubated for 8 hours at 20°C for ligation.

Date
:   10/08/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pACYCDuet-1\_dxCas9 restricted (neg control) | Restriction/Ligation | 8 | LB-Cam || *E.coli* DH5α | pACYCDuet-1\_dxCas9-Linker-Tn5 ratio 1:3 (9449 bp) | Restriction/Ligation | 5.5 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_dxCas9-Linker-Tn5 ratio 1:10 (9449 bp) | Restriction/Ligation | 3 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_Tn5-Lin restricted (neg. control) | Restriction | 8 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_dxCas9-Linker-Tn5 ratio 1:3 (9449 bp) | Restriction/Ligation | 3 | LB Cam |
| *E.coli* DH5α | pACYCDuet-1\_dxCas9-Linker-Tn5 ratio 1:10 (9449 bp) | Restriction/Ligation | 1 | LB Cam |

For the transformation 40 ng of total ligate was used. 200 μl LB medium was added as recovery medium.

Close

Saturday, 11/08/2018

Date
:   11/8/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight DH5alpha starter cultures of dxCas9(3.7)-VPR and pACYC-dxCas9-LinkerTn5 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 40 μl of pre-warmed MilliQ (2 times 20µl).

Date
:   11/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| dxCas9(3.7)-VPR | 23 | 1.82 | 1.83 |
| dxCas9(3.7)-VPR | 28 | 1.82 | 1.9 |
| dxCas9(3.7)-VPR | 24 | 1.83 | 1.44 |
| dxCas9(3.7)-VPR | 26 | 1.81 | 1.97 |
| dxCas9(3.7)-VPR | 13 | 1.59 | 0.97 |
| dxCas9(3.7)-VPR | 23 | 1.88 | 1.99 |
| dxCas9(3.7)-VPR | 26 | 1.83 | 1.59 |
| pACYC-dxCas9-LinkerTn5 | 30 | 1.83 | 1.85 |
| pACYC-dxCas9-LinkerTn5 | 31 | 1.81 | 1.88 |
| pACYC-dxCas9-LinkerTn5 | 31 | 1.78 | 1.63 |
| pACYC-dxCas9-LinkerTn5 | 31 | 1.72 | 1.69 |
| pACYC-dxCas9-LinkerTn5 | 29 | 1.85 | 1.79 |
| pACYC-dxCas9-LinkerTn5 | 27 | 1.74 | 1.71 |
| pACYC-dxCas9-LinkerTn5 | 28 | 1.81 | 1.74 |

Close

[ ]
Week 3 13/08/2018 - 19/08/2018

Sunday, 19/08/2018

Date
:   19/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the pSB1C3 BioBrick backbone, Tn5 and Linker coding sequences and T7p-Tn5-T7t expression cassette, high fidelity PCR was done. The template DNA is the BioBrick plasmid pSB1C3 (primerset 051 & 052 & 053) and pACYCDuet-1\_Tn5 (054 & 055, 058 & 059 and 060 & 061). Eight 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 051 | 052 | pSB1C3 backbone | 2068 |
| 2 | 051 | 053 | pSB1C3 backbone with extra nucleotide | 2070 |
| 3 | 054 | 055 | Tn5 | 1511 |
| 4 | 058 | 059 | Linker | 143 |
| 5 | 060 | 061 | T7p-Tn5-T7t | 1756 |

The thermocycler programme was installed to have an extension time of 1 min 30 seconds for pSB1C3 backbones, Tn5 and T7p-Tn5-T7t and 23 seconds for Linker and both an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   19/08/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons for construction of the BioBricks Tn5, T7p-Tn5-T7t and Linker into pSB13 were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/c2/T--TUDelft--2018_20180819_PCR_BB_Tn5_Linker_pSB1C3.png)

The gel image shows that the pSB1C3 BioBrick backbone is succesfully amplified. However, when the extra nucleotide is incorporated for non coding sequence BioBrick construction, this resulted not in the desired amplicon. The Tn5 was correctly amplified as well as the T7p-Tn5-T7t expression cassette. The Linker sequence is not correctly amplified as there is no band seen at 143 bp.

Close

[ ]
Week 4 20/08/2018 - 26/08/2018

Tuesday, 21/08/2018

Date
:   21/08/2018

Operator
:   Nicole Bennis

#### DpnI digestion

To eliminate any residual template DNA, a DpnI digestion was performed with the following reaction mixture:

| Component | Volume (uL) |
| --- | --- |
| 10X CutSmart buffer | 5 |
| PCR product | 44 |
| Enzyme (DpnI) | 1 |
| MilliQ | 0 |

The reaction was incubated for 30 minutes at 37 °C.
Heat inactivation was performed for 20 minutes at 80 °C.

Date
:   21/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 μL of each DpnI digestion reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   21/09/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons for construction of the BioBricks Tn5, T7p-Tn5-T7t and Linker into pSB13 were run on a 0.8% agarose gel in TAE buffer. Upon electroseparation based on size, the fragments that are undesired were separated from the desired amplicons. The desired fragments were excised and proceded for PCR Clean Up. The complete reaction mixture (50 μL) was pipetted into each lane. The gel was not imaged due to damage of UV-light.

Date
:   21/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the pSB1C3 BioBrick backboneand Linker coding sequence, high fidelity PCR was done. The template DNA are the BioBrick plasmid pSB1C3 (primerset 051 & 053) and pACYCDuet-1\_Linker-Tn5 (058 & 059). Four 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 051 | 053 | pSB1C3 backbone with extra nucleotide | 2070 |
| 4 | 058 | 059 | Linker | 143 |

The thermocycler programme was installed to have an extension time of 1 min 30 seconds for pSB1C3 backbones, Tn5 and T7p-Tn5-T7t and 23 seconds for Linker and both an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   22/09/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons for construction of the BioBrick Linker into pSB13 were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/e9/T--TUDelft--2018_20180821_pSB1C3_Linker.png)

The gel image shows that the pSB1C3 BioBrick backbone is succesfully amplified. However, the Linker sequence is not correctly amplified as there is no band seen at 143 bp.

Date
:   21/08/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

*E.coli* strains harbouring the plasmid pACYCDuet-1\_dxCas9-Lin-Tn5 and pACYCDuet-1\_Tn5 respectively were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Close

Wednesday, 22/08/2018

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of pACYCDuet-1\_Lin-Tn5 and pACYCDuet-1 were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 μL of each PCR reaction done on 16-07-2018 was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| T7p-Tn5-T7t | 94.7 | 1.84 | 1.78 |
| T7p-Tn5-T7t | 105.7 | 1.83 | 1.93 |
| Tn5 | 157.1 | 1.66 | 1.26 |
| pSB1C3 | 58.8 | 1.87 | 1.71 |
| pSB1C3 | 67.8 | 1.86 | 1.88 |
| pSB1C3 | 64.8 | 1.84 | 1.77 |

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3 with Tn5 to obtain pSB1C3\_Tn5 (3499 bp) and pSB1C3 with T7p-Tn5-T7t to obtain pSB1C3\_T7p-Tn5-T7t (3755 bp).

| Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | Gibson Assembly Mastermix 2X | 10 |
| Vector pSB1C3 | 2 (100 ng) | Vector pSB1C3 | 2 (100 ng) |
| Fragment T7p-Tn5-T7t | 3 (300 ng) | Fragment Tn5 | 2 (225 ng) |
| MilliQ | 5 | MilliQ | 4 |

The samples were incubated at 50°C for 1 hour, after which they were kept on ice for subsequent transformation.

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3\_Tn5 (3499 bp) | Gibson Assembly | 2.5 | LB-Cam || *E.coli* DH5α | pSB1C3\_T7p-Tn5-T7t (3755 bp) | Gibson Assembly | 2.5 | LB Cam |

200 μl LB medium was added as recovery medium.

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1\_dxCas9-Lin-Tn5 | 68.9 | 1.69 | 1.03 |
| pACYCDuet-1\_dxCas9-Lin-Tn5 | 74.2 | 1.68 | 1.06 |
| pACYCDuet-1\_dxCas9-Lin-Tn5 | 92.8 | 1.68 | 1.07 |
| pACYCDuet-1\_dxCas9-Lin-Tn5 | 77.8 | 1.66 | 0.92 |
| pACYCDuet-1\_Tn5 | 57.0 | 1.72 | 1.37 |
| pACYCDuet-1\_Tn5 | 52.4 | 1.75 | 1.42 |
| pACYCDuet-1\_Tn5 | 53.4 | 1.77 | 1.58 |
| pACYCDuet-1\_Tn5 | 70.9 | 1.76 | 1.742 |

Date
:   22/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

The dxCas9 coding sequence contains forbidden restriction sites to be RFC compatible. Therefore, Gblocks were ordered at IDT. The dxCas9 is to large to order as one Gblock. Therefore I ordered two Gblocks, also incorporating Linker-Tn5 coding sequences (Gblock 1 and 2). To be able to construct also a different variant of the fusion protein, two Gblocks were ordered in the order Tn5-Linker-dxCas9 (Gblock 3 and 4).

To amplify the Gblocks 1 and 2 as well as the pACYCDuet-1 backbone, high fidelity PCR was done. The template DNA are the Gblocks 1 (primerset 045 & 046) and 2 (primerset 047 & 048) and pACYCDuet-1 (primerset 049 & 050). Six 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 045 | 046 | Gblock 1 | 2892 |
| 4 | 047 | 048 | Gblock 2 | 2890 |
| 1 | 049 | 050 | pACYCDuet-1 | 3778 |

The thermocycler programme was installed to have an extension time of 2 min 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Thursday, 23/08/2018

Date
:   23/08/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons of Gblock 1 and 2 and pACYCDuet-1 were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/2c/T--TUDelft--20180823_Gblock12_pACYC.png)

The gel image shows that the pACYCDuet-1 backbone for Gblock1 and Gblock2 cloning is succesfully amplified. However, the Gblocks 1 and 2 are not correctly amplified as there is a smeer visible.

Date
:   23/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the Gblocks 1 and 2 as well as the pACYCDuet-1 backbone, high fidelity PCR was done. The template DNA are the Gblocks 1 (primerset 045 & 046) and 2 (primerset 047 & 048) and pACYCDuet-1 (primerset 049 & 050). Six 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 045 | 046 | Gblock 1 | 2892 |
| 4 | 047 | 048 | Gblock 2 | 2890 |
| 1 | 049 | 050 | pACYCDuet-1 | 3778 |

The thermocycler programme was installed to have an extension time of 1 min 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   23/08/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons of Gblock 1 and 2 and pACYCDuet-1 were run on a 0.8% agarose gel in TAE buffer. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/83/T--TUDelft--20180823_Gblock12_pACYC2.png)

The gel image shows that the pACYCDuet-1 backbone for Gblock1 and Gblock2 cloning is succesfully amplified. However, the Gblocks 1 and 2 are not correctly amplified as there is a smeer visible.

Date
:   23/08/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3\_Tn5 (3499 bp) | Gibson Assembly | 2.5 | LB-Cam || *E.coli* DH5α | pSB1C3\_T7p-Tn5-T7t (3755 bp) | Gibson Assembly | 2.5 | LB Cam |

200 μl LB medium was added as recovery medium.

Close

[ ]
Week 5 27/08/2018 - 02/09/2018

Monday, 27/08/2018

Date
:   27/08/2018

Operator
:   Nicole Bennis

#### Gel Purification

The amplicon pACYCDuet-1 backbone and Gblock 2 have a size of 3778 bp and 2890 bp respectively, but the PCR samples were contaminated with by-products. The products were loaded on a gel prior to [DNA purification from agarose gel conform protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#DNApurificationfromgel-scroll).
Elution was done in 50 µL Milli-Q.

Date
:   27/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 backbone (1) | 29.7 | 1.86 | 0.44 |
| pACYCDuet-1 backbone (2) | 25.4 | 1.88 | 1.46 |
| pACYCDuet-1 backbone (3) | 24.4 | 1.86 | 1.46 |
| Gblock 2 | 10.5 | 1.73 | 1.14 |

Date
:   27/08/2018

Operator
:   Nicole Bennis

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3 with Tn5 to obtain pSB1C3\_Tn5 (3499 bp) and pSB1C3 with T7p-Tn5-T7t to obtain pSB1C3\_T7p-Tn5-T7t (3755 bp).

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | Gibson Assembly Mastermix 2X | 10 | Gibson Assembly Mastermix 2X | 10 |
| Vector pACYCDuet-1 | 1.6 (40 ng) | Vector pSB1C3 | 2 (100 ng) | Vector pSB1C3 | 2 (100 ng) |
| Fragment Gblock 1 | 4.3 (43 ng) | Fragment T7p-Tn5-T7t | 4 (300 ng) | Fragment Tn5 | 2 (225 ng) |
| Fragment Gblock 2 | 4.3 (43 ng) |  |  |  |  |
| MilliQ | 0 | MilliQ | 4 | MilliQ | 6 |

The samples were incubated at 50°C for 1 hour, after which they were kept on ice for subsequent transformation.

Date
:   27/08/2018

Operator
:   Nicole Bennis

#### Gel Purification

The PCR reaction performed on 24/08/2018 were loaded on a gel. Since the expectation of contamination based on previous results, I decided to not use UV imaging due to the potential of creating mutations in the DNA. The amplicon pACYCDuet-1 backbone, Glock 1 and 2 have a size of 3778 bp, 2892 bp and 2890 bp respectively, but indeed the PCR samples were contaminated with by-products. The products were loaded on a gel prior to [DNA purification from agarose gel conform protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#DNApurificationfromgel-scroll).
Elution was done in 50 µL Milli-Q.

Date
:   27/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1 backbone (1) | 43.7 | 1.89 | 0.16 |
| pACYCDuet-1 backbone (2) | 48.3 | 1.88 | 0.46 |
| Gblock 1 (1) | 34.4 | 1.87 | 1.16 |
| Gblock 1 (2) | 24.8 | 1.91 | 1.08 |
| Gblock 2 (1) | 23.3 | 1.81 | 0.65 |
| Gblock 2 (2) | 23.2 | 1.85 | 0.92 |

Close

Wednesday, 29/08/2018

Date
:   29/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

The dxCas9 coding sequence contains forbidden restriction sites to be RFC compatible. Therefore, Gblocks were ordered at IDT. The dxCas9 is to large to order as one Gblock. Therefore I ordered two Gblocks, also incorporating Linker-Tn5 coding sequences (Gblock 1 and 2). To be able to construct also a different variant of the fusion protein, two Gblocks were ordered in the order Tn5-Linker-dxCas9 (Gblock 3 and 4).

To amplify the Gblocks 3 and 4 as well as the pACYCDuet-1 backbone, high fidelity PCR was done. The template DNA are the Gblocks 1 (primerset 045 & 046) and 2 (primerset 047 & 048) and pACYCDuet-1 (primerset 049 & 050). Additionally, we use the templates Gblocks 3 (primerset 079 & 080) and 4 (primerset 081 & 082) and pACYCDuet-1 (primerset 083 & 084). Multiple 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 045 | 046 | Gblock 1 | 2892 |
| 2 | 047 | 048 | Gblock 2 | 2890 |
| 3 | 049 | 050 | pACYCDuet-1 | 3778 |
| 4 | 079 | 080 | Gblock 3 |  |
| 5 | 081 | 082 | Gblock 4 |  |
| 6 | 083 | 084 | pACYCDuet-1 | 3652 |

The thermocycler programme was installed to have an extension time of 4 minutes and an annealing temperature in a gradient from 52 °C 66 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   29/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the pSB1C3 BioBrick backbone, Tn5 and Linker coding sequences and T7p-Tn5-T7t expression cassette, high fidelity PCR was done. The template DNA is the BioBrick plasmid pSB1C3 (primerset 051 & 052 & 053) and pACYCDuet-1\_Tn5 (054 & 055, 058 & 059 and 060 & 061). 54 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 051 | 052 | pSB1C3 backbone | 2068 |
| 2 | 051 | 053 | pSB1C3 backbone with extra nucleotide | 2070 |
| 3 | 054 | 055 | Tn5 | 1511 |
| 4 | 058 | 059 | Linker | 143 |
| 5 | 060 | 061 | T7p-Tn5-T7t | 1756 |

The thermocycler programme was installed to have an extension time of 2 min 30 seconds for pSB1C3 backbones, Tn5 and T7p-Tn5-T7t and 30 seconds for Linker and both an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   29/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the Gblocks 1, 2, 3 and 4 a high fidelity PCR was done, but using a 2 step PCR program. The template DNA are the Gblocks 1 (primerset 045 & 046) and 2 (primerset 047 & 048) and pACYCDuet-1 (primerset 049 & 050). Additionally, we use the templates Gblocks 3 (primerset 079 & 080) and 4 (primerset 081 & 082) and pACYCDuet-1 (primerset 083 & 084). Multiple 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 045 | 046 | Gblock 1 | 2892 |
| 2 | 047 | 048 | Gblock 2 | 2890 |
| 4 | 079 | 080 | Gblock 3 |  |
| 5 | 081 | 082 | Gblock 4 |  |

The thermocycler programme was installed to have an extension time of 4 minutes and an annealing temperature of 72 °C (2 step PCR). After the programme was finished, samples were kept at 4 °C.

Close

Thursday, 30/08/2018

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The PCR amplicons for construction of the fusion protein with Gblock 1 and 2 and Gblock 3 and 4 as well as the amplicons for BioBricks Tn5, T7p-Tn5-T7t and Linker into pSB13 were run on multiple 0.8% agarose gel in TAE buffer to check for amplification. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/c7/T--TUDelft--20180830_pACYC_pSB1C3.png)
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/18/T--TUDelft--20180830_PCR_Gblocks_gradient.png)
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/2f/T--TUDelft--20180830_PCR_linker_Tn5_pSB1C3Nuc.png)
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/5a/T--TUDelft--20180830_PCR_Tobal_Gblocks.png)

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Gel Purification

The PCR amplicons were contaminated with by-products. The products were loaded on a gel prior to [DNA purification from agarose gel conform protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#DNApurificationfromgel-scroll).
Elution was done in 50 µL Milli-Q.

Date
:   09/08/2018

Operator
:   Nicole Bennis

#### Restriction

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s) to obtain fragments Tn5-Lin-dxCas9 and pACYCDuet-1 for ligation.

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart | 5 | 10x CutSmart | 2 | 10x CutSmart | 2 |
| pSB1C3 | 43 (~2000 ng) | Tn5 | 17 (~2000 ng) | T7p-Tn5-T7t | 17 (~2000 ng) |
| Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 0.5 | Enzyme (EcoRI) | 0.5 |
| Enzyme (PstI) | 1 | Enzyme (PstI) | 0.5 | Enzyme (PstI) | 0.5 |
| MilliQ | 0 | MilliQ | 0 | MilliQ | 0 |

The samples were incubated for 4 hours at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 or 20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pSB1C3 cut with EcoRI and PstI | 49.0 | 1.82 | 1.55 |
| Tn5 cut with EcoRI and PstI | 39.4 | 1.86 | 1.79 |
| T7p-Tn5-T7t cut with EcoRI and PstI | 34.9 | 1.79 | 1.30 |

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pSb1C with Tn5 or T7p-Tn5-T7t ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pSB1C3 | 2068 | EcoRI and PstI |
| Fragment Tn5 | 1511 | EcoRI and PstI |
| Fragment T7p-Tn5-T7t | 1756 | NotI and PstI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector pSB1C3 (~200 ng) | 4 | DNA vector pSB1C3 (~200 ng) | 13 |
| DNA Tn5 (~450 ng) | 0.7 | DNA T7p-Tn5-T7t (~600 ng) | 13 |
| MilliQ | 0 | MilliQ | 0 |

The samples were incubated overnight at 4 °C for ligation.

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the Gblocks 1 and 2 and Gblock 3 and 4 as one fragment, high fidelity overlap PCR was done. The template DNA are the Gblocks 1 and 2 (primerset 045 & 048) and Gblocks 3 and 4 (primerset 079 & 082). two 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 045 | 048 | Gblock 1&2 | 5741 |
| 2 | 079 | 082 | Gblock 3&4 | 5897 |

The thermocycler programme was installed to have an extension time of 6 minutes 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Friday, 31/08/2018

Date
:   31/08/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3\_Tn5 (3499 bp) | Restriction/Ligation | 5 | LB-Cam || *E.coli* DH5α | pSB1C3\_T7p-Tn5-T7t (3755 bp) | Restriction/Ligation | 5 | LB Cam |

200 μl LB medium was added as recovery medium.

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The excised gel fragments that were cleaned using PCR Clean up were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pSB1C3 | 740.3 | 1.84 | 2.08 |
| pACYCDuet-1 backbone Gblock 1,2 | 150.9 | 1.92 | 1.93 |
| pACYCDuet-1 backbone Gblock 3,4 | 173.0 | 1.91 | 1.98 |
| Gblock 2 | 21.4 | 1.88 | 1.00 |
| Gblock 3 | 113.6 | 1.90 | 1.83 |
| Gblock 4 | 11.4 | 1.91 | 0.53 |

Date
:   31/08/2018

Operator
:   Nicole Bennis

#### DNA Gel Electrophoresis

The overlap PCR amplicons for construction of the fusion protein with Gblock 1 and 2 and Gblock 3 and 4 during overlap PCR were run on multiple 0.8% agarose gel in TAE buffer to check for amplification. Also, the ligation reaction mixture was loaded on the gel, to check if the desired plasmids were already assembled and ligated. 4 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a7/T--TUDelft--20180831_PCR_overlap_ligationcheck.png)

Date
:   31/08/2018

Operator
:   Nicole Bennis

#### Plasmid drying

In order to sent our pACYCDuet-1\_dxCas9, pACYCDuet-1\_Tn5 and pACYCDuet-1\_dxCas9-Lin-Tn5 to Marburg for our [collaboration](https://2018.igem.org/Team%3ATUDelft/Collaborations), I dried the plasmids in a 96 wells plate.

Close

### September

[ ]
Week 1 03/09/2018 - 09/09/2018

Monday, 03/09/2018

Date
:   03/09/2018

Operator
:   Nicole Bennis

#### Restriction

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s) to obtain fragments Tn5-Lin-dxCas9 and pACYCDuet-1 for ligation.

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart | 5 | 10x CutSmart | 2 | 10x CutSmart | 2 |
| pSB1C3 | 6 (~3000 ng) | Tn5 | 4 (~3000 ng) | T7p-Tn5-T7t | 4 (~3000 ng) |
| Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 0.5 | Enzyme (EcoRI) | 0.5 |
| Enzyme (PstI) | 1 | Enzyme (PstI) | 0.5 | Enzyme (PstI) | 0.5 |
| MilliQ | 10 | MilliQ | 12 | MilliQ | 12 |

The samples were incubated for 4 hours at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pSB1C3 cut with EcoRI and PstI | 56.3 | 1.85 | 0.43 |
| Tn5 cut with EcoRI and PstI | 45.1 | 1.76 | 0.62 |
| T7p-Tn5-T7t cut with EcoRI and PstI | 157.8 | 1.76 | 0.71 |

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pSb1C with Tn5 or T7p-Tn5-T7t ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pSB1C3 | 2068 | EcoRI and PstI |
| Fragment Tn5 | 1511 | EcoRI and PstI |
| Fragment T7p-Tn5-T7t | 1756 | NotI and PstI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector pSB1C3 (~200 ng) | 5 | DNA vector pSB1C3 (~200 ng) | 5 |
| DNA Tn5 (~450 ng) | 12 | DNA T7p-Tn5-T7t (~600 ng) | 12 |
| MilliQ | 0 | MilliQ | 0 |

The samples were incubated overnight at 4 °C for ligation.

Close

Tuesday, 04/09/2018

Date
:   04-09-2018

Operator
:   Susan Bouwmeester

#### Restriction

Restriction cloning was done for assembling both Tn5 (157.1ng/uL) and T7-Tn5-T7 (94.7ng/uL) into with previous with PCR linearized pSB1C3 backbone (64.8ng/uL). The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (Tn5) (uL) | Volume (T7p.Tn5.T7t) (uL) | Volume (pSB1C3) (uL) || 10x CutSmart buffer | 2 | 2 | 2 |
| Fragment | 1.6 | 2.6 | 3.8 |
| Enzyme (EcoRI) | 0.5 | 0.5 | 0.5 |
| Enzyme (PstI) | 0.5 | 0.5 | 0.5 |
| MilliQ | 14.4 | 15.4 | 13.2 |

The samples were incubated for 30 minutes at 37 °C, and followed by 20 minutes heat inactivation of the restriction enzymes at 80°C.

Date
:   04/09/2018

Operator
:   Susan Bouwmeester

#### Ligation

In order to assemble cut fragments both Tn5 and T7p.Tn5.T7t to the pSB1C3 backbone, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector pSB1C3 | 2068 | EcoRI and PstI |
| Fragment 1 Tn5 | 1503 | EcoRI and PstI |
| Fragment 2 T7p.Tn5.T7t | 1756 | EcoRI and PstI |

A 10uL ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume Tn5 (uL) | Volume T7p.Tn5.T7t (uL) || Ligase Buffer (10x) | 1 | 1 |
| T4 DNA Ligase | 0.5 | 0.5 |
| DNA vector | 2 | 2 |
| DNA fragment | 2 | 2 |
| MilliQ | 4.5 | 4.5 |

The samples were incubated for 4 hours at 16°C for ligation and followed by 20 minutes heat inactivation of the ligase at 80°C.

Date
:   04/09/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify the 3 and 4 as one fragment, high fidelity overlap PCR was done. The template DNA are Gblocks 3 and 4 (primerset 079 & 082). Eight 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 079 | 082 | Gblock 3&4 | 5897 |

The thermocycler programme was installed to have an extension time of 6 minutes 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   04/09/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3\_Tn5 (3499 bp) | Restriction/Ligation | 2.5 | LB-Cam || *E.coli* DH5α | pSB1C3\_T7p-Tn5-T7t (3755 bp) | Restriction/Ligation | 2.5 | LB Cam |

200 μl LB medium was added as recovery medium.

Close

Wednesday, 05/09/2018

Date
:   05/09/2018

Operator
:   Susan Bouwmeester

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | [pSB1C3-Tn5] (3499bp) | Restriction/ligation cloning | 2uL | LBcam |

950 uL LB medium was added as recovery medium.

Close

Thursday, 06/09/2018

Date
:   06/09/2018

Operator
:   Susan Bouwmeester

#### DNA Purification of linker from Agarose Gel

The amplicon of interest, the linker, has a size of 69bp, but the PCR samples were contaminated with by-products. The products were loaded on a 2% agarose gel prior to DNA purification from agarose gel conform protocol.
Elution was done in 30 µL Milli-Q.

#### DNA Purification of pSB1C3, with extra nucleotide, from Agarose Gel

The amplicon of interest, pSB1C3 with extra nucleotide, has a size of around 2000bp, but the PCR samples were contaminated with by-products. The products were loaded on a 0.8% agarose gel prior to DNA purification from agarose gel conform protocol.
Elution was done in 30 µL Milli-Q.

Date
:   06/09/2018

Operator
:   Susan Bouwmeester

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble both Tn5 and T7p.Tn5.T7t and pSB1C3 to obtain pSB1C3-Tn5 (3499bp) and pSB1C3-T7p.Tn5.T7t (3605bp).

| Component | Volume (uL) pSB1C3-Tn5 | Volume (uL) pSB1C3-T7p.Tn5.T7t |
| --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 |
| Vector | 1.54uL (100 ng) | 1.54uL (100 ng) |
| Fragment | 1.34 uL (210.6 ng) | 2.3 uL (2107.9 ng) |
| MilliQ | 7.12 | 6.2 |

The samples were incubated at 50°C for 60 minutes, after which they were kept on ice for subsequent transformation.

Date
:   06/09/2018

Operator
:   Susan Bouwmeester

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | [pSB1C3-Tn5] (3499bp) | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α | [pSB1C3-T7p.Tn5.T7t] (3605bp) | Gibson Assembly | 2 | LB Cam |

950 uL LB medium was added as recovery medium.

Close

Friday, 07/09/2018

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pSB1C3extranuc1 | 366.7 | 1.88 | 2.04 |
| pSB1C3extranuc2 | 415.5 | 1.88 | 2.17 |
| linker1 | 41.7 | 1.83 | 1.43 |
| linker2 | 39.3 | 1.83 | 1.44 |
| Fusionduet1 | 120.3 | 1.82 | 1.61 |
| Fusionduet2 | 49.7 | 1.68 | 0.81 |
| Fusionbb | 294.9 | 1.85 | 2.10 |

Date
:   9/7/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify multiple parts required for strain construction, high fidelity PCR was done using the following primer and template combinations. The sample preparation was done according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s):

| Template\* | Fw & Rv primer | Amplicon name & size in bp | Elongation time (min) |
| --- | --- | --- | --- |
| Gblock1 & Gblock2 | 054 & 057 | Gblock1-Gblock2 (~6kb) | 6.5 |
| Gblock1 & Gblock2 | 048 & 088 | Gblock1-Gblock2 (~6kb) | 6.5 |
| Gblock3-Gblock4 | 079 & 082 | Gblock3-Gblock4 (~6kb) | 6.5 |
| Gblock3 & Gblock4 | 056 & 055 | Gblock3-Gblock4~6kb | 6.5 |
| Gblock1 | 088 & 089 | Gblock 1 (~3kb) | 4 |
| pACYC | 079 & 082 | pACYC backbone (~4kb) | 4 |
| pACYC | 083 & 084 | pACYC backbone (~4kb) | 4 |

\*when the template consists of two molecules (like Gblock1 & Gblock2), this is indicated with an & sign, and means overlap PCR. A hyphen (-) indicates that the two molecules are already connected.

The thermocycler programme was installed to have an an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Saturday, 08/09/2018

Date
:   08-09-2018

Operator
:   Susan Bouwmeester

#### Restriction

Restriction cloning was done for five different constructs. The following table shows an overview of what fragments were cut with what restriction enzymes.

| Component | Volume (pSB1C3en) (uL) | Volume (Linker1) (uL) | Volume (Tn5) (uL) | Volume (T7p.Tn5.T7t) (uL) | Volume (pSB1C3en) (uL) | Volume (fusionbb) (uL) | Volume (Pacyc) (uL) | Volume (fusionduet) | Volume (fusionduet2) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Fragment | 5.4 (~2000ng) | 5.99 (~250ng) | 2 (~1500ng) | 10.6 (~1000ng) | 3 (~2000ng) | 10 (~3000ng) | 10 (~1500ng) | 10 (500ng) | 10 (1200ng) |
| Enzyme | EcoRI 0.5uL | EcoRI 0.5uL | EcoRI 0.5uL | EcoRI 0.5uL | EcoRI 0.5uL | EcoRI 0.5uL | KpnI 0.5uL | KpnI 0.5uL | KpnI 0.5uL |
| Enzyme | PstI 0.5uL | PstI 0.5uL | PstI 0.5uL | PstI 0.5uL | PstI 0.5uL | PstI 0.5uL | NcoI 0.5uL | NcoI 0.5uL | NcoI 0.5uL |
| MilliQ | 11.6 | 12 | 15 | 6.4 | 14 | 7 | 7 | 7 | 7 |

The samples were incubated for 4h at 37 °C. PCR Clean-Up is done, according to the protocol.

Date
:   08-09-2018

Operator
:   Susan Bouwmeester

#### Nanodrop DNA Quantification

Nanodrop is done after restriction and PCR cleanup. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| linker | 3.3 | 1.7 | 0.2 |
| pSB1C3extranuc | 39.9 | 1.8 | 0.9 |
| Tn5 | 48.9 | 1.8 | 1.2 |
| Pycduet | 3.9 | 1.5 | 0.3 |
| pSB1C3 | 46.9 | 1.8 | 0.9 |
| Fusionbb | 54.1 | 1.8 | 1.3 |
| Fusionduet1 | 6.2 | 1.5 | 0.3 |
| oldpSB1C3 | 46.9 | 1.8 | 0.9 |
| T7p.Tn5.T7t | 16.5 | 1.7 | 0.4 |
| Fusionduet2 | 13.1 | 1.7 | 0.8 |

Date
:   08/09/2018

Operator
:   Susan Bouwmeester

#### Colony PCR

After carrying out the transformation protocol on 06/09/2018, the plates were left for overnight incubation. In total five colonies were observed.

Colony PCR was done to verify propagation of pSB1C3-Tn5 and pSB1C3-T7p.Tn5.T7t after transformation from 06/09/2018. This plasmid contains Tn5 or T7.Tn5.T7, which can be checked with primers VF2 and VR

|  |  |  |
| --- | --- | --- |
| Template DNA | pSB1C3-Tn5 | pSB1C3-T7p.Tn5.T7t |
| Forward primer | VF2 | VF2 |
| Reverse primer | VR | VR |
| Expected amplicon size (bp) | ~1700bp | ~1800bp |

The thermocycler programme was installed to have an extension time of 2:20 and an annealing temperature of 52 °C. After the programme was finished, samples were kept at 4°C.

Date
:   08/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

The products of the colony PCR were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

s
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/cb/T--TUDelft--2018_180908Susan.jpg)
**Figure: Colony PCR pSB1C3-Tn5 and pSB1C3-T7p.Tn5.T7t** 0.8% agarose. Expected hight ~1700 bp. Lane 1: 500bp ladder; Lane 2: Colony1 (Tn5); Lane 3: Colony2 (Tn5); Lane 4: Colony 3 (Tn5); Lane 5: Colony 4 (Tn5); Lane 6: Colony 5 (T7p.Tn5.T7t)

Date
:   08/09/2018

Operator
:   Susan Bouwmeester

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble the linker and pSB1C3en to obtain pSB1C3-linker, the fusion protein with pSB1C3 to obtain pSB1C3-fusion.

| Component | Volume (uL) pSB1C3-linker | Volume (uL) pSB1C3-fusion |
| --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 |
| Fragment | 2uL (50 ng) | 6.8 uL(2000 ng) |
| Vector | 2.7 uL (1000 ng) | 2.3 uL (2107.9 ng) |
| MilliQ | 5.3 uL | 1.5 uL |

The samples were incubated at 50°C for 20 minutes, after which they were kept on ice for subsequent transformation.

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR products for fusion protein construction were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/7/71/T--TUDelft--20180908kAVISHMILLIONPCRsHAHAHAHAHA.jpg)

The gel shows:

+ connecting Gblock 1 & 2 did not work (lanes 2-17)
+ Amplifying gblock 3 & 4, connected, did work without byproducts (lanes 18-34)
+ amplifying Gblock 1 on itself did not work (lanes 35-43)
+ pACYC backbone for cloning with Gblock 1 & 2 was succesful (lanes 44-51)
+ pACYC backbone for cloning with Gblock 3 & 4 was only successful in 1 sample (lane 52)

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

Based on the obtained gel, it was decided to PCR clean-up amplicons "Gblock3&4 gibson pACYC" and "pACYC backbone for Gblock 3&4" was subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were verified to be around 1.8~1.9.

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Isolate | Concentration (ng/uL) || Gblock3&4 gibson pACYC | 455 |
| Gblock3&4 gibson pACYC | 294 |
| pACYC backbone for Gblock 3&4 | 113 |
|  |

Close

Sunday, 09/09/2018

S

Date
:   09/09/2018

Operator
:   Susan Bouwmeester

#### Overnight culture

An overnight culture of the five colonies as result of the transformation 05/09 and 06/09 is started. 3mL LBmedium with Cam resistance is used. Cultures overnight 37°C.

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

PCR products "Gblock1&2 Gibson pSB1C3", "Gblock3&4 Gibson pSB1C3" and "pACYC (for cloning with Gblock 1&2)" (all dating from 7/9/2018) were subjected to PCR Clean-Up, according to the protocol. Isolation was done by gel cut-out, due to byproducts visible on the gel (see 8/9/2018Elution was done in 40 µL Milli-Q.

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Gblock1&2 Gibson pSB1C3 | 16 | 1.35 | 0.75 |
| Gblock1&2 Gibson pSB1C3 | 15 | 1.8 | 0.6 |
| Gblock1&2 Gibson pSB1C3 | 13.5 | 2 | 0.31 |
| Gblock3&4 Gibson pSB1C3 | 61 | 1.9 | 0.9 |
| Gblock3&4 Gibson pSB1C3 | 69 | 1.9 | 0.2 |
| pACYC (for cloning with Gblock 1&2) | 108.8 | 1.9 | 1.4 |
| pACYC (for cloning with Gblock 1&2) | 145 | 1.9 | 1.5 |
| pACYC (for cloning with Gblock 1&2) | 163 | 1.9 | 1.6 |

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify Gblock1&2 Gibson pSB1C3 and Gblock3&4 Gibson pSB1C3 (both ~6kb), high fidelity PCR was done using PCR cleanup isolates as template DNA. PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

| Template | Fw primer | Rv primer | expected size (bp) |
| --- | --- | --- | --- |
| Gblock1&2 Gibson pSB1C3 | 054 | 057 | ~6kb |
| Gblock3&4 Gibson pSB1C3 | 055 | 056 | ~6kb |

The thermocycler programme was installed to have an extension time of 4 minutes and an annealing temperature of 65°C. After the programme was finished, samples were kept at 4 °C.

Close

[ ]
Week 2 10/09/2018 - 16/09/2018

Monday, 10/09/2018

Date
:   10/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR products "Gblock1&2 Gibson pSB1C3" and "Gblock3&4 Gibson pSB1C3" (9/9/2018) were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/ca/T--TUDelft--20180910kavishSTRAINCONSTRUCTIONpcr.jpg)

According to this gel, neither of the two fragments is properly amplified.

Date
:   10/9/2018

Operator
:   Kavish Kohabir

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble Gblock 3&4 (insert) into pACYC (vector).

| Component | Amount |
| --- | --- |
| Gibson Assembly Mastermix 2X | 10µl |
| Vector | ~0.35pmol |
| Fragment | ~0.35pmol |
| MilliQ | until 20µl |

The samples were incubated at 50°C for 20 minutes, after which they were kept on ice for subsequent transformation.

Date
:   10/9/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pACYC-Gblock3&4 | Gibson Assembly | 20 | LB+Cam || *E.coli*DH5α | - | - | - | LB+Cam |

200 uL LB medium was added as recovery medium.

Date
:   10/9/2018

Operator
:   Kavish Kohabir

#### Electroporation

The following transformations were carried out according to the protocol for electroporation:

| Strains | Plasmid/fragment | Amount | Plate medium |
| --- | --- | --- | --- |
| *E. coli* BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA | MEpKantME | 200ng | LB+X-gal+IPTG+Cam+Kan |
| *E. coli* BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA | pKant | 200ng | LB+X-gal+IPTG+Cam+Kan |
| *E. coli* BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA | - | - | LB+X-gal+IPTG+Cam+Kan |

200 μl LB medium+0.1mM IPTG was added as recovery medium.

Close

Wednesday, 12/09/2018

Date
:   12/09/2018

Operator
:   Susan Bouwmeester

#### High Fidelity PCR

To amplify dxCas9 (~4207bp), high fidelity PCR was done using the fusionbb as template DNA and fw\_bb\_dxcas9 and rv\_bb\_dxcas9\_2 as forward and reverse primers respectively. 6 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 2:30 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

Date
:   12/09/18

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of Tn5 and T7p.Tn5.T7t after transformation from 06/09 and 07/09 and plasmid isolation from 10/9. Primers VF2 and VR were used.

| Component | Volume (uL) |
| --- | --- |
| Template DNA | 0.5 |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |
| Expected amplicon size (bp) | ~1700 bp |

5 tubes were prepared for colony PCR, containing purified plasmids as template DNA. The thermocycler programme was installed to have an extension time of 2:30 and an annealing temperature of 52 °C. After the programme was finished, samples were kept at 12°C.

Date
:   12/09/18

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of the fusion protein for four colonies. Primers dxcas9\_mut2 and dxcas9\_mut4 were used. Expected size is 1470bp.

| Component | Volume (uL) |
| --- | --- |
| Template DNA | 0.5 |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |
| Expected amplicon size (bp) | ~1470 bp |

8 tubes were prepared for colony PCR, containing directly the colony or first boyled colony as template DNA. The thermocycler programme was installed to have an extension time of 2 minutes and an annealing temperature of 50 °C. After the programme was finished, samples were kept at 12°C.

Close

Thursday, 13/09/2018

Date
:   13/09/2018

Operator
:   Susan Bouwmeester

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells. All transformations were previous made samples.

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | [pSB1C3-T7p.Tn5.T7t] (3605bp) | Restriction/ligation | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-linker1] (~2000bp) | Restriction/ligation | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-linker2] (~2000bp) | Restriction/ligation | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-fusion1] (7699bp) | Restriction/ligation | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-fusion2] (7699bp) | Restriction/ligation | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-Tn5] (3499bp) | Restriction/ligation | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-fusion] (7699) | Gibson Assembly | 18 | LB Cam |
| *E.coli*DH5α | [pSB1C3-linker] (~2000bp) | Gibson Assembly | 18 | LB Cam |

950 uL LB medium was added as recovery medium.

Date
:   13/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

dxCas9 amplification product was run on a 0.8% agarose gel in TEA buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f4/T--TUDelft--2018_1309108_dxcas9amp.png)
**Figure dxCas9 amplification** 0.8% Agarose; Lane 1: 500bp ladder; Lane 2-7: PCR product dxCas9 amplification

Conclusion: dxCas9 is produced, but not pure. Gelcutout is needed.

Date
:   13/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

PCR Tn5 product from pure plasmid was run on a 0.8% agarose gel in TEA buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/bd/T--TUDelft--2018_180913_tn5.png)
**Figure PCR Tn5 and T7p.Tn5.T7t in pSB1C3 on pure plasmid** 0.8% agarose. Lane 1:Tn5 Colony 1; Lane 2: Tn5 Colony 2; Lane 3: Tn5 Colony 3; Lane 4: Tn5 Colony 4; Lane 5: T7p.Tn5.T7t Colony 5; Lane 6: 500bp ladder

No product, no correct Tn5 or T7p.Tn5.T7t integration

Date
:   13/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

Colony PCR of four colonies after transformation with pSB1C-fusion was run on a 0.8% agarose gel in TEA buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/e4/T--TUDelft--2018_180913_fusioncol.png)
**Figure Colony PCR pSB1C3-fusion**0.8% agarose. Expected size ~1500 bp. Lane 1: 500bp ladder; Lane 2: Fusion Colony 1; Lane 3: Fusion Colony 2; Lane 4: Fusion Colony 3; Lane 5: Fusion Colony 4; Lane 6: Fusion Colony 1 preboiled; Lane 7: Fusion Colony 2 preboiled; Lane 8: Fusion Colony 3 preboiled; Lane 9: Fusion Colony 4 preboiled;

Fusion Colony 4 preboiled have product. Start overnight culture in 3mL with cam. of this colony.

Date
:   13/09

Operator
:   Susan Bouwmeester

#### High Fidelity PCR

To amplify fusionbb (~6000bp) and fusionduet (~6000) , high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively for fusionbb and fw\_3\_dxCas9-L-Tn5\_Gblock and rv\_4\_dxCas9-L-Tn5\_Gblock forward and reverse primers respctively for fusionduet. 16 50µL PCR mixtures were prepared, 8 for fusionbb and 8 for fusionduet, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| fusionbb | fw\_bb\_dxCas9 | rv\_bb\_Tn5 | fusionbb | ~6000bp |
| fusionduet | fw\_3\_dxCas9-L-Tn5\_Gblock | rv\_4\_dxCas9-L-Tn5\_Gblock | fusionduet | ~6000bp |

The thermocycler programme was installed to have an extension time of 4 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Friday, 14/09/2018

Date
:   14/09

Operator
:   Susan Bouwmeester

#### High Fidelity PCR

To amplify pSB1C3 linear , high fidelity PCR was done using linear pSB1C3 as template DNA and fw\_BB\_pSB1C3 and rv\_BB\_pSB1C3 forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| pSB1C3 | fw\_bb\_pSB1C3 | rv\_bb\_pSB1C3 | pSB1C3 | ~2000bp |

The thermocycler programme was installed to have an extension time of 2:30 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   14/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

The products of the PCR 13/09 to amplify fusionbb and fusion duet was run on a 0.8 % agarose gel in TAE buffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/d5/T--TUDelft--2018_180914fusionbb.png)
**Figure PRC product fusionbb** Lane 1: 500bp ladder; Lane 2-9: PCR product fusion bb

There is fusionbb made. There are a lot of side products made. There must be done a gelcut.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/82/T--TUDelft--2018_180914_fusionduet.png)
**Figure PRC product fusionduet** Lane 1: 500bp ladder; Lane 2-9: PCR product fusionduet

There is no fusionduet made

#### DNA gel purification

The fusionbb has a size of ~6000bp, but the PCR samples were contaminated with by-products. All the PCR product is loaded on a 0.8% agarose gel. DNA purification from agarose gel conform protocol
Elution was done in 30 µL Milli-Q.

Date
:   14/09/2018

Operator
:   Susan Bouwmeester

#### Plasmid Isolation

Overnight starter cultures of Tn5, T7p.Tn5.T7t, fusion are subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   14/09/2018

Operator
:   Susan Bouwmeester

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Tn5 Col1 | 23.2 | 1.57 | 0.53 |
| Tn5 Col3 | 11.8 | 1.69 | 0.49 |
| Tn5 Col4 | 133.5 | 1.52 | 0.58 |
| T7p.Tn5.T7t Col5 | 118 | 1.7 | 1 |
| T7p.Tn5.T7t Col5 | 118 | 1.7 | 1 |
| T7p.Tn5.T7t OldCol6 | 315 | 1.87 | 1.9 |
| Fusion Col1 | 211 | 1.68 | 0.92 |
| Fusion Col2 | 283.3 | 1.66 | 0.92 |
| Fusin Col3 | 357.3 | 1.6 | 0.8 |
| Fusion Col4 | 358.1 | 1.6 | 0.7 |
| Fusion Col5 | 121.4 | 1.8 | 1.65 |

Close

[ ]
Week 3 17/09/2018 - 23/09/2018

Monday, 17/09/2018

Date
:   17/09

Operator
:   Susan Bouwmeester

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble linker and pSB1C3 to obtain pSB1C3-linker; dxCas9 and pSB1C3 to obtain pSB1C3-dxCas9; fusionduet and PACYCduet to obtain PACYCduet-fusion .

| Component | Volume (uL) |
| --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 | 10 |
| Vector | pSB1C3en, 3 (1100 ng) | pSB1C3, 1.9 (1400 ng) | PACYCduet, 2.7 (2700 ng) |
| Fragment | linker, 3.6 (147 ng) | dxCas9, 7 (1400 ng) | fusionduet, 4.4 (2000) |
| MilliQ | 3.4 | 1.1 | 2.9 |

The samples were incubated at 50°C for 60 minutes, after which they were kept on ice for subsequent transformation.

Date
:   17/09/2018

Operator
:   Susan Bouwmeester

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | [pSB1C3-linker] (~2000bp) | Gibson Assembly | 2 | LB Cam || *E.coli*DH5α | [pSB1C3-linker] (~2000bp) | Gibson Assembly | 18 | LB Cam || *E.coli*DH5α | [pSB1C3-dxCas9] (6217bp) | Gibson Assembly | 2 | LB Cam || *E.coli*DH5α | [pSB1C3-dxCas9] (6217bp) | Gibson Assembly | 18 | LB Cam || *E.coli*DH5α | [PACYC-fusion] (9432bp) | Gibson Assembly | 2 | LB Cam || *E.coli*DH5α | [PACYC-fusion] (9432bp) | Gibson Assembly | 18 | LB Cam |

950 uL LB medium was added as recovery medium.

Date
:   17/09/18

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusion and pSB1c3-Tn5. This plasmid contains pSB1C3 and or the fusion protein or Tn5, which both can be checked with primers VR and VF2

| Component | Volume (uL) |
| --- | --- |
| Template | Colony pSB1C3-fusion | Colony pSB1C3-Tn5 || Forward primer | 0.6 | 0.6 |
| Reverse primer | 0.6 | 0.6 |
| Expected amplicon size (bp) | 7699 | 3499 |

12 tubes were prepared for colony PCR. The thermocycler programme was installed to have an extension time of 2:30 for pSB1C3-Tn5 and 7:00 for pSB1C-fusion and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

The PCR product of the colony PCR was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/64/T--TUDelft--2018_180917_fusioncolpcr.png)
**Figure Colony PCR pSB1C3-fusion** expected size is 7699bp. 0.8% agarose. Lane 1: 500bp ladder; Lane 2: Fusion Col1; Lane 3: Fusion Col2; Lane 4: Fusion Col3; Lane 5: Fusion Col4; Lane 6: Fusion Col5; Lane 7: Negative control, GFP; Lane 8: Negative control, no template

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/1b/T--TUDelft--2018_180917_tn5.png)
**Figure Colony PCR pSB1C3-Tn5** expected size is 3499bp. 0.8% agarose. Lane1: Tn5 Col1; Lane 2: Tn5 Col2; Lane 3: Tn5 Col3; Lane 4: Tn5 Col4; Lane 5: Tn5 Col5; Lane 6: Negative control, GFP; Lane 7: Negative control, no template

Date
:   17/09/18

Operator
:   Susan Bouwmeester

#### High Fidelity PCR

To eliminate the forbidden sites, high fidelity PCR was done using PACYCduet-fusionfs as template DNA and fw\_dxCas9\_mut4\_BL and rv\_dxCas9\_mut4\_BL as forward and reverse phosphorylated primers respectively. This makes the product compitable for blunt end ligation. 50µL PCR mixture were prepared, according to the High Fidelity PCR protocol. Expected size of the product PACYCduet-fusion-BL4 is 9432 bp.

The thermocycler programme was installed to have an extension time of 5:00 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

Date
:   17/09/2018

Operator
:   Susan Bouwmeester

#### Ligation

In order to assemble cut fragments linker and pSB1C3en, Tn5 and pSB1C3, ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| Vector | ~2000bp | EcoRI and PstI |
| linker | ~60bp | EcoRI and PstI |
| Tn5 | ~1500 bp |

A 29 uL ligation reaction mixture was prepared with the following composition

| Component | Volume Linker1(uL) | Volume Linker2 | Volume Tn51(uL) | Volume Tn52 |
| --- | --- | --- | --- | --- |
| Ligase Buffer (10x) | 2 | 2 | 2 | 2 |
| T4 DNA Ligase | 1 | 1 | 1 | 1 |
| DNA vector (~100 ng) | pSB1C3en 2.5 uL | pSB1C3en 2.5 uL | pSB1C3 2.1 uL | pSB1C3 2.1 uL |
| DNA fragment | linker 2.6 uL (8.7ng) | linker 7.5 uL (26ng) | Tn5 4.4 uL | Tn5 10 uL |
| MilliQ | 11.9 uL | 7 uL | 10.5 uL | 4.9 uL |

The samples were incubated for overnight at room temperature for ligation.

Close

Tuesday, 18/09/2018

Date
:   18/09/2018

Operator
:   Susan Bouwmeester

#### Restriction

Restriction cloning was done for different constructs. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

| Component | Volume (uL) - pSB1C3 linear | Volume (uL) - Tn5 | Volume (uL) - fusionbb | Volume (uL) - dxCas9 | Volume (uL) - linker | Volume (uL) - pSB1C3en | Volume (uL) - pSB1C3RFP |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Fragment | 2.7 (~2000 ng) | 2 (~2000 ng) | 10 (~600 ng) | 10 (~2000 ng) | 5 (~200 ng) | 4.8 (~2000 ng) | 17 (~2000 ng) |
| Enzyme (EcoRI) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| Enzyme (PstI) | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| MilliQ | 14.3 | 15 | 7 | 7 | 13 | 12.2 | 0 |

The samples were incubated for 2 hours in the incubator 37 °C and 2 hours in the shaker 37 °C , and followed by PCR cleanup.

#### PCR clean-up

PCR clean-up is according to the protocol for all the restricted framgents and PACYCduet-fusion-mut4. Elution was done in 30 µL Milli-Q.

Date
:   18/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

Restricted backbone was run on a 0.8% agarose gel in TAEuffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/fc/T--TUDelft--2018_180918_restrictioncheck.png)
**Figure Restriction** Restriction check. 0.8% Agarose. Lane 1: pSB1C3-RFP restricted; Lane 2: linear pSB1C3 restriced; Lane 3: pSB1C3-RFP uncut; Lane 4: 500bp ladder

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. Also two plasmids were measered. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio |
| --- | --- | --- |
| Tn5 plasmid 1 | 13.3 | 1.63 |
| Tn5 plasmid 2 | 100.5 | 1.86 |
| linker | - | - |
| pSB1C3en | 73.7 | 1.91 |
| pSB1C3 | 24.4 | 2.00 |
| pSB1C3 RFP | 10 | 1.99 |
| dxCas9 | 34.8 | 1.84 |
| Tn5 | 73.7 | 1.91 |
| fusion | 11.65 | 1.93 |
| Bl4 | 68.5 | 1.96 |
| Bl- | 17.0 | 1.96 |

Date
:   18/09/2018

Operator
:   Susan Bouwmeester

#### Ligation

In order to assemble cut several constructs, ligation was performed.

A 10ul ligation reaction mixture was prepared with the following composition

| Component | pSB1C3-linker Volume (uL) | pSB1C3-Fusion Volume (uL) | pSB1C3RFP-Fusion Volume (uL) | pSB1C3-Tn5 Volume (uL) | pSB1C3RFP-Tn5 Volume (uL) | pSB1C3-dxCas9 Volume (uL) | pSB1C3RFP-dxCas9 Volume (uL) | PACYCduet-fusion-mut4 Volume (uL) | PACYCduet-fusion (-) Volume (uL) |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Ligase Buffer (10x) | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| T4 DNA Ligase | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| DNA vector | 1.5 uL (~100ng) | 8.5 uL (~200ng) | 8.5 uL (~85ng) | 4.2 uL (~100ng) | 8.5 uL (~85ng) | 6.4 uL (~150ng) | 8.5 uL (~85ng) | 1.5 uL (~100ng) | 6.0 ul (~100ng) |
| DNA fragment | 14 uL (~8.7ng) | 8.5 uL (~100ng) | 8.5 uL (~100ng) | 3.4 uL (~225ng) | 3.4 uL (~225ng) | 10.6 uL (~100 ng) | 8.5 uL (~100ng) | - | - |
| MilliQ | 1.5 uL | 0 uL | 0 uL | 9.4 | 5.1 uL | 0 uL | 0 uL | 5.5 uL | 11.0 |

The samples were incubated overnight on room temperature for ligation.

Close

Wednesday, 19/09/2018

Date
:   19/09/2018

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-Tn5 after transformation in 2 colonies. This plasmid contains Tn5 and pSB1C3, which can be checked with primers VR and VF2.

| Component | Volume (uL) |
| --- | --- |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |
| Expected amplicon size (bp) | ~1700 bp |

2 tubes were prepared for colony PCR. The thermocycler programme was installed to have an extension time of 2:30 and an annealing temperature of 52 °C. After the programme was finished, samples were kept at 12°C.

Overnight starter cultures of PACYCduet-Fusion was subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   19/09/2018

Operator
:   Susan Bouwmeester

#### Transformation of chemically competent cells

Transformations were carried out according to the protocol for transformation of chemically competent cells. DNA used for the transformation is the result of overnight DNA Ligation of 17/09 and 18/09. 20uL ligation product is used.

#### DNA Gel Electrophoresis

The product of the colony-PCR was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/7/72/T--TUDelft--2018_180919Tn5.png)
**Figure colonyPCR** Result colony PCR. Expected size ~1700bp. 0.8% Agarose. Lane 1: 500bp ladder; Lane 2: Colony 1, Tn5 8/9; Lane 3: Colony 2, Tn5 13/9

Close

Thursday, 20/09/2018

Date
:   20/09

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

The different fragments were run on a 0.8% agarose gel in TAE buffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a2/T--TUDelft--2018_180920_BL.png)
**Figure** Different fragments. 0.8% Agarose. Lane 1: 500 bp ladder; Lane 2: product of blunt end ligation PACYCduet-fusion-mut4 19/9 (plasmid); Lane 3: product of blunt end ligation 19/9 negative control (plasmid); Lane 4: 500bp ladder; Lane 5: pSB1C3-RFP (plasmid); Lane 6: pSB1C3 linear, expected size ~2000bp; Lane 7: pSB1C3en restricted with EcoRI and PstI

#### Transformation Results

After carrying out the transformation protocol on 19/09, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3-linker | LB+Cam | no growth |
| *E.coli* DH5α | pSB1C3-Fusion | LB+Cam | no growth |
| *E.coli* DH5α | pSB1C3RFP-Fusion | LB+Cam | only red colonies |
| *E.coli* DH5α | pSB1C3-Tn5 | LB+Cam | no growth |
| *E.coli* DH5α | pSB1C3RFP-Tn5 | LB+Cam | 10 white colonies; several red colonies |
| *E.coli* DH5α | pSB1C3-dxCas9 | LB+Cam | no growth |
| *E.coli* DH5α | pSB1C3RFP-dxCas9 | LB+Cam | 14 white colonies; several red colonies |
| *E.coli* DH5α | PACYCduet-fusion-mut4 | LB+Cam | ~200 colonies || *E.coli* DH5α | PACYCduet-fusion-mut4 | LB+Cam | no colonies |

Close

Friday, 21/09/2018

Date
:   21/09/2018

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-Tn5 in 10 colonies and pSB1C3-dxCas9 in 14 colonies after transformation 19/9. This plasmid contains Tn5 or dxCas9 and pSB1C3, which can be checked with primers VR and VF2.

| Component | Volume (uL) |
| --- | --- |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |
| Expected amplicon size (bp) | ~1700 bp |

30 tubes were prepared for colony PCR. The thermocycler programme was installed to have an extension time of 2:30 for Tn5 and 5:00 for dxCas9 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

Date
:   21/09/18

Operator
:   Susan Bouwmeester

#### High Fidelity PCR

To eliminate the forbidden sites, high fidelity PCR was done using PACYCduet-fusionfs as template DNA and fw\_dxCas9\_mut4\_BL and rv\_dxCas9\_mut4\_BL as forward and reverse phosphorylated primers respectively. This makes the product compitable for blunt end ligation. 50µL PCR mixture were prepared, according to the High Fidelity PCR protocol. Expected size of the product PACYCduet-fusion-BL4 is 9432 bp.

The thermocycler programme was installed to have an extension time of 5:30 and a gradient in anealing temperature (55.1; 56.8; 58.7; 61.5; 63.7; 65.2) After the programme was finished, samples were kept at 12 °C.

#### High Fidelity PCR

To eliminate the forbidden sites, high fidelity PCR was done using PACYCduet-fusionfs as template DNA. To check the primers, all needed eliminations were performed parallel. The primers used can be found in the table. Phosphorylated primers were used. This makes the product compitable for blunt end ligation. 6x 50µL PCR mixture were prepared, according to the High Fidelity PCR protocol. Expected size of the product PACYCduet-fusion-mutation is 9432 bp.

| Primer |
| --- |
| fw\_dxCas9\_mut1\_BL |
| rv\_dxCas9\_mut1\_BL |
| fw\_dxCas9\_mut2\_BL |
| rv\_dxCas9\_mut2\_BL |
| fw\_dxCas9\_mut3\_BL |
| rv\_dxCas9\_mut3\_BL |
| fw\_dxCas9\_mut4\_BL |
| rv\_dxCas9\_mut4\_BL |
| fw\_dxCas9\_mut5\_BL |
| rv\_dxCas9\_mut5\_BL |

The thermocycler programme was installed to have an extension time of 8:00 and an anealing temperature of 60°C. After the programme was finished, samples were kept at 12 °C.

#### DNA Gel Electrophoresis

The different fragments were run on a 0.8% agarose gel in TAE buffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/65/T--TUDelft--2018_180921_dxCas9ColPCR.png)
**Figure dxCas9 colony PCR**0.8% Agarose. Expected size = ~4000bp. Lane 1: 500 bp ladder; Lane 2 t/m 14: Colony 1 t/m 13 pSB1C3-dxCas9; Lane 14: 500bp ladder; Lane 15 500bp ladder; Lane 16: Colony 15; Lane 17: Negative control GFP; Lane 18: Negative control, no template

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/1e/T--TUDelft--2018_180921_Tn5.png)
**Figure Tn5 colony PCR**0.8% Agarose. Lane 1: 500 bp ladder; Lane 2 t/m 11: Colony 1 t/m 10 pSB1C3-Tn5; Lane 11: Negative control GFP; Lane 12: Negative control, no template

Conclusion: No correct integrated dxCas9, 9 times possible integrated Tn5.

Close

Saturday, 22/09/2018

Date
:   18/09/2018

Operator
:   Susan Bouwmeester

#### Restriction

Restriction cloning was done for different constructs. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

| Component | Volume (uL) - fusionbb | Volume (uL) - dxCas9 | Volume (uL) - linker | Volume (uL) - pSB1C3en | Volume (uL) - pSB1C3RFP |
| --- | --- | --- | --- | --- | --- |
| 10x CutSmart buffer | 2 | 2 | 2 | 2 | 2 |
| Fragment | 16 (~976 ng) | 5 (~1000 ng) | 16 (~624 ng) | 4.8 (~2000 ng) | 16 (~1600 ng) |
| Enzyme (EcoRI-HF) | 1 | 1 | 1 | 1 | 1 |
| Enzyme (PstI-HF) | 1 | 1 | 1 | 1 | 1 |
| MilliQ | 0 | 11 | 0 | 11.2 | 0 |

The samples were incubated for 3.5 hours in the incubator 37 °C, followed by PCR cleanup. The enzymes were heat inactivated for 10 minutes 80 °C.

#### PCR clean-up

PCR clean-up is according to the protocol for all the restricted framgents. Elution was done in two times 15uL Milli-Q.

#### High Fidelity PCR

To amplify dxCas9 (~4207bp), high fidelity PCR was done using the fusionbb as template DNA and fw\_bb\_dxcas9 and rv\_bb\_dxcas9\_2 as forward and reverse primers respectively. 6 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 2:30 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

#### DNA Gel Electrophoresis

The different PCR products from PACYCduet-fusionfs (21/9) were run on a 0.8% agarose gel in TAE buffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a4/T--TUDelft--2018_180922BLligation.png)
**Figure PCR PACYCduet-fusionfs**0.8% Agarose. Expected size = ~10000bp. Lane 1: 500 bp ladder; Lane 2: annealing temperature PCR 65.2°C; Lane 3: annealing temperature PCR 63.7°C; Lane 4: annealing temperature PCR 61.5°C; Lane 5: annealing temperature PCR 58.7°C; Lane 6: annealing temperature PCR 56.8°C; Lane 7: annealing temperature PCR 55.1°C; Lane 8: 500 bp ladder; Lane 9: primers mutation 1; Lane 10: primers mutation 2; Lane 11: primers mutation 3; Lane 12: primers mutation 4; Lane 13: primers mutation 5; Lane 14: primers mutation 6; Lane 15: negative control pSB1C3RFP

Close

[ ]
Week 4 24/09/2018 - 30/09/2018

Monday, 24/09/2018

Date
:   24/09/2018

Operator
:   Susan Bouwmeester

#### Restriction

Restriction cloning was done for pSB1C3 and the fusion protein. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) - pSB1C3 linear | Volume (uL) - fusionbb || 10x CutSmart buffer | 5 | 5 |
| Fragment | 42 | 13 |
| Enzyme (EcoRI) | 1.5 | 0.75 |
| Enzyme (PstI) | 1.5 | 0.75 |
| MilliQ | 0 | 3.5 |

The samples were incubated for 4.5 hours in the incubator 37 °C and followed by 20 minutes heat inactivation 80 °C.

#### High Fidelity PCR

To amplify fusionbb (~6000bp), high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively. 16 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6:30 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

#### DNA gel purification

The fusionbb has a size of ~6000bp, but the PCR samples were contaminated with by-products. All the PCR product is loaded on a 0.8% agarose gel. DNA purification from agarose gel conform protocol
Elution was done in 30 µL Milli-Q.

#### PCR clean-up

100 μL of PCR product PACYCduet-fusionfs-mut4 was subjected to PCR Clean-Up, according to the protocol. Elution was done in 2 times 15 µL Milli-Q.

#### DpnI Digestion

To eliminate any residual template DNA, a DpnI digestion was performed with the following reaction mixture:

| Component | Volume (uL) |
| --- | --- |
| 10x CutSmart buffer | 4 |
| PCR product | 23 |
| Enzyme (DpnI) | 1 |
| MilliQ | 12 |

The reaction was incubated for 2 hours at 37 °C.
Heat inactivation was performed for 20 minutes at 80 °C.

#### Plasmid Isolation

Overnight starter cultures of pSB1C3-T7.Tn5.T7, pSB1C3-Tn5colony5 and pSB1C3-Tn5colony8 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with two times 15 μL of pre-warmed MilliQ.

#### Nanodrop DNA Quantification

After plasmid isoaltion, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 230/280 ratio |
| --- | --- | --- | --- |
| pSB1C3-T7p.Tn5.T7t | 134.8 | 1.74 | 1.81 |
| pSB1C3-Tn5Col5 | 146.7 | 1.96 | 1.8 |
| pSB1C3-Tn5Col8 | 1.8 | 1.85 |

#### Sequence Verification

##### Option 1: Sequence from plasmid

For verification of T7-Tn5.T7 biobrick and Tn5 biobrick, primers VF2 and VR were used for sequencing. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | T7.Tn5.T7 Volume (uL) | Tn5 Col5 Volume (uL) | Tn5 Col8 Volume (uL) | Final concentration |
| --- | --- | --- | --- | --- |
| Plasmid DNA (500ng) | 3.71 | 3.41 | 2.61 | 50 ng/uL |
| 10uM primer XXX | 2.5 | 2.5 | 2.5 | 25 pmol |
| MilliQ | 3.79 | 4.09 | 4.89 |

The result of the sequencing: Only Tn5Col5 and Tn5Col8 turned out to be correct. T7.Tn5.T7 missed a part.

#### DNA gel purification

The dxCas9bb has a size of ~4000bp, but the PCR samples were contaminated with by-products. All the PCR product is loaded on a 0.8% agarose gel. DNA purification from agarose gel conform protocol. Elution was done in two times 15 µL Milli-Q.

#### PCR clean-up

Product of restriction of pSB1C3 and the fusion protein with EcoRI and PstI was subjected to PCR Clean-Up, according to the protocol. Elution was done in two times 15 µL Milli-Q.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples and some older restricted samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 230/280 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/280 ratio |
| --- | --- | --- | --- |
| pSB1Cen 18/9 | 71.5 | 0.99 | 1.87 |
| Linker 18/9 | 7.3 | 0.23 | 1.42 |
| pSB1C3en 22/9 | 68 | 1.38 | 1.88 |
| pSB1C3RFP 22/9 | 85.3 | 0.84 | 1.82 |
| dxCas9 22/9 | 17 | 0.39 | 1.79 |
| linker 22/9 | 7.7 | 0.25 | 1.70 |
| fusion 22/9 | 11.3 | 0.46 | 1.79 |
| pSB1C3RFP 24/9 | 179.2 | 1.96 | 1.88 |
| fusion | 32.8 | 1.17 | 1.83 |

#### Ligation

In order to assemble several cut constructs, ligation was performed.

A 10ul ligation reaction mixture was prepared with the following composition

| Component | pSB1C3-Fusion Volume(uL) | pSB1C3-dxCas9 Volume (uL) | pSB1C3-linker(uL) | pSB1C3-negative Volume (uL) |
| --- | --- | --- | --- | --- |
| Ligase Buffer (10x) | 2 | 2 | 2 | 2 |
| T4 DNA Ligase | 1 | 1 | 1 | 1 |
| DNA vector | 0.6 uL (~100ng) | 0.6 uL (~100ng) | 1.47 uL (~100ng) | 0.6 uL (~100ng) |
| DNA fragment | 17uL (~500ng) | 16.4 uL (~270ng) | 15.5 uL(~ng) | 0 uL |
| MilliQ | 0 uL | 0.6 uL | 0 uL | 16.4 |

The samples were incubated overnight on 16°C for ligation. After 13h 10minutes 90°C heat inactivation.

#### PCR clean-up

After DpnI digestion PACYCduet-fusionfs-mut4 was subjected to PCR Clean-Up, according to the protocol. Elution was done in 2 times 15 µL Milli-Q.

#### Ligation

ligation was performed for blunt end ligation of PACYCduet-fusionfs-mut4.

A 10ul ligation reaction mixture was prepared with the following composition:

| Component | PACYCduet-fusionfs-mut4 |
| --- | --- |
| Ligase Buffer (10x) | 2 |
| T4 DNA Ligase | 1 |
| DNA fragment | 2.4 uL (~200ng) |
| MilliQ | 14.6 uL |

The samples were incubated overnight on 16°C for ligation. After 13h 10minutes 90°C heat inactivation.

Close

Tuesday, 25/09/2018

Date
:   25/09/2018

Operator
:   Susan Bouwmeester

#### High Fidelity PCR

To amplify dxCas9 (~4207bp), high fidelity PCR was done using the dxCas9bb as template DNA and fw\_bb\_dxcas9 and rv\_bb\_dxcas9\_2 as forward and reverse primers respectively. 6 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 2:30 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

#### DNA Gel Electrophoresis

PCR product for the amplification of dxCas9bb was run on a 0.8% agarose gel in TAE buffer. 4μL was pipetted into the gel. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/0/02/T--TUDelft--2018_180925_dxCas9amp.png)
**Figure dxCas9 amplification**. Expected size (~4000bp). 0.8% agarose. Lane 1: 500bp ladder; Lane 2 PCR product

No dxCas9bb produced during the PCR.

#### DNA Gel Electrophoresis

PCR product for the amplification of fusionbb was run on a 0.8% agarose gel in TAE buffer. All PCR-product was loaded on the gel. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/ba/T--TUDelft--2018_180925_fusionamp.png)
**Figure fusionbb amplification**. Expected size (~6000bp). 0.8% agarose. Lane 1: 500bp ladder; Lane 2t/m 9 PCR product fusionbb

fusionbb produced during the PCR.

#### High Fidelity PCR

To amplify linker(~60bp), high fidelity PCR was done using the previous produced linker as template DNA and fw\_bb\_linker and rv\_bb\_linker forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 0:23 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12 °C.

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pSB1C3-fusion | restiction/ligation | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-fusion | restiction/ligation | 18 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas9 | restiction/ligation | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas9 | restiction/ligation | 18 | LB Cam |
| *E.coli*DH5α | pSB1C3-linker | restiction/ligation | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-linker | restiction/ligation | 18 | LB Cam |
| *E.coli*DH5α | PACYCduet-fusionfs-mut4 | restiction/ligation | 2 | LB Cam |
| *E.coli*DH5α | PACYCduet-fusionfs-negative | restiction/ligation | 18 | LB Cam |

800 uL LB medium was added as recovery medium.

#### High Fidelity PCR

To amplify fusionbb (~6000bp), high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively. 4 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6:30 minutes and an annealing temperature of 70 °C. After the programme was finished, samples were kept at 4 °C.

#### High Fidelity PCR

To amplify dxCas9 (~4207bp), high fidelity PCR was done using the fusionbb as template DNA and fw\_bb\_dxcas9 and rv\_bb\_dxcas9\_2 as forward and reverse primers respectively. 4 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 3:00 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

Close

Wednesday, 26/09/2018

Date
:   26/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

PCR product of the amplification of the linker was run on a 2% agarose gel in TAE buffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/2a/T--TUDelft--2018_180926_linkeramp.png)
**Figure** linker amplification. 2% agarose. Expected size is ~60bp. Lane 1: 100bp ladder; Lane 2: PCR product linker

#### Transformation Results

After carrying out the transformation protocol on 25/09 the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3-dxCas9-1 | LB+Cam | 1 white; 8 red |
| *E.coli* DH5α | pSB1C3-dxCas9-2 | LB+Cam | 16 red |
| *E.coli* DH5α | pSB1C3-dxCas9-3 | LB+Cam | 1 white; ~100 red |
| *E.coli* DH5α | pSB1C3-dxCas9-4 | LB+Cam | ~100 red |
| *E.coli* DH5α | pSB1C3-fusion-1 | LB+Cam | 10 red |
| *E.coli* DH5α | pSB1C3-fusion-2 | LB+Cam | 15 red |
| *E.coli* DH5α | pSB1C3-fusion-3 | LB+Cam | 6 white; ~100 red |
| *E.coli* DH5α | pSB1C3-dxCas9-4 | LB+Cam | 5 white; ~100 red |
| *E.coli* DH5α | PACYCduet-fusionfs-mut4 | LB+Cam | ~100 |

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusion in 2 colonies and pSB1C3-dxCas9 in 1 colony after transformation. This plasmid contains the fusion(~6000bp) or dxCas9 (~4000bp) and pSB1C3, which can be checked with primers VR and VF2.

| Component | Volume (uL) |
| --- | --- |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |

5 tubes were prepared for colony PCR. The thermocycler programme was installed to have an extension time of 8:00and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of dxCas9 and fusion was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/ba/T--TUDelft--2018_180926_dxcas9colPCR.png)
**Figure** Colony PCR fusion and dxCas9. 0.8% agarose. Expected size fusion ~6000 bp. Expected size dxCas9 ~4000bp. Lane 1: 500bp ladder; Lane 2: fusion colony 1; Lane 3: fusion colony 2; Lane 4: dxCas9 colony 1

The amplified product colony PCR is around the correct hight. An overnight culture dxCas9 colony1 in 3mL with Cam is made.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of dxCas9 and fusion was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/80/T--TUDelft--2018_180926_amp.png)
**Figure**Amplification dxCas9bb and fusionbb. 0.8% agarose. Expected size fusion ~6000 bp. Expected size dxCas9 ~4000bp. Lane 1: 500bp ladder; Lane 2: fusionbb; Lane 4: dxCas9bb

#### DNA purification agarose gel

The amplicon of interest has a size of ~4000bp or ~6000bp, but the PCR samples were contaminated with by-products. The products were loaded on a gel prior to [DNA purification from agarose gel conform protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#DNApurificationfromgel-scroll).
Elution was done in 2 times 15 µL Milli-Q.

#### PCR clean-up

200 ul of PCR product of the linker was subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 ratios and 260/230) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| linker | 262.2 | 1.84 | 1.91 |
| dxCas9 | 111.4 | 1.84 | 1.77 |
| fusion | 88.8 | 1.88 | 1.66 |

#### High Fidelity PCR

To amplify fusionbb (~6000bp), high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6:30 minutes and an annealing temperature of 70 °C. After the programme was finished, samples were kept at 4 °C.

#### High Fidelity PCR

To amplify dxCas9 (~4207bp), high fidelity PCR was done using the fusionbb as template DNA and fw\_bb\_dxcas9 and rv\_bb\_dxcas9\_2 as forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 3:00 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

Close

Thursday, 27/09/2018

Date
:   27/09/2018

Operator
:   Susan Bouwmeester

#### Plasmid Isolation

Overnight starter cultures of pSB1C3-dxCas9 subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with two times 15 μL of pre-warmed MilliQ.

#### Nanodrop DNA Quantification

After plasmid isoaltion, the sample was measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 230/280 ratio |
| --- | --- | --- | --- |
| pSB1C3-dxCas9 | 292.7 | 1.87 | 2.09 |

#### Sequence Verification

##### Sequence from plasmid

For verification of dxCas9 biobrick primers VF2, VR, fw\_dxCas9\_mut\_EcoRI, fw\_Tn5\_beforeC, fw\_dxCas9\_mut2\_GA and rv\_dxCas9\_mut3\_GA were used for sequencing. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | Volume (uL) | Final concentration |
| --- | --- | --- |
| Plasmid DNA (500ng) | 1.71 | 50 ng/uL |
| 10uM primer XXX | 2.5 | 25 pmol |
| MilliQ | 5.79 |

The result of the sequencing: dxCas9 is only partly integrated. It is not the complete sequence for dxCas9bb.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusion in 12 colonies and after transformation. This plasmid contains the fusion (~6000bp) and pSB1C3, which can be checked with primers VR and VF2.

| Component | Volume (uL) |
| --- | --- |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |

12 tubes were prepared for colony PCR of 12 pSB1C3-fusion colonies. The thermocycler programme was installed to have an extension time of 8:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of fusion was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f8/T--TUDelft--2018_180927_fusion.png)
**Figure** Colony PCR dxCas9. 0.8% agarose. Expected size dxCas9 ~4000bp. Lane 1: 500bp ladder; Lane 2: colony 1; Lane 3: colony 2; Lane 4: colony 3; Lane 5: colony 4; Lane 6: colony 5; Lane 7: colony 6; Lane 8: colony 7; Lane 9: colony 8; Lane 10: colony 9; Lane 11: colony 10; Lane 12: colony 11; Lane 13: colony 12; Lane 14: Negative control, no template; Lane 15: 500bp ladder

The amplified product colony PCR is not around the correct hight. No succesful integration of dxCas9 in pSB1C3

Close

Friday, 28/09/2018

Date
:   28/09/2018

Operator
:   Susan Bouwmeester

#### DNA Gel Electrophoresis

PCR product of PCR for fusion amplification was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/66/T--TUDelft--2018_180928_dxCas9.png)
**Figure**Fusion amplification. 0.8% agarose. Expected size fusion ~6000bp. Lane 1: 500bp ladder; Lane 2: amplified fusion

#### DNA gel purification

The fusionbb has a size of ~6000bp, but the PCR samples were contaminated with by-products. All the PCR product is loaded on a 0.8% agarose gel. DNA purification from agarose gel conform protocol. Elution was done in two times 15 µL Milli-Q.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples and some older restricted samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 230/280 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/280 ratio |
| --- | --- | --- | --- |
| fusionbb | 173.2 | 1.87 | 0.8 |

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble fusionbb and pSB1C3 to obtain pSB1C3-fusion.

| Component | Volume (uL) |
| --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 | 10 |
| Vector | pSB1C3, 0.3 (200 ng) |
| Fragment | fusionbb, 9.7 (1800) |
| MilliQ | 3.4 | 1.1 | 2.9 |

The samples were incubated at 55°C for 60 minutes, after which they were stored at -20°C.

#### Restriction

Restriction cloning was done for pSB1C3 and the fusion protein. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) - pSB1C3RFP | Volume (uL) - fusionbb || 10x CutSmart buffer | 5 | 5 |
| Fragment | 41 (~5000ng) | 20.4 (~2000ng) |
| Enzyme (EcoRI) | 2 | 2 |
| Enzyme (PstI) | 2 | 2 |
| MilliQ | 0 | 20.6 |

The samples were incubated for 4 hours in the incubator 37 °C and followed by 20 minutes heat inactivation 80 °C.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusion in 10 colonies and after transformation. This plasmid contains the fusion (~6000bp) and pSB1C3, which can be checked with primers VR and VF2.

| Component | Volume (uL) |
| --- | --- |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |

10 tubes were prepared for colony PCR of 10 pSB1C3-fusion colonies. The thermocycler programme was installed to have an extension time of 8:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of fusion was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/d0/T--TUDelft--2018_180928_colPCRfusion.png)
**Figure** Colony PCR fusion. 0.8% agarose. Expected size fusion ~6000bp. Lane 1: 500bp ladder; Lane 2: colony 1; Lane 3: colony 2; Lane 4: colony 3; Lane 5: colony 4; Lane 6: colony 5; Lane 7: colony 6; Lane 8: colony 7; Lane 9: colony 8; Lane 10: colony 9; Lane 11: colony 10; Lane 12: Negative control, no template;

The amplified product colony PCR is not around the correct hight. No succesful integration of fusion in pSB1C3

#### High Fidelity PCR

To eliminate the forbidden sites, high fidelity PCR was done using PACYCduet-mut4-1, PACYCduet-mut4-2 and PACYCduet-mut4-3 as template DNA. Primer fw\_dxCas9\_mut5\_BL and rv\_dxCas9\_mut5\_BL were used. Phosphorylated primers were used. This makes the product compitable for blunt end ligation. 3x 50µL PCR mixture was prepared, according to the High Fidelity PCR protocol. Expected size of the product PACYCduet-fusion-mutation is 9432 bp.

The thermocycler programme was installed to have an extension time of 8:00 and an anealing temperature of 60°C. After the programme was finished, samples were kept at 12 °C.

#### DNA Gel Electrophoresis

the PCR product of the amplification of PACYCduet-mut5 was run on a 0.8% agarose gel in TAE buffer to check the pCR reaction. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/68/T--TUDelft--2018_180928_BL5.png)
**Figure** Colony PCR fusion. 0.8% agarose. Expected size fusion ~10000bp. Lane 1: 500bp ladder; Lane 2: PACYCduet-mut5-1; Lane 3: PACYCduet-mut5-2; Lane 4: PACYCduet-mut5-3;

#### High Fidelity PCR

To amplify fusionbb (~6000bp), high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6:30 minutes and an annealing temperature of 70 °C. After the programme was finished, samples were kept at 4 °C.

#### DNA Gel Electrophoresis

Restriction product of fusion and pSB1C3RFP was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/c6/T--TUDelft--2018_180928_res_fus.png)
**Figure**Restriction fusionbb and pSB1C3RFP. 0.8% agarose. Expected size fusion ~6000bp; Expected size pSB1C3 restricted is two fragments of ~2000bp and ~1500bp. Lane 1: 500bp ladder; Lane 2: restricted fusionbb; Lane 3: restricted pSB1C3RFP

#### PCR clean-up

PCR product of the PACYCduet-fusion-mut5 was subjected to PCR Clean-Up, according to the protocol. Elution was done in one time 15uL and one time 20uL Milli-Q.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 ratios and 260/230) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| PACYCduet-fusion-mut5a | 100.7 | 1.97 | 0.15 |
| PACYCduet-fusion-mut5b | 130.7 | 1.87 | 0.16 |
| PACYCduet-fusion-mut5c | 225.0 | 1.89 | 0.27 |

#### DpnI Digestion

To eliminate any residual template DNA, a DpnI digestion was performed for PACYCduet-fusion-mut5a, PACYCduet-fusion-mut5b, PACYCduet-fusion-mut5c, with the following reaction mixture:

| Component | Volume (uL) PACYCduet-fusion-mut5a | Volume (uL) PACYCduet-fusion-mut5b | Volume (uL) PACYCduet-fusion-mut5c |
| --- | --- | --- | --- |
| 10x CutSmart buffer | 5 | 5 | 5 |
| PCR product | 32 | 32 | 32 |
| Enzyme (DpnI) | 1.5 | 1.5 | 1.5 |
| MilliQ | 12 | 12 | 12 |

The reaction was incubated for 4 hours at 37 °C.
Heat inactivation was performed for 20 minutes at 80 °C.

#### DNA Gel Electrophoresis

Restriction product of fusion and pSB1C3RFP was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/c6/T--TUDelft--2018_180928_res_fus.png)
**Figure**Restriction fusionbb and pSB1C3RFP. 0.8% agarose. Expected size fusion ~6000bp; Expected size pSB1C3 restricted is two fragments of ~2000bp and ~1500bp. Lane 1: 500bp ladder; Lane 2: restricted fusionbb; Lane 3: restricted pSB1C3RFP

#### DNA Gel Electrophoresis

Restriction product of fusion and pSB1C3RFP was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/85/T--TUDelft--2018_180928_dxcas9amp.png)
**Figure** Amplification dxCas9. Expected size ~4000bp. 0.8% agarose. Expected size fusion ~6000bp; Expected size pSB1C3 restricted is two fragments of ~2000bp and ~1500bp. Lane 1: 500bp ladder; Lane 2: restricted fusionbb; Lane 3: restricted pSB1C3RFP

Close

Saturday, 29/09/2018

Date
:   29/09/2018

Operator
:   Susan Bouwmeester

#### PCR clean-up

DpnI digested PCR products of the PACYCduet-fusion-mut5 were subjected to PCR Clean-Up, according to the protocol. Elution was done in one time 15uL and one time 20uL Milli-Q.

#### DNA gel purification

The EcoRI and PstI restricted fusionbb and pSB1C3 contains RFP and other flanks. To clean this, the restricted product is loaded on a 0.8% agarose gel. DNA purification from agarose gel conform protocol. Elution was done in one time 15 µL Milli-Q and one time 20 µL.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 230/280 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/280 ratio |
| --- | --- | --- | --- |
| PACYCduet-fusion-mut5-a | 15.3 | 1.7 | 0.45 |
| PACYCduet-fusion-mut5-b | 15.1 | 1.8 | 0.72 |
| PACYCduet-fusion-mut5-c | 16.2 | 1.6 | 0.07 |
| pSB1C3-restricted | 57.4 | 1.88 | 1.35 |
| fusion-resticted | 26.7 | 1.5 | 0.78 |

#### Blunt end Ligation

ligation was performed for blunt end ligation of PACYCduet-fusionfs-mut5.

A 10ul ligation reaction mixture was prepared with the following composition:

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | PACYCduet-fusion-mutation5 || Ligase Buffer (10x) | 2 |
| T4 DNA Ligase | 1 |
| DNA fragment | 2.4 uL (~200ng) |
| MilliQ | 14.6 uL |

The samples were incubated overnight on 16°C for ligation.

#### Ligation

In order to assemble pSB1C3-fusion, ligation was performed.

A 10ul ligation reaction mixture was prepared with the following composition

| Component | pSB1C3-Fusion Volume(uL) |
| --- | --- |
| Ligase Buffer (10x) | 2 |
| T4 DNA Ligase | 1 |
| DNA vector | 0.9 uL (~100ng) |
| DNA fragment | 16.1 uL (~500ng) |
| MilliQ | 0 uL |

The samples were incubated overnight on 16°C for ligation.

#### High Fidelity PCR

To amplify fusionbb (~6000bp), high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6:30 minutes and an annealing temperature of 70 °C. After the programme was finished, samples were kept at 4 °C.

#### High Fidelity PCR

To amplify dxCas9 (~4207bp), high fidelity PCR was done using the fusionbb as template DNA and fw\_bb\_dxcas9 and rv\_bb\_dxcas9\_2 as forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 3:00 and an annealing temperature of 63 °C. After the programme was finished, samples were kept at 12 °C.

Date
:   29/09/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

*E.coli* strain harbouring the plasmids pACYCDuet-1\_Tn5, pACYCDuet-1 and pSB1C3 were used for inoculation of 10 mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Date
:   29/09/2018

Operator
:   Nicole Bennis

#### High Fidelity PCR

To amplify pSB1C3 backbone, dxCas9 (BioBrick cloning), dxCas9-Lin-Tn5 (BioBrick cloning), dxCas9 (pACYCDuet-1 cloning), dxCas9-Lin-Tn5 (pACYCDuet-1 cloning) high fidelity PCR was done. The template DNA are the Gblocks 3 and 4 combined (primerset 056 & 057 and 056 & 055) and pACYCDuet-1 (primerset 083 & 084) and pSB1C3 (primerset 051 & 052). Multiple 50µL PCR mixtures were prepared, according to the [High Fidelity PCR protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#highfidelityPCR-scroll).
Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Expected fragment | Expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 051 | 052 | pSB1C3 | 2068 |
| 2 | 056 | 057 | dxCas9 (BB) | 4226 |
| 3 | 056 | 055 | dxCas9-Lin-Tn5 (BB) | 5703 |
| 4 | 001 | 002 | dxCas9 (pACYCDuet-1) | 4172 |
| 5 | 079 | 082 | Gblock 3 4 | 5897 |
| 6 | 083 | 084 | pACYCDuet-1 | 3652 |

The thermocycler programme was installed to have an extension time of 6 minutes 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Sunday, 30/09/2018

Close

### October

[ ]
Week 1 01/10/2018 - 07/10/2018

Monday, 01/10/2018

Date
:   01/10/2018

Operator
:   Susan Bouwmeester

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pSB1C3-fusion | restiction/ligation | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-fusion | restiction/ligation | 18 | LB Cam |
| *E.coli*DH5α | pSB1C3-fusion | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-fusion | Gibson Assembly | 18 | LB Cam |

800 uL LB medium was added as recovery medium.

#### DNA purification agarose gel

The dxCas9 and the fusion protein have a size of ~4000bp or ~6000bp, but the PCR samples were contaminated with by-products. The products were loaded on a gel prior to [DNA purification from agarose gel conform protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#DNApurificationfromgel-scroll).
Elution was done in 30 µL Milli-Q.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 230/280 ratios) was measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/280 ratio |
| --- | --- | --- | --- |
| Fusion 1 | 72.7 | 1.84 | 0.14 |
| Fusion 2 | 6.09 | 1.4 | 0.08 |
| Fusion 3 | 64.9 | 1.84 | 0.07 |
| Fusion 4 | 4.4 | 1.17 | 0.13 |
| Fusion 5 | 23.2 | 2.1 | 0.13 |
| dxCas9 1 | 279.7 | 1.85 | 1.21 |
| dxCas9 2 | 3.17 | 1.11 | 0.24 |
| dxCas9 3 | 120.1 | 1.87 | 1.09 |
| dxCas9 4 | 2.5 | 1.26 | 0.32 |
| dxCas9 5 | 36.6 | 1.9 | 0.9 |

Date
:   01/10/2018

Operator
:   Nicole Bennis

#### Liquid Starter Culture Preparation

*E.coli* strain harbouring the plasmids pACYCDuet-1\_dxCas9, pACYCDuet-1\_dxCas9-Lin-Tn5, pSB1C3-mRFP, colony 3 pSB1C3\_intron1, colony 8 pSB1C3\_intron2, colony 10 pSB1C3\_intron 1 and 2, pSB1C3\_EPO and pSB1C3\_gRNALacZ were used for inoculation of 4 times 10 mL per strain liquid starter culture (Luria Broth, complemented with chloramphenicol) following the [Liquid Starter Culture Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#liquidstarterculture-scroll).

Close

Tuesday, 02/10/2018

Date
:   02/10/2018

Operator
:   Susan Bouwmeester

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3-dxCas9.

| Component | Volume (uL) pSB1C3-dxCas9-1 | Volume (uL) pSB1C3-dxCas9-2 | Volume (uL) pSB1C3-dxCas9-3 |
| --- | --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 | 10 |
| Vector (pSB1C3) | 1.5 (200 ng) | 7.35 (980 ng) | 2.5 (330 ng) |
| Fragment (dxCas9) | 4.3 (1200 ng) | 2.2 (600 ng) | 7.08 (1900 ng) |
| MilliQ | 4.2 | 0.45 | 0.42 |

The samples were incubated at 55°C for 60 minutes, after which they were stored at -20°C.

#### Restriction

Restriction cloning was done for pSB1C3 and the dxCas9. The following table shows an overview of what fragments were cut with what restriction enzymes.

| Component | Volume (uL) - dxCas9bb |
| --- | --- |
| 10x CutSmart buffer | 5 |
| Fragment | 30 |
| Enzyme (EcoRI) | 2 |
| Enzyme (PstI) | 2 |
| MilliQ | 11 |

The samples were incubated for 4 hours in the incubator 37 °C and followed by 20 minutes heat inactivation 80 °C.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-dxCas9 in 20 colonies after transformation. This plasmid contains the fusion (~6000bp) and pSB1C3, which can be checked with primers VR and VF2.

| Component | Volume (uL) |
| --- | --- |
| Forward primer | 0.6 |
| Reverse primer | 0.6 |

20 tubes were prepared for colony PCR of pSB1C3-dxCas9 colonies. The thermocycler programme was installed to have an extension time of 5:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pSB1C3-dxCas9-1 | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas9-1 | Gibson Assembly | 18 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas9-2 | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas9-2 | Gibson Assembly | 18 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas9-3 | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α | pSB1C3-dxCas-9 | Gibson Assembly | 18 | LB Cam |

800 uL LB medium was added as recovery medium.

Date
:   02/10/2018

Operator
:   Nicole Bennis

#### Plasmid Isolation

Overnight starter cultures of *E.coli* strains harbouring the plasmids pACYCDuet-1\_dxCas9, pACYCDuet-1\_dxCas9-Lin-Tn5, pSB1C3-mRFP, colony 3 pSB1C3\_intron1, colony 8 pSB1C3\_intron2, colony 10 pSB1C3\_intron 1 and 2, pSB1C3\_EPO and pSB1C3\_gRNALacZ were subjected to plasmid isolation following the [Plasmid Isolation Protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#plasmidisolation-scroll). Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   02/10/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYCDuet-1\_dxCas9 (1) | 1447.8 | 2.06 | 2.17 |
| pACYCDuet-1\_dxCas9 (2) | 1867 | 2.09 | 2.38 |
| pACYCDuet-1\_dxCas9-Lin-Tn5 (1) | 1256 | 2.06 | 2.10 |
| pACYCDuet-1\_dxCas9-Lin-Tn5 (2) | 1361.4 | 1.99 | 1.86 |
| pSB1C3-mRFP (1) | 738 | 1.98 | 2.12 |
| pSB1C3-mRFP (2) | 737 | 2.01 | 2.18 |
| pSB1C3\_EPO\_intron 1 | 495.9 | 1.83 | 1.72 |
| pSB1C3\_EPO\_intron 2 | 422.2 | 1.79 | 1.38 |
| pSB1C3\_EPO\_intron 1 and 2 | 391.8 | 1.87 | 1.99 |
| pSB1C3\_EPO | 462.7 | 2.01 | 1.70 |
| pSB1C3\_LacZgRNA | 347.6 | 2.01 | 1.68 |

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

50 μL of each PCR mixture performed on 29/09/2018was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   02/10/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| dxCas9 (pACYCDuet-1) | 62.8 | 1.79 | 0.99 |
| dxCas9 (BioBrick) | 197.1 | 1.84 | 2.09 |
| dxCas9-Lin-Tn5 (pACYCDuet-1) | 277.2 | 1.84 | 2.21 |
| dxCas9-Lin-Tn5 (BioBrick) | 340.6 | 1.83 | 2.16 |
| pSB1C3 backbone (1) | 396.4 | 1.85 | 1.83 |
| pACYCDuet-1 backbone | 553.1 | 1.84 | 2.19 |
| pSB1C3\_EPO\_intron 1 | 495.9 | 1.83 | 1.72 |
| pSB1C3\_EPO\_intron 2 | 422.2 | 1.79 | 1.38 |
| pSB1C3\_EPO\_intron 1 and 2 | 391.8 | 1.87 | 1.99 |
| pSB1C3\_EPO | 462.7 | 2.01 | 1.70 |
| pSB1C3\_LacZgRNA | 347.6 | 2.01 | 1.68 |

Close

Wednesday, 03/10/2018

Date
:   03/10/2018

Operator
:   Susan Bouwmeester

#### PCR clean-up

PCR clean-up is done according to the protocol for pSB1C3, dxCas9fs and fusionfs previous made. Elution was done in 30 µL Milli-Q

#### DNA Gel Electrophoresis

PCR product of the colony PCR of fusion was run on a 0.8% agarose gel in TAE buffer. Only 4 colonies turned out to be white. A total of 9 colony PCR results were put on a gel. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/fe/T--TUDelft--2018_181003.png)
**Figure** Colony PCR fusion. 0.8% agarose. Expected size fusion ~6000bp. Lane 1: 500bp ladder; Lane 2: colony 2; Lane 3: colony 5; Lane 4: colony 9; Lane 5: colony 14; Lane 6: positive control; Lane 7: Empty; Lane 8: colony 1 (red); Lane 9: colony 3 (red); Lane 10: colony 6 (red); Lane 11: colony 10 (red); Lane 12: colony 15 (red); Lane 13: negative control (no template)

The amplified product colony PCR is not around the correct hight. No succesful integration of fusion in pSB1C3

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-dxCas9 in 8 colonies after transformation. This plasmid contains the dxcas9 (~4000bp) and pSB1C3, which can be checked with primers VR and VF2.

20 tubes were prepared for colony PCR of pSB1C3-fusion colonies. The thermocycler programme was installed to have an extension time of 8:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### Restriction

Restriction cloning was done for pSB1C3 and the fusion protein. The following table shows an overview of what fragments were cut with what restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) - pSB1C3-linear | Volume (uL) - fusionbb | Volume (uL) - pSB1C3-RFP | Volume (uL) - dxCas9bb || 10x CutSmart buffer | 5 | 5 | 5 | 5 |
| Fragment | 41 (~3700ng) | 41 (~3000ng) | 20 (~8480 ng) | 20 (~2220 ng) |
| Enzyme (EcoRI) | 2 | 2 | 2 | 2 |
| Enzyme (SpeI) | 2 | 2 | 2 | 2 |
| MilliQ | 0 | 0 | 21 | 21 |

The samples were incubated for 4 hours in the incubator 37 °C.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration was measured.

| Isolate | Concentration (ng/uL) |
| --- | --- |
| pSB1C3 (1.1.1) | 2290 |
| pSB1C3 (1.2.1) | 2215 |
| pSB1C3 (1.1.2) | 430 |
| pSB1C3 (1.2.2) | 770.6 |
| dxCas9 (2.1) | 2541.4 |
| dxCas9 (2.2) | 690 |
| dxCas9 (4.1) | 1823 |
| dxCas9 (4.2) | 668.8 |

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3-dxCas9fs and pSB1C3-fusionfs.

| Component | Volume (uL) pSB1C3-fusionfs-5 | Volume (uL) pSB1C3-fusionfs-10 | Volume (uL) pSB1C3-dxCas9fs-5 | Volume (uL) pSB1C3-dxCas9fs-10 |
| --- | --- | --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 | 10 | 10 |
| Vector (pSB1C3) | 1.5 (670 ng) | 1 (140 ng) | 1.5 (670 ng) | 1 (140 ng) |
| Fragment | 5.5 (1823 ng) | 6.3 (668 ng) | 2.6 (2541 ng) | 4.1 (690 ng) |
| MilliQ | 3 | 2.7 | 5.9 | 4.9 |

The samples were incubated at 55°C for 60 minutes.

After cooling down, 1uL DpnI is added. The samples were incubated at 37°C for 60 minutes. Heatinactivation for 20 minutes at 60°C

#### Transformation of chemically competent cells

Transformations was carried out according to the protocol. The 2 and 18 uL of product of Gibson Assembly was transformed into *E. coli*DH5α

800 uL LB medium was added as recovery medium.

#### DNA gel purification

DNA purification of fusionbb from agarose gel conform protocol. Elution was done in one time 15 µL Milli-Q and one time 20 µL.

#### Colony PCR

9 tubes were prepared for colony PCR of pSB1C3-dxCas9 colonies. The thermocycler programme was installed to have an extension time of 5:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### PCR clean-up

PCR clean-up is done according to the protocol for restriced pSB1C3-RFP, restricted fusion, restricted pSB1C3 and restricted dxCas9. Elution was done in 30 µL Milli-Q

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity (280/260 and 230/260 ratio) was measured.

| Isolate | Concentration (ng/uL) | 280/260 ratio | 230/260 ratio |
| --- | --- | --- | --- |
| restricted pSB1C3RFP | 98.6 | 1.6 | 1.8 |
| restricted fusionbb | 10.7 | 0.4 | 1.7 |
| restricted pSB1C3lin | 27.3 | 1 | 1.9 |
| restricted dxCas9bb | 15.0 | 0.6 | 1.68 |

#### Ligation

In order to assemble pSB1C3-fusion and pSB1C3-dxCas9, ligation was performed.

A 10ul ligation reaction mixture was prepared with the following composition

| Component | pSB1C3-Fusion Volume(uL) | pSB1C-dxCas9 |
| --- | --- | --- |
| Ligase Buffer (10x) | 2 | 2 |
| T4 DNA Ligase | 1 | 1 |
| DNA vector | 0.25 uL (~25 ng) | 0.40 uL (~40 ng) |
| DNA fragment | 16.7 uL (~170 ng) | 16.6 uL (~225 ng) |
| MilliQ | 0 uL | 0 uL |

The samples were incubated overnight on room temperature for ligation.

Date
:   03/10/2018

Operator
:   Nicole Bennis

#### Restriction

The following table shows an overview of what fragment(s) were cut with what restriction enzyme(s) to obtain fragments Tn5-Lin-dxCas9 and pACYCDuet-1 for ligation.

| Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) | Component | Volume (uL) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 10x CutSmart | 5 | 10x CutSmart | 2 | 10x CutSmart | 2 | 10x CutSmart | 2 |
| pSB1C3-mRFP | 3 (~2000 ng) | pSB1C3 backbone | 6 (~2000 ng) | dxCas9 | 12 (~2000 ng) | dxCas9-Lin-Tn5 | 8 (~2000 ng) |
| Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 | Enzyme (EcoRI) | 1 |
| Enzyme (PstI) | 1 | Enzyme (PstI) | 1 | Enzyme (PstI) | 1 | Enzyme (PstI) | 1 |
| MilliQ | 13 | MilliQ | 10 | MilliQ | 4 | MilliQ | 8 |

The samples were incubated for 4 hours at 37 °C, and followed by 20 minute heat inactivation of the restriction enzyme(s).

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### PCR Clean-Up

20 μL of each restriction reaction mixture was subjected to PCR Clean-Up, according to the [protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#PCRcleanup-scroll). Elution was done in 30 µL Milli-Q.

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Nanodrop DNA Quantification

The following isolates were quantified according to the [Nanodrop DNA Quantification protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#NanodropDNA-scroll). The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pSB1C3-mRFP cut with EcoRI and PstI | 15.0 | 1.91 | 0.48 |
| pSB1C3 backbone cut with EcoRI and PstI | 63.3 | 1.85 | 1.11 |
| dxCas9 cut with EcoRI and PstI | 52.3 | 1.85 | 0.75 |
| dxCas9-Lin-Tn5 cut with EcoRI and PstI | 67.1 | 1.86 | 0.90 |

Date
:   30/08/2018

Operator
:   Nicole Bennis

#### Ligation

In order to assemble cut fragments pSB1C3 with dxCas9 or dxCas9-Lin-Tn5 ligation was performed. This table gives an overview of which fragments were cut with which enzymes.

| DNA | Size (bp) | Cut with enzymes |
| --- | --- | --- |
| pSB1C3 | 2068 | EcoRI and PstI |
| Fragment dxCas9 | 4226 | EcoRI and PstI |
| Fragment dxCas9-Lin-Tn5 | 5703 | EcoRI and PstI |

A 20 μL ligation reaction mixture was prepared with the following composition:

| Component | Volume (μL) | Component | Volume (μL) |
| --- | --- | --- | --- |
| Ligase Buffer (10X) | 2 | Ligase Buffer (10X) | 2 |
| T4 DNA Ligase | 1 | T4 DNA Ligase | 1 |
| DNA vector pSB1C3 (~100 ng) | 2 | DNA vector pSB1C3 (~100 ng) | 2 |
| DNA dxCas9 (~600 ng) | 12 | DNA dxCas9-Lin-Tn5 (~700 ng) | 14 |
| MilliQ | 3 | MilliQ | 0 |

The samples were incubated overnight at 4 °C for ligation.

Close

Thursday, 04/10/2018

Date
:   04/10/2018

Operator
:   Susan Bouwmeester

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity (280/260 and 230/260 ratio) was measured.

| Isolate | Concentration (ng/uL) | 280/260 ratio | 230/260 ratio |
| --- | --- | --- | --- |
| fusion A | 230.4 | 1.88 | 1.87 |
| fusion B | 97.8 | 1.87 | 1.84 |

#### Restriction

Restriction cloning was done for pSB1C3 and the fusion B. The following table shows an overview of what fragments were cut with what restriction enzymes. Restriction enzymes from the kitchen are used.

| Component | Volume (uL) - pSB1C3 | Volume (uL) - fusion B |
| --- | --- | --- |
| 10x CutSmart buffer | 5 | 5 |
| Fragment | 25 (~2500 ng) | 25 (~3500 ng) |
| Enzyme (EcoRI-HF) | 2 | 2 |
| Enzyme (PstI-HF) | 2 | 2 |
| MilliQ | 16 | 16 |

The samples were incubated for 4 hours in the incubator 37 °C and followed by 20 minutes heat inactivation 80 °C.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-dxCas9 and pSB1C3-fusion in after transformation (transformation done 2/10 and 1/10). This plasmid contains the dxcas9 (~4000bp) or fusion (~6000bp) and pSB1C3 (~2000bp), which can be checked with primers VR and VF2.

6 tubes were prepared for colony PCR of pSB1C3-fusion colonies. The thermocycler programme was installed to have an extension time of 10:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3-dxCas9 and pSB1C3-fusion.

| Component | Volume (uL) pSB1C3-fusionfs-5 | Volume (uL) pSB1C3-fusionfs-10 |
| --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 |
| Vector (pSB1C3) | 0.34 (146 ng) | 0.52 (72.8 ng) |
| Fragment (fusion) | 9.66 (2200 ng) | 9.48 (2200 ng) |
| MilliQ | 0 | 0 |

The samples were incubated at 55°C for 60 minutes.

After cooling down, 1uL DpnI is added. The samples were incubated at 37°C for 60 minutes. Heatinactivation for 20 minutes at 60°C

#### PCR clean-up

PCR clean-up is done according to the protocol for restricted fusion and pSB1C3. Elution was done in 30 µL Milli-Q

#### Ligation

In order to assemble pSB1C3-fusion, ligation was performed.

A 10ul ligation reaction mixture was prepared with the following composition

| Component | pSB1C3-Fusion Volume(uL) | pSB1C3-Fusion Volume(uL) |
| --- | --- | --- |
| Ligase Buffer (10x) | 2 | 2 |
| T4 DNA Ligase | 1 | 1 |
| DNA vector | 0.33 uL (~30 ng) | 0.28 uL (~25 ng) |
| DNA fragment | 15 uL (~200 ng) | 15 uL (~750 ng) |
| MilliQ | 2 uL | 2 uL |

The samples were incubated overnight on room temperature for ligation.

#### High Fidelity PCR

To amplify fusionbb (~6000bp), high fidelity PCR was done using fusionbb1 as template DNA and fw\_BB\_dxCas9 and rv\_BB\_Tn5 forward and reverse primers respectively. 16 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 6:30 minutes and an annealing temperature of 70 °C. After the programme was finished, samples were kept at 4 °C.

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α NEB | pSB1C3-fusion | restiction/ligation (2/10) | 2 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-fusion | restiction/ligation (2/10) | 18 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-dxCas9 | restiction/ligation (2/10) | 2 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-dxCas9 | restiction/ligation (2/10) | 18 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-fusion-1 | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-fusion-1 | Gibson Assembly | 18 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-fusion-2 | Gibson Assembly | 2 | LB Cam |
| *E.coli*DH5α NEB | pSB1C3-fusion-2 | Gibson Assembly | 18 | LB Cam |

800 uL LB medium was added as recovery medium.

Date
:   04/10/2018

Operator
:   Nicole Bennis

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid and size (bp) | Cloning method | Volume added (μl) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli* DH5α | pSB1C3\_dxCas9 (6217 bp) | Restriction/Ligation | 2.5 | LB-Cam || *E.coli* DH5α | pSB1C3\_dxCas9-Lin-Tn5 (7699 bp) | Restriction/Ligation | 2.5 | LB Cam |
| *E.coli* DH5α | pSB1C3 cut with EcoRI and PstI (neg control) (2060 bp) | Restriction/Ligation | 2.5 | LB Cam |

200 μl LB medium was added as recovery medium.

Close

Friday, 05/10/2018

Date
:   05/10/2018

Operator
:   Susan Bouwmeester

#### Restriction

DpnI digestion is done for pSB1C3, fusionfs and dxCas9fs.

| Component | Volume (uL) pSB1C3 | Volume (uL) fusionfs | Volume (uL) dxCas9fs |
| --- | --- | --- | --- |
| 10x CutSmart buffer | 5 | 5 | 5 |
| Fragment | 42 | 42 | 42 |
| Enzyme (EcoRI) | 2 | 2 | 2 | 2 |
| Enzyme (DpnI) | 3 | 3 | 3 |

The samples were incubated for 4 hours in the incubator 37 °C.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusionfs in 20 colonies after transformation. This plasmid contains the fusionfs (~6000bp) and pSB1C3, which can be checked with primers VR and VF2.

20 tubes were prepared for colony PCR of pSB1C3-fusionfs colonies. The thermocycler programme was installed to have an extension time of 8:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusionfs in 20 colonies after transformation. This plasmid contains the dxCas9fs (~4000bp) and pSB1C3, which can be checked with primers VR and VF2.

20 tubes were prepared for colony PCR of pSB1C3-dxCas9fs colonies. The thermocycler programme was installed to have an extension time of 5:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of fusion was run on a 0.8% agarose gel in TAE buffer. A total of 20 colony PCR results were put on a gel. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://2018.igem.org/File:T--TUDelft--2018_181020_colPCRfusion.png)
**Figure** Colony PCR fusionfs. 0.8% agarose. Expected size fusion ~6000bp. Lane 1: 200bp ladder; Lane 2: colony 1; Lane 3: colony 2; Lane 4: colony 3; Lane 5: colony 4; Lane 6: colony 5; Lane 7: colony 6; Lane 8: colony 7; Lane 9: colony 8; Lane 10: colony 9; Lane 11: colony 10; Lane 12: colony 11; Lane 13: colony 12; Lane 14: colony 13; Lane 15: colony 14; Lane 16: 200bp ladder. Lane 17: colony 15; Lane 18: colony 16; Lane 19: colony 17; Lane 20: colony 18; Lane 21: colony 19; Lane 22: colony 20

Overnight culture is started for colony number 1, 2 and 8

#### DNA Gel Electrophoresis

PCR product of the colony PCR of dxCas9fs was run on a 0.8% agarose gel in TAE buffer. A total of 20 colony PCR results were put on a gel. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/thumb/d/d4/T--TUDelft--2018_181005.png/1199px-T--TUDelft--2018_181005.png)
**Figure** Colony PCR dxCas9fs. 0.8% agarose. Expected size dxCas9 ~4000bp. Lane 1: 200bp ladder; Lane 2: colony 1; Lane 3: colony 2; Lane 4: colony 3; Lane 5: colony 4; Lane 6: colony 5; Lane 7: colony 6; Lane 8: colony 7; Lane 9: colony 8; Lane 10: colony 9; Lane 11: colony 10; Lane 12: colony 11; Lane 13: colony 12; Lane 14: colony 13; Lane 15: colony 14; Lane 16: 200bp ladder. Lane 17: colony 15; Lane 18: colony 16; Lane 19: colony 17; Lane 20: colony 18; Lane 21: colony 19; Lane 22: colony 20

Overnight culture is started for colony number 1, 7, 8 and 15

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-dxcas9 in 17 colonies after transformation. This plasmid contains the dxcas9 (~4000bp) and pSB1C3, which can be checked with primers VR and VF2.

17 tubes were prepared for colony PCR of pSB1C3-dxcas9 colonies. The thermocycler programme was installed to have an extension time of 8:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-fusion in 33 colonies after transformation. This plasmid contains the fusion (~6000bp) and pSB1C3, which can be checked with primers VR and VF2.

33 tubes were prepared for colony PCR of pSB1C3-fusion colonies. The thermocycler programme was installed to have an extension time of 8:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA gel purification

DNA purification of fusionbb from agarose gel conform protocol. Elution was done in one time 15 µL Milli-Q and one time 20 µL.

#### Nanodrop DNA Quantification

After PCR cleanup, all te samples were measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity (280/260 and 230/260 ratio) was measured.

| Isolate | Concentration (ng/uL) | 280/260 ratio | 230/260 ratio |
| --- | --- | --- | --- |
| Cas | 698.0 | 1.84 | 2.01 |
| Cas 2 | 298.4 | 1.89 | 1.95 |
| Fusion | 345.2 | 1.88 | 1.76 |
| Fusion 2 | 165.4 | 1.8 | 1.6 |

Close

Saturday, 06/10/2018

Date
:   06/10/2018

Operator
:   Susan Bouwmeester

#### Restriction

Restriction cloning was done for pSB1C3 and the fusion protein. The following table shows an overview of what fragments were cut with what restriction enzymes.

| Component | Volume (uL) - pSB1C3en | Volume (uL) - linker |
| --- | --- | --- |
| 10x CutSmart buffer | 3 | 3 |
| Fragment | 8.5 | 8.5 |
| Enzyme (EcoRI) | 1 | 1 |
| Enzyme (PstI) | 1 | 1 |
| MilliQ | 16.5 | 16.5 |

The samples were incubated for 4 hours in the incubator 37 °C.

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3-linker.

| Component | Volume (uL) pSB1C3-linker-10 | Volume (uL) pSB1C3-linker-50 | Volume (uL) pSB1C3-linker-100 |
| --- | --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 | 10 |
| Vector (pSB1C3) | 1.44 (600ng) | 1.44 (600ng) | 0.7 (300ng) |
| Fragment | 1.45 (180 ng) | 3.7 (900 ng) | 3.7 (900 ng) |
| MilliQ | 7.11 | 4.9 | 5.6 |

The samples were incubated at 55°C for 20 minutes.

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3-dxCas9.

| Component | Volume (uL) pSB1C3-dxCas9-5 Volume (uL) pSB1C3-dxCas9-10 | Volume (uL) pSB1C3-dxCas9-25 | | |
| --- | --- | --- | --- | --- |
| Gibson Assembly Mastermix 2X | 10 | 10 | 10 |
| Vector (pSB1C3) | 0.34 (576ng) | 0.18 (300ng) | 0.06 (50ng) |
| Fragment | 8.2 (5760 ng) | 8.6 (6000 ng) | 7.1 (2560)/td> |
| MilliQ | 1.46 | 1.22 | 2.9 |

The samples were incubated at 55°C for 60 minutes.

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble pSB1C3-fusion

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) pSB1C3-fusion-5 Volume (uL) pSB1C3-fusion-10 || Gibson Assembly Mastermix 2X | 10 | 10 | | Vector (pSB1C3) | 0.12 (200ng) | 0.6 (100ng) | | Fragment | 8.6 (3000 ng) | 8.6 (3000 ng) | | MilliQ | 1.3 | 0.8 | | |

The samples were incubated at 55°C for 60 minutes.

#### Transformation of chemically competent cells

Transformations was carried out according to the protocol. The 2 and 18 uL of product of Gibson Assembly was transformed into *E. coli*DH5α

800 uL LB medium was added as recovery medium.

Close

Sunday, 07/10/2018

Date
:   07/10/2018

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-linker in 10 colonies after transformation. This plasmid contains the linker (~60bp) and pSB1C3, which can be checked with primers VR and VF2.

15 tubes were prepared for colony PCR of pSB1C3-linker colonies. The thermocycler programme was installed to have an extension time of 1:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of linker was run on a 0.8% agarose gel in TAE buffer. A total of 10 colony PCR results were put on a gel. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/thumb/0/09/T--TUDelft--2018_linker.png/1200px-T--TUDelft--2018_linker.png)
**Figure** Colony PCR fusion. 0.8% agarose. Lane 1: 500bp ladder; Lane 2: colony 1; Lane 3: Colony 2; Lane 4: Colony 3; Lane 5: Colony 4; Lane 6: Colony 5; Lane 7: Colony 6; Lane 8: Colony 7; Lane 9: Colony 8; Lane 10; Colony 9; Lane 11; Colony 10

Close

[ ]
Week 2 08/10/2018 - 14/10/2018

Monday, 08/10/2018

Date
:   08/10/2018

Operator
:   Susan Bouwmeester

#### Colony PCR

Colony PCR was done to verify propagation of pSB1C3-linker in 8 colonies after transformation. This plasmid contains the linker (~60bp) and pSB1C3, which can be checked with primers VR and VF2.

15 tubes were prepared for colony PCR of pSB1C3-linker colonies. The thermocycler programme was installed to have an extension time of 1:00 and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 12°C.

#### DNA Gel Electrophoresis

PCR product of the colony PCR of linker was run on a 2% agarose gel in TAE buffer. A total of 7 colony PCR results were put on a gel. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/b7/T--TUDelft--2018_linker_2.png)
**Figure** Colony PCR fusion. 0.8% agarose. Lane 1: 500bp ladder; Lane 2: colony 1; Lane 3: Colony 2; Lane 4: Colony 3; Lane 5: Colony 4; Lane 6: Colony 5; Lane 7: Colony 6; Lane 8: Colony 7;

Overnight culture of colony 1 is started.

Close

Tuesday, 09/10/2018

Date
:   09/10/2018

Operator
:   Susan Bouwmeester

#### Plasmid Isolation

Overnight starter culture of pSB1C3-linker was subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

#### Nanodrop DNA Quantification

After PCR cleanup, te sample was measered with nanodrop quantification. The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity (280/260 and 230/260 ratio) was measured.

| Isolate | Concentration (ng/uL) | 280/260 ratio | 230/260 ratio |
| --- | --- | --- | --- |
| pSB1C3-linker | 223.6 | 1.65 | 0.86 |

Close

Wednesday, 03/10/2018

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Thursday, 04/10/2018

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Friday, 05/10/2018

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Saturday, 06/10/2018

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Saturday, 06/10/2018

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* July
  + [Week: 02/07/2018 - 08/07/2018](#JulyInVitro1)
  + [Week: 09/07/2018 - 15/07/2018](#JulyInVitro2)
  + [Week: 16/07/2018 - 22/07/2018](#JulyInVitro3)
* August
  + [Week: 30/07/2018 - 05/08/2018](#AugustInVitro1)
  + [Week: 06/08/2018 - 12/08/2018](#AugustInVitro2)
  + [Week: 13/08/2018 - 19/08/2018](#AugustInVitro3)
  + [Week: 20/08/2018 - 26/08/2018](#AugustInVitro4)
  + [Week: 27/08/2018 - 02/09/2018](#AugustInVitro5)
* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberInVitro1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberInVitro2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberInVitro3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberInVitro4)
* October
  + [Week: 01/10/2018 - 07/10/2018](#OctoberInVitro1)
  + [Week: 08/10/2018 - 14/10/2018](#OctoberInVitro2)

### July

[ ]
Week 2 09/07/2018 - 15/07/2018

Tuesday, 10/07/2018

Date
:   10/07/2018

Operator
:   Venda Mangkusaputra

#### GBlock resuspension

GBlock EPO was resuspended in 50 µL sterile TE buffer (10mM Tris, 1mM EDTA, pH8) to a final concentration of 10 ng/µL. GBlock sgRNA for EPO was resuspended in 25 µL sterile TE buffer (10mM Tris, 1mM EDTA, pH8) to a final concentration of 10 ng/µL.

Date
:   10/07/2018

Operator
:   Venda Mangkusaputra

#### Primer working stock preparation

The following primers were resuspended in sterile Milli-Q water, in order to make 100µM stock solutions.

| Primer name | Amount (nmol) | Volume (uL) |
| --- | --- | --- |
| 033 | 25.8 | 258 |
| 034 | 25.1 | 251 |
| 038 | 32.6 | 326 |
| 039 | 32.0 | 320 |

The primers in these 100 µM stocks were heated to 60 °C for 20 minutes and centrifuged at maximum speed (~17,000 x g) for 2 minutes. 10µM primer working stock solutions were prepared by making 10x dilution aliquots with sterile Milli-Q, each in a total volume of 100µl.

Date
:   10/07/2018

Operator
:   Venda Mangkusaputra

#### High Fidelity PCR

To amplify EPO gBlocks (632bp), high fidelity PCR was done using EPO gBlocks as template DNA and 038 and 039 as forward and reverse primers respectively. To amplify sgRNA for EPO gBlocks (125bp), high fidelity PCR was done using sgRNA for EPO gBlocks as template DNA and 033 and 034 as forward and reverse primers respectively. 2 50µL PCR mixtures were prepared for each amplification of EPO and sgRNA for EPO gBlocks, according to the High Fidelity PCR protocol. 1 50µl of PCR mixtures with no template and primer 033 and 034 were prepared in parallel for negative control.

Primer combination(s) and expected fragment(s)

| Sample | fw primer | rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 033 | 034 | sgRNA for EPO gBlocks | 125 |
| 2 | 033 | 034 | sgRNA for EPO gBlocks | 125 |
| 3 | 038 | 039 | EPO gBlocks | 632 |
| 4 | 038 | 039 | EPO gBlocks | 632 |
| 5 | 033 | 034 | negative control | none |

The thermocycler programme was installed to have an extension time of 16 seconds and an annealing temperature of 60°C. After the programme was finished, samples were run on 2% TAE agarose gel. Figure 1 displays the result of the gel imaged with GelDoc system.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/ed/T--TUDelft--venda_07_10_fig1.png)

**Figure 1. 2% TAE Agarose gel of PCR amplification of gBlocks**  *. Lanes are (1) DNA Ladder, (2) and (3) amplified sgRNA template for EPO1 gBlocks, (4) and (5) amplified EPO gBlocks.
.*

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Thursday, 12/07/2018

Date
:   12/07/2018

Operator
:   Venda Mangkusaputra

#### sgRNA Transcription

To produce sgRNAs that target the EPO gene, sgRNA transcription was done with sgRNA for EPO as a template. the following components were assembled at room temperature in the following order:

| Component | Volume (uL) |
| --- | --- |
| MilliQ | 17.35 (fill to 30 µl) |
| 10x Reaction Buffer | 2 uL |
| NTP (25mM each) | 6 uL |
| sgRNA for EPO template | 1.5 uL (0.2 - 1 µg) |
| RNase inhibitor | 2 ul (1U/mL final) |
| T7 RNA polymerase | 1 uL |
| MgCl2 | 0.15 uL |
| Final Volume | 30 uL |

The reaction was incubated at 37 °C overnight.

Date
:   12/07/2018

Operator
:   Venda Mangkusaputra

#### Tn5 protein functionality testing: integration assay

Integration assay was performed to test the integration of adapter DNA by Tn5.

The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter.

| Name | Sequence |
| Upper strand | CTAGCTGTCTCTTATACACATCTATTGGCGAAATGGATGAACTGTTTACCATGC |
| Lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAGCTAG |

To anneal the two oligos, equamolar amount of each strand is mixed, incubated at 95°C and cooled slowly to room temperature.

**Tn5:adapter DNA complex formation**
Since this is the first time this experiment was done, the idea is to get the idea of the required amount of adapter needed in the reaction. Therefore 3 different adapter concentrations were added, namely 5µM, 25µM and 50µM. Tn5 was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | 5µM reaction (µl) | 25µM reaction (µl) | 50µM reaction (µl) |
| Buffer (250mM Tris, pH 7.5) | 2.5 | 2.5 | 2.5 |
| Tn5 (0.5m/ml) | 9 | 9 | 9 |
| Adapter | 2.5 | 2.5 | 2.5 |
| Glycerol | 11 | 11 | 11 |
| Total | 25 | 25 | 25 |

The samples were incubated at 37°C for 45 minutes.

Next, EPO (130ng/µl) was added in two different transposome:EPO volume ratios, namely 1:1 and 3:1 in total volume of 10µM. Finally 12.5mM final MgCl2 concentration were added. The samples were incubated at 55°C for 10 minutes and stopped by adding final 1% SDS. The integration products were analysed on 2% TAE agarose gel. The gel was stained with sybersafe and imaged with GelDoc system. The resulting gel is shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/7/7e/T--TUDelft--venda_07_12_fig1.png)

**Figure 1. 2% TAE Agarose gel of Tn5 integration assay.**  *. Lanes are (1) DNA ladder (100bp-1000bp), (2) 5µM adapter. 1:3 EPO:transposome, (3) 25µM adapter. 1:3 EPO:transposome, (4) 50µM adapter. 1:3 EPO:transposome, (6) 5µM adapter. 1:1 EPO:transposome, (7) 25µM adapter. 1:1 EPO:transposome, (8) 50µM adapter. 1:1 EPO:transposome.*

Figure 1 suggested that the staining of the gel was poorly done as bands that was supposed to be present on the first and last lane were not visible. The smear on the rest of the lanes suggested the possible integration of adapter, creating bands of smaller sizes. However, more negative controls should be done to confirm this further.

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Friday, 13/07/2018

Date
:   13/07/2018

Operator
:   Venda Mangkusaputra

#### RNA cleanup

After the *in vitro* transcription, transcripts were isolated following the RNA Cleanup protocol. The isolated RNA were measured with nanodrop. The measure concentration of sgRNA for EPO is 11.3ng/µl.

Close

### August

[ ]
Week 2 06/08/2018 - 12/08/2018

Friday, 10/07/2018

Date
:   10/08/2018

Operator
:   Venda Mangkusaputra

#### Tn5 protein functionality testing 2: integration assay

Integration assay was performed to test the integration of adapter DNA by Tn5.

The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter.

| Name | Sequence |
| Upper strand | CTAGCTGTCTCTTATACACATCTATTGGCGAAATGGATGAACTGTTTACCATGC |
| Lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAGCTAG |

**Tn5:adapter DNA complex formation**
From previous result, we learned that:

+ More negative control should be done to confirm integration
+ 50µM adapter concentration was too high. In this experiment 10µM and 25µM adapter will be used.
+ 50% glycerol might be too high. As the transposase already contain glycerol. No addition glycerol will be added in this experiment

Two different amount of Tn5 was loaded with adapter DNA by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | No Tn5 (µl) | Low Tn5 (µl) | High Tn5 (µl) |
| Buffer (250mM Tris, pH 7.5) | 2 | 2 | 2 |
| Tn5 (0.5mg/ml) | 0 | 10 | 15 |
| Adapter (10µM/25µM) | 2.5 | 2.5 | 2.5 |
| Water | 15.5 | 5.5 | 0.5 |
| Total | 25 | 25 | 25 |

The samples were incubated at 37°C for 45 minutes.

Next, 1µl or 2µl of EPO (130ng/µl) was added to 5µl of each transposome reaction. 1µl of 125mM MgCl2 was added. Water was added to make final volume of 10µM. The samples were incubated at 55°C for 10 minutes and stopped by adding final 1% SDS. The integration products were analysed on 2% TAE agarose gel. The gel was stained with sybersafe and imaged with GelDoc system. The resulting gel is shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/93/T--TUDelft--venda_08_10_fig1.png)

**Figure 1. TAE Agarose gel of PCR amplification of gBlocks.**  *. Lanes are (1) low adapter, low Tn5, (2) high adapter, low Tn5, (3) low EPO, low adapter, low Tn5, (4) low EPO, high adapter, low Tn5, (5) low EPO, low adapter, high Tn5, (6) low EPO, high adapter, high Tn5, (7) DNA ladder, (8) low adapter, high Tn5, (9) high adapter, high Tn5, (10) high EPO, low adapter, low Tn5, (11) lohighw EPO, high adapter, low Tn5, (12) high EPO, low adapter, high Tn5, (13) high EPO, high adapter, high Tn5, (14) high Tn5, (15) high EPO, high Tn5, (16) empty, (17) high Tn5, (18) high adapter, (19) empty, (20) DNA ladder.*

Comparing our result with the negative control. It seemed like no (or very little) integration took place.

Close

Sunday, 11/08/2018

Date
:   12/08/2018

Operator
:   Venda Mangkusaputra

#### gRNA Transcription

For producing gRNAs that target EPO gene, gRNA transcription was done with sgRNA for EPO as a template. the following components were assembled at room temperature in the following order:

| Component | Volume (uL) |
| --- | --- |
| MilliQ | 4.5ul |
| 10x Reaction Buffer | 2 uL |
| NTP (25mM each) | 4 uL |
| sgRNA for EPO | 5ul (0.3 µg) |
| RNase inhibitor | 0.5ul (1U/mL final) |
| DTT | 2ul (5mM final) |
| T7 RNA polymerase | 2 uL |
| Final Volume | 20 uL |

The reaction was incubated at 37 °C overnight.

Date
:   12/08/2018

Operator
:   Venda Mangkusaputra

#### Primer working stock preparation

The following primers were resuspended in sterile Milli-Q water, in order to make 100µM stock solutions.

| Primer name | Amount (nmol) | Volume (uL) |
| --- | --- | --- |
| 035 | 29.8 | 298 |

The primers in these 100 µM stocks were heated to 60 °C for 20 minutes and centrifuged at maximum speed (~17,000 x g) for 2 minutes. 10µM primer working stock solutions were prepared by making 10x dilution aliquots with sterile Milli-Q, each in a total volume of 100µl.

Date
:   12/08/2018

Operator
:   Venda Mangkusaputra

#### High Fidelity PCR

This PCR reaction was done to amplify sgRNA template for EPO. There are two different sgRNA that will target slightly different position of EPO cDNA. The two templates will be addressed as sgRNA for EPO1 and sgRNA for EPO2. To amplify sgRNA for EPO1 and EPO2 (125bp), high fidelity PCR was done using sgRNA for EPO gBlocks as template DNA and either 033 and 034 or 035 and 034 as forward and reverse primers respectively. 2 50µL PCR mixtures were prepared for each amplification of sgRNA for EPO, according to the High Fidelity PCR protocol. 1 50µl of PCR mixtures with no template and primer 035 and 034 were prepared in parallel for negative control.

Primer combination(s) and expected fragment(s)

| Sample | fw primer | rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| 1 | 033 | 034 | sgRNA for EPO1 | 125 |
| 2 | 033 | 034 | sgRNA for EPO1 | 125 |
| 3 | 035 | 034 | sgRNA for EPO2 | 632 |
| 4 | 035 | 034 | sgRNA for EPO2 | 632 |
| 5 | 035 | 034 | negative control | none |

The thermocycler programme was installed to have an extension time of 16 seconds and an annealing temperature of 60°C. After the programme was finished, samples were run on 2% TAE agarose gel.Figure 1 displays the result of the gel imaged with GelDoc system.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/3/3b/T--TUDelft--venda_08_12_fig1.png)

**Figure 1. 2% TAE Agarose gel of PCR products.** *. Lanes are (1) DNA ladder, (2) and (3) amplified sgRNA template for EPO 1, (4) and (5) amplified sgRNA template for EPO2.*

Close

[ ]
Week 3 13/08/2018 - 19/08/2018

Monday, 13/08/2018

Date
:   13/08/2018

Operator
:   Venda Mangkusaputra

#### RNA cleanup

After the *in vitro* transcription on the previous day, transcripts were isolated following the RNA Cleanup protocol.
Following the clean up, the concentration of RNA is measured with nanodrop. The concentration achieved after clean up was 181.7 ng/µl. To visualize it, the samples before and after clean up, along with the template DNA were run on 2% TBE agarose gel.

RNA loading buffer was added to samples following the RNA gel electrophoresis protocol. The agarose gel was run on 80 Voltage. The image of the gel is shown in Figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/18/T--TUDelft--venda_08_13_fig1.png)

**Figure 1. 2% TBE Agarose gel of *in vitro* transcription products.**  *. Lanes are (1) RNA ladder, (2) sgRNA for EPO 1 before RNA clean up, (3) sgRNA for EPO 1 after RNA clean up, (4) DNA ladder (5) sgRNA DNA template*

Close

Wednesday, 15/08/2018

Date
:   15/08/2018

Operator
:   Venda Mangkusaputra

#### Tn5 protein functionality testing 3: integration assay

Integration assay was performed to test the integration of adapter DNA by Tn5.

The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter.

| Name | Sequence |
| Short adapter upper strand | CTAGCTGTCTCTTATACACATCTATTGGCGAAATGGATGAACTGTTTACCATGC |
| Short adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAGCTAG |
| Long adapter upper strand | BBa\_K2643011 |
| Long adapter lower strand | BBa\_K2643011 |

From previous result, we learned that:

+ High amount of glycerol might be needed for integration since previous experiment without glycerol did not result in any integration.
+ Since finally Tn5 has to function together with dxCas9, we will try to do the reaction in lower temperature, since dxCas9 might be denatured at 55°C.Therefore, in this experiment, the adapter loading and integration experiment will be done in one go, at 37°C for 2 hours.
+ New adapter will be used, which has ME sequence on both sides to test whether this type of integration (integration without splitting the fragment into two) would work better.

The reaction was done following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for integration reaction.

|  | Reaction with short adapter (µl) | Reaction with long adapter (µl) | No EPO, short adapter (µl) | No EPO, long adapter(µl) | No Tn5, short adapter (µl) | No Tn5, long adapter (µl) | No adapter (µl) |
| Buffer (250mM Tris, pH 7.5) | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Tn5 (0.5m/ml) | 3 | 3 | 3 | 3 | 0 | 0 | 3 |
| Adapter | 0.5 | 1.5 | 0.5 | 1.5 | 0.5 | 1.5 | 0 |
| Glycerol | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 | 2.5 |
| MgCl2 (125mM) | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| EPO (170ng/ul) > | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| Water | 1 | 0 | 2 | 1 | 4 | 3 | 1.5 |
| Total | 10 | 10 | 10 | 10 | 10 | 10 | 10 |

The samples were incubated at 37°C for 2 hours.

The reaction was stopped by adding final 1% SDS. The integration products were analysed on 2% TAE agarose gel. The gel was stained with sybersafe and imaged with GelDoc system. The resulting gel is shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/e2/T--TUDelft--venda_08_15_fig1.png)

**Figure 1. 2% TAE Agarose gel of Tn5 integration assay.**  *. Lanes are (1) integration with short adapter, (2) integration with long adapter, (3) integration with short adapter, no EPO, (4) integration with long adapter, no EPO, (5) integration with short adapter, no Tn5, (6) integration with long adapter, no Tn5*

The presence of upper bands on lane xx and xx suggested that integration occurs. However, since some negative controls are missing, it might be difficult to draw any conclusive result.

Close

Thursday, 16/08/2018

Date
:   16/08/2018

Operator
:   Venda Mangkusaputra

#### High Fidelity PCR

PCR amplification for the long adapter (KanR adapter), high fidelity PCR was done using KanR adapter as template DNA and XXX and XXX as forward and reverse primers respectively. 10 50µl PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

| Sample | fw primer | rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| 1-10 | IV005 | IV006 | KanR adapter | 800 |

The thermocycler programme was installed to have an extension time of 30 seconds and an annealing temperature of 60 °C. After the programme was finished, samples were run on 2% TAE agarose gel. Figure 1 displays the result of the gel imaged with GelDoc systemr.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/96/T--TUDelft--venda_08_16_fig1.png)

**Figure 1. 0.8% TAE Agarose gel of long adapter amplification.**  *. Lanes are (1) DNA ladder, (2-11) amplification of the long adapter*

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Friday, 17/07/2018

Date
:   17/08/2018

Operator
:   Venda Mangkusaputra

#### Tn5 protein functionality testing 4: integration assay

Integration assay was performed to test the integration of adapter DNA by Tn5.

The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter.

| Name | Sequence |
| Long adapter upper strand | BBa\_K2643011 |
| Long adapter lower strand | BBa\_K2643011 |

From previous result, we learned that:

+ Integration could be seen with the longer adapter.
+ More negative control has to be done

In this reaction, we tried reproduce the previous result. Moreover, the effect of Tn5 concentration was tested. Two different Tn5 batches was used, one with 0.5mg/ml concentration, one with 1.5mg/ml concentration.

The reaction was done following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for integration reaction.

|  | Reaction (µl) | Reaction without adapter (µl) | Reaction without EPO (µl) |
| Buffer (250mM Tris, pH 7.5) | 12 | 8 | 8 |
| Adapter | 12 | 0 | 8 |
| Glycerol | 30 | 20 | 20 |
| MgCl2(125mM) | 12 | 8 | 8 |
| EPO (170ng/ul) > | 6 | 4 | 0 |
| Water | 0 | 8 | 4 |
| Total | 10 | 10 | 10 |

12µl of each master mix shown on Table two was added to 0 / 3 / 5 / 8 µl of low concentration Tn5, or 8µl of high concentration Tn5. Water was added to each reaction to make final volume of 20µl. The samples were incubated at 37°C for 2 hours.

The reaction was stopped by adding final 1% SDS. The integration products were analysed on 2% TAE agarose gel. The gel was stained with sybersafe and imaged with GelDoc system. The resulting gel is shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/db/T--TUDelft--venda_09_17_fig1.png)

**Figure 1. 2% TAE Agarose gel of Tn5 integration assay.**  *. Lanes are (1) reaction, no Tn5, (2) reaction 1µM Tn5, (3) reaction 1.5µM Tn5, (4) reaction 3µM Tn5, (5) reaction 6µM Tn5, (6) DNA ladder, (7) DNA ladder, (8) 50nM EPO, (9) 2.5µM adapter, (10) 3µM Tn5, (11) 6µM, (12) no adapter, no Tn5, (13) no adapter, 1µM Tn5, (14) no adapter, 3µM Tn5, (15) no adapter, 6µM Tn5, (16) no EPO, no Tn5, (17) no EPO, 1µM Tn5, (18) no EPO, 3µM Tn5, (19) no EPO, 6µM Tn5,*

The presence of smearing, even when no adapter was added suggested that the Tn5 might be preloaded already with DNA.

Close

[ ]
Week 4 20/08/2018 - 26/08/2018

Thursday, 23/08/2018

Date
:   23/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| colony 1 (EPO) | 20.4 | 1.62 | 0.95 |
| colony 2 (EPO) | 22 | 1.60 | 0.93 |
|

Date
:   23/8/2018

Operator
:   Kavish Kohabir

#### Sequence Verification

##### Sequence from plasmid

For verification of EPO in pSB1C3, primers VF2 and VR were used for sequencing. The sequencing starts from the biobrick backbone in the direction of the insert. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | Volume (uL) | Final concentration |
| --- | --- | --- |
| Plasmid DNA (500ng) | 7.5 | >50 ng/uL |
| 10uM primer | 2.5 | 25 pmol |

Within 7 days, the sequence was retrievable online and alignment of colony 1 with the in silico designed molecule was compared by Venda, and showed 100% match of the 1050 bp downstream of the sequencing primer binding site.

Close

[ ]
Week 5 27/08/2018 - 02/09/2018

Monday, 27/08/2018

Date
:   27/08/2018

Operator
:   Venda Mangkusaputra

#### Tn5 protein functionality testing 5: integration assay

Integration assay was performed to test the integration of adapter DNA by Tn5.

The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter.

| Name | Sequence |
| Long adapter lower strand | BBa\_K2643011 |
| Short adapter lower strand | BBa\_K2643011 |

From previous result, we learned that:

+ Our Tn5 might be preloaded. Therefore we treated our Tn5 with heparin column chromatography to get rid of all the DNA on it.
+ We will treat the reaction with protease to make sure that the protein will release the DNA and allow DNA to enter the gel.

The reaction was done following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for integration reaction.

|  | No EPO, No adapter (µl) | Complete reaction (µl) | No adapter (µl) | No EPO (µl) |
| Buffer (250mM Tris, 125mM MgCl2, pH 7.5) | 4 | 4 | 4 | 4 |
| Adapter | 0 | 4 | 0 | 4 |
| Glycerol | 4 | 4 | 4 | 4 |
| EPO (170ng/ul) > | 0 | 4 | 4 | 0 |
| Water | 20 | 12 | 16 | 16 |
| Total | 28 | 28 | 28 | 28 |

14µl of each master mix shown on Table 1 was added to 6µl of Tn5 (1.8mg/ml) or water. The samples were incubated at 37°C for 2 hours. 1µl of proteinase K were added to one set of the reaction

The integration products were analysed on 2% TAE agarose gel. The gel was stained with sybersafe and imaged with GelDoc system. The resulting gel is shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/b4/T--TUDelft--venda_08_27_fig1.png)

**Figure 1. 2% TAE Agarose gel of Tn5 integration assay.**  *. Lanes are (1) DNA ladder, (2) buffer, (3) Tn5 (2.5µM), (4) Tn5(2.5µM) + protease K, (5) reaction without Tn5, (6) reaction with Tn5 (2.5µM), (7) reaction with Tn5(2.5µM) + protease K, (8) DNA ladder, (9) reaction without Tn5, without adapter (10) reaction without adapter, with Tn5 (2.5µM), (11) reaction without adapter, with Tn5(2.5µM) + protease K, (12) reaction without Tn5, without EPO (13) reaction without EPO, with Tn5 (2.5µM), (14) reaction without EPO, with Tn5(2.5µM) + protease K, (15) EPO only, (16) adapter only.*

When Tn5 was added All bands are missing, which could be the result of two possible reasonings. First, our Tn5 protein might be contaminated with DNase, degrading all of our DNA. Second, our Tn5 might be really active that it degrade our DNA to very small pieces that already ran off the gel.

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Thursday, 30/08/2018

Date
:   30/8/2018

Operator
:   Kavish Kohabir

#### Cell banking

Strain *E. coli* DH5alpha pSB1C3-EPO was stocked (colony 1) and stored at -80oC following the cell banking protocol.

Close

### SEPTEMBER

[ ]
Week 1 03/09/2018 - 09/09/2018

Thursday, 06/09/2018

Date
:   06/09/2018

Operator
:   Venda Mangkusaputra

#### Tn5 functionality testing: Electrophoretic Mobility Shift Assay

Since the Tn5 integration assay has been showing inconsistent result. We took a step further and first focus on checking whether our Tn5 is able to load the adapter DNA. EMSA was performed to test this.

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

**Tn5:adapter DNA complex formation**
Tn5 was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | Reaction (µl) | No adapter (µl) |
| Buffer | 1 | 1 |
| Tn5 (~8µM) | 0/1/2/3/5/8 | 8 |
| Adapter (25µM) | 1 | 0 |
| Water | 8/7/6/5/3/0 | 0 |
| Total | 10 | 10 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, Tn5 or dxCas9-Tn5, and adapter. The samples were incubate at 23°C for 1 hour and analysed on 8% native TBE polyacrylamide gel. The resulting gel is either stained with ethidium bromide and imaged with GelDoc system, or directly imaged with Typhoon Imaging System. The resulting gels are shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/d0/T--TUDelft--venda_09_06_fig1.png)

**Figure 1. % TBE native PAGE on Tn5 EMSA.**  *. Lanes are (1) DNA ladder, (2) 2.5µM adapter + 0µM Tn5 (3) 2.5µM adapter + 0µM Tn5, (4) 2.5µM adapter + 0.8µM Tn5, (5) 2.5µM adapter + 1.5µM Tn5, (6) 2.5µM adapter + 2.5µM Tn5, (7) 2.5µM adapter + 4µM Tn5 , (8) 2.5µM adapter + 6µM Tn5, (9) 2.5µM adapter + 6µM Tn5 + SDS, (10) 0µM adapter + 6µM Tn5, (11) 0µM adapter + 6µM Tn5, (12) 2.5µg protein control albumin, (13) 5µg protein control albumin, (14) protein ladder, (15) 2.5µM adapter
.*

In order to see the position of the protein in the gel, and therefore confirmed the mobility shift further, the gel were stained with silver quest staining. The resulting gel is shown on figure 2.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/fe/T--TUDelft--venda_09_06_fig2.png)

**Figure 2. % TBE native PAGE on Tn5 EMSA with silver stain.**  *. Lanes are (1) DNA ladder, (2) 2.5µM adapter + 0µM Tn5 (3) 2.5µM adapter + 0µM Tn5, (4) 2.5µM adapter + 0.8µM Tn5, (5) 2.5µM adapter + 1.5µM Tn5, (6) 2.5µM adapter + 2.5µM Tn5, (7) 2.5µM adapter + 4µM Tn5 , (8) 2.5µM adapter + 6µM Tn5, (9) 2.5µM adapter + 6µM Tn5 + SDS, (10) 0µM adapter + 6µM Tn5, (11) 0µM adapter + 6µM Tn5, (12) 2.5µg protein control albumin, (13) 5µg protein control albumin, (14) protein ladder, (15) 2.5µM adapter*

Close

[ ]
Week 2 10/09/2018 - 16/09/2018

Thursday, 13/09/2018

Date
:   13/09/2018

Operator
:   Venda Mangkusaputra

#### Fusion protein functionality testing: Electrophoretic Mobility Shift Assay

From the previous test, it was shown that Tn5 by itself were able to load the adapter DNA. Since the Tn5 portion of the fusion protein might behave differently, we did the same experiment with the fusion protein.

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

**Fusion protein:adapter DNA complex formation**
Fusion protein was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | Reaction (µl) | No adapter (µl) |
| Buffer | 1 | 1 |
| Fusion protein(~3µM) | 0/0.5/2/4/8 | 8 |
| Adapter (25µM) | 1 | 0 |
| Water | 8/7.5/6/2/0 | 0 |
| Total | 10 | 10 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, fusion protein and adapter. The samples were incubate at 23°C for 1 hour and analysed on 5% native TBE polyacrylamide gel. One of the 8µl fusion protein reaction was treated with heat (90oC) to denature the fusion protein. The resulting gel is either stained with ethidium bromide and imaged with GelDoc system, or directly imaged with Typhoon Imaging System. The resulting gels are shown on figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/ce/T--TUDelft--venda_09_13_fig1.png)

**Figure 1. 5% TBE native PAGE on Tn5 EMSA stained with EtBr, imaged by a) GelDoc and b) Typhoon Imaging.**  *. Lanes are (1) DNA ladder, (2)2.5µM fusion protein, (3) 2.5µM adapter + 0µM fusion protein (4) 2.5µM adapter + 0µM fusion protein, (5) 2.5µM adapter + 0.15µM fusion protein, (6) 2.5µM adapter + 0.5µM fusion protein, (7) 2.5µM adapter + 1.5µM fusion protein, (8) 2.5µM adapter + 2.5µM fusion protein, (9) 2.5µM adapter + 2.5µM fusion protein + heat, (10) 2.5µM adapter
.*

The observed higher bands on figure 1 confirmed the ability of the fusion protein to load the adapter DNA.

In order to see the position of the protein in the gel, and therefore confirmed the mobility shift further, the gel were stained with silver quest staining. The resulting gel is shown on figure 2. It can be seen the observed bands on Figure 2 is consistent with the ones shown on Figure 1

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/89/T--TUDelft--venda_09_13_fig2.png)

**Figure 2. 5% TBE native PAGE on Tn5 EMSA with silver stain, imaged by a) GelDoc and b) Typhoon Imaging.**  *. Lanes are (1) DNA ladder, (2)2.5µM fusion protein, (3) 2.5µM adapter + 0µM fusion protein (4) 2.5µM adapter + 0µM fusion protein, (5) 2.5µM adapter + 0.15µM fusion protein, (6) 2.5µM adapter + 0.5µM fusion protein, (7) 2.5µM adapter + 1.5µM fusion protein, (8) 2.5µM adapter + 2.5µM fusion protein, (9) 2.5µM adapter + 2.5µM fusion protein + heat, (10) 2.5µM adapter*

Close

[ ]
Week 3 17/09/2018 - 23/09/2018

Thursday, 19/09/2018

Date
:   19/09/2018

Operator
:   Venda and Monique

#### Fusion protein functionality testing: integration assay

EMSA on both the Tn5 and dxCas9 portion of the fusion showed that these portion individually were able to load adapter or sgRNA and EPO. Following this, integration assay was performed to test the integration of adapter DNA by fusion protein.

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

**Fusion protein:adapter DNA complex formation**
Tn5-dxCas9 protein was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | 1x Fusion (µl) | 1x Negative (no adapter) (µl) |
| Buffer | 1 | 1 |
| Fusion (~3µM) | 0/4/6/8 | 8 |
| Adapter (25µM) | 1 | 0 |
| Water | 8/4/2/0 | 0 |
| Total | 10 | 10 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, Tn5 or dxCas9-Tn5, and adapter. The samples were incubated at 23°C for 1 hour.

To each of the transposome reaction, reaction with sgRNA was done in parallel with the negative control where no sgRNA were added. To add appropriate amount of sgRNA, the pipetting scheme on Table 3 was followed. sgRNA J2.1 signifies version 1 of sgRNA targeting juntion 2 of EPO gene:

**Table 3:** Pipetting scheme for sgRNA loading.

|  | With sgRNA J2.1 (µl) | Without sgRNA (µl) |
| Transposome | 10 | 10 |
| sgRNA (100ng/µl) | 2 | 0 |
| MgCl2 Buffer | 0.5 | 0.5 |
| Water | 0 | 2 |

The samples were incubated at 37°C for 10 minutes.
Target DNA (EPO cDNA) was added to make final concentration of ~5ng/µl.

The samples were incubated at 37°C for 1 hour and analysed on 5% TBE native PAGE

![GEL PICTURE 2](https://static.igem.org/mediawiki/2018/1/18/T--TUDelft--2018_Fusion_.png)

**Figure 1:** TBE native PAGE stained with EtBr and imaged by GelDoc (left) or directly imaged with Typhoon Imaging (right). The gel was loaded as follow: Lane 1 molecular ladder (bp), lane 2 0um fusion w/out EPO, lane 3 2.1um fusion w/out EPO, lane 4 0uM fusion w/o gRNA (1:1) w/ EPO, lane 5 1uM fusion w/o gRNA (1:1) w/ EPO, lane 6 1.6uM fusion w/o gRNA (1:1) w/ EPO, lane 7 2.1uM fusion w/o gRNA (1:1) w/ EPO, lane 8 2.1uM fusion no adapter, lane 9 0uM fusion w gRNA (1:1) w/ EPO, lane 10 1uM fusion w/ gRNA (1:1) w/ EPO, lane 11 1.6uM fusion w/ gRNA (1:1) w/ EPO, lane 12 2.1uM fusion w/ gRNA (1:1) w/ EPO, lane 13 2.1uM fusion no adapter, lane 14 2.1uM fusion w/o gRNA (boiled), lane 15 2.1uM fusion w/ gRNA (boiled).

Figure 1 shows that no integration were visible as there is not visible bands other than the ones also found on the negative controls. Further evaluation is required.

Close

Saturday, 22/09/2018

Date
:   22/09/2018

Operator
:   Venda and Monique

#### Fusion protein functionality testing: integration assay

Previous integration assay did not show any integration product on the gel. In this section, we will perform PCR amplication with primer sets that will specifically bind to integration product (primer will bind on the target DNA on one side, and to the adapter on the other side). Since this is the first time this type of experiment was carried out. Different variables were tested:

+ Two different reaction time were tested, namely 1 and 16 hours to see the effect of reaction duration on integration.
+ Some of the reaction were stopped with heat or SDS to release DNA from the protein. PCR clean up were carried out right after the integration to get rid of excess SDS before PCR.
+ For the rest of the reaction, PCR reaction was done directly after integration.
+ The PCR product was either directly run on the gel, or was first subjected to PCR clean up to get rid of small background DNA fragment before running it on the gel.
+ Second PCR was done to reamplify the first PCR product, in case very little product was visualised.

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

**Fusion protein:adapter DNA complex formation**
Tn5 or fusion protein was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | Reaction (µl) | Reaction no fusion protein (µl) | Reaction no adapter (µl) |
| Buffer | 12 | 12 | 12 |
| Fusion (~3µM) | 96 | 0 | 16 |
| Adapter (25µM) | 12 | 12 | 0 |
| Water | 0 | 96 | 2 |
| Total | 120 | 120 | 20 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, Tn5 or dxCas9-Tn5, and adapter. The samples were incubated at 23°C for 1 hour.

To add appropriate amount of sgRNA, the pipetting scheme on Table 3 was followed. sgRNA J2.1 signifies version 1 of sgRNA targeting juntion 2 of EPO gene:

**Table 3:** Pipetting scheme for sgRNA loading.

|  | Reaction with sgRNA J2.1 (µl) | Reaction no fusion protein (µl) | Reaction no adapter (µl) || Transposome | 120 | 120 | 20 |
| sgRNA (100ng/µl) | 24 | 24 | 4 |
| MgCl2 Buffer | 6 | 6 | 1 |

The samples were incubated at 37°C for 10 minutes.
Target DNA (EPO cDNA) was added to make final concentration of ~5ng/µl.

The samples were incubated at 37°C for 1 hour. 10µl of samples were taken to be analysed on 5% native TBE polyacrylamide gel (Figure 1). Based on Figure 1a, only uncleaved target DNA at 635bp and Donor DNA at 50pb are observed. No integration or high order complexes are visible. On Figure 1b, it showed that after 16h integration time, the target DNA disappears when Fusion protein is present, and significant smearing is observed, indicating functionality of the fusion protein. No specific cleavage bands nor big complex bands are observed.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/91/T--TUDelft--venda_09_22_fig1.png)

**Figure 1. 2% TAE Agarose gel of Tn5 integration assay.**  *. Lanes are (1) DNA ladder (100bp-1000bp), (2) 5µM adapter. 1:3 EPO:transposome, (3) 25µM adapter. 1:3 EPO:transposome, (4) 50µM adapter. 1:3 EPO:transposome, (6) 5µM adapter. 1:1 EPO:transposome, (7) 25µM adapter. 1:1 EPO:transposome, (8) 50µM adapter. 1:1 EPO:transposome.
.*

25µl of samples were taken and deactivated by either only heat or heat + SDS. This was followed by PCR clean up.

To amplify the integration products, PCR was done, both to the cleaned up sample and to the original itegration product. PCR mix and cycle can be found on protocol.

The PCR products were analysed on 5% native TBE polyacrylamide gel. The resulting gels are shown on Figure 2.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a4/T--TUDelft--venda_09_22_fig2.png)

**Figure 2. 5% TBE native PAGE on fusion protein integration assay after PCR of 1 hour of integration duration.**  *Primer set 1 are Fw EPO + Rv adapter. Primer set 2 are Rv EPO + Rv EPO. Lanes are (1) DNA ladder, (2) PCR with primer set 1 (3) PCR with primer set 2, (4) No fusion, primer set 1, (5)no fusion, primer set 2, (6) DNA ladder, (7) PCR after PCR clean up with primer set 1, (8) PCR after PCR clean up with primer set 2, (9) without primer, PCR after PCR clean up with primer set 1, (10) without primer, PCR after PCR clean up with primer set 2, (11) protease treated PCR with primer set 1, (12) protease treated PCR with primer set 2, (13) no fusion, protease treated PCR with primer set 1, (14) no fusion, protease treated PCR with primer set 2.*

Figure 2 shows 2 interesting PCR products observed (lane 2 and 3). Note the 2 PCR products are not observed in the negative controls (lane 4 and 5). No PCR products observed for lane 1-14 (when PCR clean up was done before PCR), most likely due to too little product, which got lost during PCR clean up.

A second PCR was done from the product of the first PCR to amplify the product further. The PCR product wan run on 5% TBE native PAGE shown on Figure 3.

A more intense band are shown after the second PCR cycle. More intense unspecific bands are shown more on the negative control

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/e0/T--TUDelft--venda_09_22_fig3.png)

**Figure 3. 5% TBE native PAGE on fusion protein integration assay after second PCR of 1 hour (a) and 16 hour (b) of integration duration.**  *. Primer set 1 are Fw EPO + Rv adapter. Primer set 2 are Rv EPO + Rv EPO. Lanes are (1) DNA ladder, (2) PCR with primer set 1 (3) PCR with primer set 2, (4) No fusion, primer set 1, (5)no fusion, primer set 2, (6) PCR after PCR clean up with primer set 1, (7) PCR after PCR clean up with primer set 2, (8) without primer, PCR after PCR clean up with primer set 1, (9) without primer, PCR after PCR clean up with primer set 2, (10) protease treated PCR with primer set 1, (11) protease treated PCR with primer set 2, (12) no fusion, protease treated PCR with primer set 1, (13) no fusion, protease treated PCR with primer set 2, (14) DNA ladder.*

The same steps were done with the 16 hours incubated samples. The results of the first PCR are shown on Figure 4.

Figure 4 shows consistent result between the 1h and 16h integration duration.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/df/T--TUDelft--venda_09_22_fig4.png)

**Figure 4. 5% TBE native PAGE on fusion protein integration assay after PCR after 16 hours of incubation.**  *Primer set 1 are Fw EPO + Rv adapter. Primer set 2 are Rv EPO + Rv EPO. Lanes are (1) PCR with primer set 1, EPO 4ng/µl (2) PCR with primer set 2, EPO 4ng/µl, (3) No fusion, primer set 1, EPO 4ng/µl, (4)no fusion, primer set 2, EPO 4ng/µl, (5) DNA ladder, (6) PCR with primer set 2, EPO 40ng/µl, (7) PCR with primer set 2, EPO 40ng/µl, (8) No fusion, primer set 1, EPO 40ng/µl, (9)no fusion, primer set 2, EPO 40ng/µl.*

Close

[ ]
Week 4 24/09/2018 - 30/09/2018

Friday, 28/09/2018

Date
:   28/09/2018

Operator
:   Venda and Monique

#### Fusion protein functionality testing: integration assay

From previous reaction, we saw that we were able to amplify some integration product, eventhough it was difficult to see whether or not it's specifically targetted. Therefore, we included more negative control in this reaction. Moreover, it was shown than performing PCR clean up prior to PCR leads no amplification of the integration product, perhaps due to the loss of products that might have been in very little amount during clean up process. Therefore, the following will be implemented in this reaction

+ The course of integration over time will be observed by stoping the integration reaction at 4 different time points, namely 1 hour, 4 hours, 8 hour, and over night.
+ PCR clean up procedure will not be implemented prior or post PCR reaction.
+ Second round PCR will be not implemented.
+ More negative control will be implemented. This includes reaction without adapter (to see whether there will be integration), without EPO (to see whether there will still be amplification product), without sgRNA (to compare the supposedly targetted integration guided by sgRNA vs non targetted integration), reaction with Tn5 (to compare the supposedly targetted integration by our fusion protien vs random integration by Tn5)

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

**Fusion protein:adapter DNA complex formation**
Tn5 or fusion protein was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | Reaction with fusion protein (µl) | Reaction no fusion protein (µl) | Reaction no adapter (µl) Reaction with Tn5 (µl) | |
| Buffer | 5.2 | 1.8 | 1.8 | 1.8 |
| Fusion (~3µM) | 41.6 | 0 | 14.4 | 0 |
| Tn5 (~8µM) | 0 | 0 | 0 | 5.4 |
| Adapter (25µM) | 5.2 | 1.8 | 0 | 1.8 |
| Water | 0 | 14.4 | 1.8 | 9 |
| Total | 52 | 18 | 18 | 18 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, Tn5 or dxCas9-Tn5, and adapter. The samples were incubated at 23°C for 1 hour.

To add appropriate amount of sgRNA, the pipetting scheme on Table 3 was followed. sgRNA J2.1 signifies version 1 of sgRNA targeting juntion 2 of EPO gene:

**Table 3:** Pipetting scheme for sgRNA loading.

|  | Reaction with sgRNA J2.1(µl) | Reaction no sgRNA/with Tn5 (µl) | Reaction no fusion/no adapter with sgRNA J2.1(µl) || Transposome | 35 | 18 | 18 |
| sgRNA (100ng/µl) | 7 | 0 | 3.6 |
| MgCl2 Buffer | 1.75 | 0.9 | 0.9 |
| Water | 0 | 3.6 | 0 |

The samples were incubated at 37°C for 10 minutes.
Target DNA (EPO cDNA) was added to make final concentration of ~5ng/µl. One negative control was done where water instead of EPO cDNA was added.

The samples were incubated at 37°C. At incubation time = 1h, 4h, 8h, and over night, 2.5 µl of sample were taken from each reaction and was subjected to PCR. PCR mix and cycle can be found on protocol.

The PCR products were analysed on 5% native TBE polyacrylamide gel. The resulting gel is either stained with ethidium bromide and imaged with GelDoc system, or directly imaged with Typhoon Imaging System. The resulting gels are shown on Figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f8/T--TUDelft--venda_09_28_fig1.png)

**Figure 1. 5% TBE native PAGE stained by EtBr and imaged by GelDoc system.Integration reaction after 1 hours.**  *Lanes are (1) DNA ladder (100-1000bp), (2) amplified product with fw EPO (3) amplified product with rv EPO, (4) amplified product with fw EPO without sgRNA, (5) amplified product with rv EPO without sgRNA, (6) amplified product with fw EPO without fusion protein, (7) amplified product with rv EPO without fusion protein, (8) amplified product with fw EPO without adapter DNA, (9) amplified product with rv EPO without adapter DNA, (10) amplified product with fw EPO with Tn5, (11) amplified product with rv EPO with Tn5, (12) amplified product with fw EPO without target DNA, (13) amplified product 2 without target DNA.*

Figure 1 showed the result of PCR. Hyphothetically, sgRNA would steer the fusion protein to the target EPO (~600bp), specifically to the area close to the 160th bp. When specific integration occurs, target EPO will be split into two fragments of around 280 and 480. PCR reactions were done with primer sets that would amplify these fragments seperately. Lane 2 shows the PCR of the first fragment. A very intense band were indeed present around 230bp, confirming the presence of this integration product. Lane 3 shows the PCR of second fragment. An intense band were also present around 480bp, which also confirmed the presence of the second integration product. On the negative control lanes (without sgRNA or with Tn5), random bands were present, and none of these intense bands shown on lane 2 and 3 were observed here . This confirmed further that targeted integration indeed occur due to our fusion protein that is loaded with sgRNA. The absence of amplification bands on the other negative controls confirmed our result further.

Results of the longer incubation time shows similar result. However, it seems like the specific bands loses intensity with increasing time, suggesting perhaps re-integration events done by excess fusion protein. The higher intensity of unspecific lower bands over time strengthen this analysis further. Figure 2,3 and 4 displays the result of PCR after 4, 8, and overnight incubation time

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/6d/T--TUDelft--venda_09_28_fig2.png)

**Figure 2. 5% TBE native PAGE stained by EtBr and imaged by GelDoc system. Integration reaction after 4 hours.**  *Lanes are (1) DNA ladder (100-1000bp), (2) amplified product with fw EPO (3) amplified product with rv EPO, (4) amplified product with fw EPO without sgRNA, (5) amplified product with rv EPO without sgRNA, (6) amplified product with fw EPO without fusion protein, (7) amplified product with rv EPO without fusion protein, (8) amplified product with fw EPO without adapter DNA, (9) amplified product with rv EPO without adapter DNA, (10) amplified product with fw EPO with Tn5, (11) amplified product with rv EPO with Tn5, (12) amplified product with fw EPO without target DNA, (13) amplified product 2 without target DNA
.*
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f5/T--TUDelft--venda_09_28_fig3.png)

**Figure 3. 5% TBE native PAGE stained by EtBr and imaged by GelDoc system. Integration reaction after 8 hours.**  *. Lanes are (1) DNA ladder (100-1000bp), (2) amplified product with fw EPO (3) amplified product with rv EPO, (4) amplified product with fw EPO without sgRNA, (5) amplified product with rv EPO without sgRNA, (6) amplified product with fw EPO without fusion protein, (7) amplified product with rv EPO without fusion protein, (8) amplified product with fw EPO without adapter DNA, (9) amplified product with rv EPO without adapter DNA, (10) amplified product with fw EPO with Tn5, (11) amplified product with rv EPO with Tn5, (12) amplified product with fw EPO without target DNA, (13) amplified product 2 without target DNA
.*
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/0/05/T--TUDelft--venda_09_28_fig4.png)

**Figure 4. 5% TBE native PAGE stained by EtBr and imaged by GelDoc system. Integration reaction overnight.**  *. Lanes are (1) amplified product with fw EPO (2) amplified product with rv EPO, (3) amplified product with fw EPO without sgRNA, (4) amplified product with rv EPO without sgRNA, (5) DNA ladder (100-1000bp), (6) amplified product with fw EPO without fusion protein, (7) amplified product with rv EPO without fusion protein, (8) amplified product with fw EPO without adapter DNA, (9) amplified product with rv EPO without adapter DNA, (10) amplified product with fw EPO with Tn5, (11) amplified product with rv EPO with Tn5, (12) amplified product with fw EPO without target DNA, (13) amplified product 2 without target DNA
.*

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Sunday, 30/09/2018

Date
:   30/09/2018

Operator
:   Venda and Monique

#### Fusion protein functionality testing: integration assay

From previous reaction, we saw that we were able to amplify the specific integration product that we expected, which, together with the results of the negative control, confirmed the ability of our fusion protein to perform specific integration. In this experiment, we would like to confirm this further by using a longer target DNA. If specific integration indeed occur, one of the new amplified fragment should also get longer by the same increase. Moreover, more sgRNA were tested to check whether other parts of the target EPO is also accessible. The following will therefore be done:

+ Incubation time of 1 hour will be implemented
+ 3 different EPO fragments were used. The original EPO cDNA, longer EPO with "intron" on the third junction, longer EPO with "intron" on the fourth junction
+ 4 different sgRNA was used, targeting junction 1-4
+ reaction without gRNA, and reaction with Tn5 will be done in parallel as negative controls

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay is shown on table 1.

**Table 1:** Primer sequence to be annealed for the adapter. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

**Fusion protein:adapter DNA complex formation**
Tn5 or fusion protein was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | Reaction (µl) | Reaction no fusion protein (µl) | Reaction with Tn5 (µl) |
| Buffer | 4.5 | 1 | 1 |
| Fusion (~3µM) | 36 | 0 | 0 |
| Tn5 (~8µM) | 0 | 0 | 3 |
| Adapter (25µM) | 4.5 | 1 | 1 |
| Water | 0 | 8 | 5 |
| Total | 45 | 10 | 10 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, Tn5 or dxCas9-Tn5, and adapter. The samples were incubated at 23°C for 1 hour.

To add appropriate amount of sgRNA, the pipetting scheme on Table 3 was followed:

**Table 3:** Pipetting scheme for sgRNA loading. sgRNA J2.1 signifies version 1 of sgRNA targeting juntion 2 of EPO gene, etc.

|  | sgRNA J1.1 (µl) | sgRNA J2.1 (µl) | sgRNA J3.1 (µl) | sgRNA J4.1 (µl) | no sgRNA (µl) | no fusion, J2.1 (µl) | Tn5 (µl) || Transposome | 4 | 12 | 8 | 8 | 12 | 10 | 10 |
| sgRNA (100ng/µl) | 0.8 | 2.4 | 1.6 | 1.6 | 0 | 2 | 0 |
| MgCl2 Buffer | 0.2 | 0.6 | 0.4 | 0.4 | 0.6 | 0.5 | 0.5 |
| Water | 0 | 0 | 0 | 0.9 | 2.4 | 0 | 2 |

The samples were incubated at 37°C for 10 minutes.
Target DNA (EPO cDNA) was added to make final concentration of ~5ng/µl. Three different types of EPO are present. The following reactions were done:

+ Normal EPO was targetted by sgRNAJ1.1, J2.1, J3.1, J4.1 to see whether all junctions are equality accessible
+ EPO with intron on Junction 3 was targetted by sgRNA 2.1 to see the increase of the size of the second fragment in comparison to the previous successfull experiments.
+ EPO with intron on Junction 3 was targetted by J3.1 to see whether EPO can be targetted even though the target site is disrupted by an intron sequence.
+ EPO with intron on Junction 4 was targetted by J4.1 to see whether EPO can be targetted even though the target site is disrupted by an intron sequence.
+ As a negative control reaction without sgRNA with normal EPO or EPO with intron on junction 3 or 4 were carried out in parallel

The samples were incubated at 37°C. At incubation time = 30 mins, 2.5 µl of sample from reaction with normal EPO + sgRNA J2.1 were taken along with the necessary negative control reactions to see whether we can decrease off target even more by deacreasing reaction time. These samples were then subjected to PCR based on the protocol. At incubation time=1 hours, 2.5µl sample from each reaction were subjected to PCR. PCR mix and cycle can be found on protocol.

The PCR products were analysed on 5% native TBE polyacrylamide gel. The resulting gel is either stained with ethidium bromide and imaged with GelDoc system. The resulting gels for intergration with sgRNA J1.1 - J4.1 are shown on figure 1-4.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/9c/T--TUDelft--venda_09_30_fig1.png)

**Figure 1. 5% TBE Native PAGE of integration with J1.1.**   *Primer sets 1 will amplify the first integration product. Primer sets 2 will amplify the second integration product. Lanes are (1) DNA ladder, (2) sgRNA J1.1, with primer set 1, (3) sgRNA J1.1, with primer set 2, (4) DNA ladder, (5) no sgRNA with primer set 1, (6) no sgRNA with primer set 2.*

Figure 1 showed uncleaved target DNA at 635bp and no integration fragments at 148bp and 548bp are observed (lane 2 and 3). Very little background is observed from ~150bp to ~50bp, probably due to off target events from the fusion protein.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/59/T--TUDelft--venda_09_30_fig2.png)

**Figure 2. 5% TBE Native PAGE of integration with J2.1.**  *Primer sets 1 will amplify the first integration product. Primer sets 2 will amplify the second integration product. Lanes are (1) DNA ladder, (2) 30 mins, sgRNA J2.1, with primer set 1, with EPO (3) 30 mins, sgRNA J2.1, with primer set 2, with EPO ,(4) 1h, sgRNA J2.1, with primer set 1, with EPO (5) 1h, sgRNA J2.1, with primer set 2, with EPO , (6) 1h, sgRNA J2.1, with primer set 1, with EPO\_intron3 (7) 1h, sgRNA J2.1, with primer set 2, with EPO\_intron3, (8) DNA ladder, (9) no sgRNA, primer set 1, with EPO (10) no sgRNA, primer set 2, with EPO, (11) no sgRNA, primer set 1, with EPO\_intron3 (12) no sgRNA, primer set 2, with EPO\_intron3*

Figure 2 Uncleaved target DNA at 635bp and 1247bp and 2 integration fragments at 280bp and 480/1068bp are observed (lane 1-7). Note the 2 PCR products are not observed in the negative controls (lane 9-12). Very little background is observed from ~150bp to ~50bp, probably due to off target events from the fusion protein. Note with the intron at junction 3, the integrated fragment is 1063, and the target DNA is 1247, causing the bands to un very close to each other.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/14/T--TUDelft--venda_09_30_fig3.png)

**Figure 3. 5% TBE Native PAGE of integration with J3.1.**  *Primer sets 1 will amplify the first integration product. Primer sets 2 will amplify the second integration product. Lanes are (1) DNA ladder, (2) 1h, sgRNA J3.1, with primer set 1, with EPO (3) 1h, sgRNA J3.1, with primer set 2, with EPO , (4) 1h, sgRNA J3.1, with primer set 1, with EPO\_intron3 (5) 1h, sgRNA J4.1, with primer set 2, with EPO\_intron3, (6)DNA Ladder, (7) no sgRNA, primer set 1, with EPO (8) no sgRNA, primer set 2, with EPO, (9) no sgRNA, primer set 1, with EPO\_intron3 (10) no sgRNA, primer set 2, with EPO\_intron3.*

Figure 3 snows uncleaved target DNA at 635bp and 1247bp and no specific integration products can be seen, and no difference in bands can be observed between the sample with and without gRNA (lane 2,3 vs lane 7,8 and lane 4,5 vs lane 9,10).

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/bd/T--TUDelft--venda_09_30_fig4.png)

**Figure 4. 5% TBE Native PAGE of integration with J4.1.**  *Primer sets 1 will amplify the first integration product. Primer sets 2 will amplify the second integration product. Lanes are (1) DNA ladder, (2) 1h, sgRNA J4.1, with primer set 1, with EPO (3) 1h, sgRNA J4.1, with primer set 2, with EPO , (4) 1h, sgRNA J4.1, with primer set 1, with EPO\_intron4 (5) 1h, sgRNA J4.1, with primer set 2, with EPO\_intron4, (6) no sgRNA, primer set 1, with EPO (7) no sgRNA, primer set 2, with EPO, (8) no sgRNA, primer set 1, with EPO\_intron4 (9) no sgRNA, primer set 2, with EPO\_intron4*

Figure 4 snows uncleaved target DNA at 635bp and no integration fragments at 284bp and 448/1053bp are observed (lane 2-5). Very little background is observed from ~150bp to ~50bp, probably due to off target events from the fusion protein. EPO\_I3 in late 5 and 9 has an interesting amplification pattern.

Close

### October

[ ]
Week 3 15/10/2018 - 17/10/2018

Monday, 15/10/2018

Date
:   15/10/2018

Operator
:   Venda Mangkusaputra

#### Sequence Verification of Integration Products

In order to confirm that the integration products of the functionality test were the ones we expected, we attempted to sequence verify them. We load the whole PCR product visualized on lane 2 and 3 from functionality assay on 28/09/2018 on 2% agarose gel and isolated the bands from the gel and purify them. Since a very low concentration was achieved, another round of PCR was done using the isolated fragment as template. We cleaned up the PCR product and achieved concentration of 250ng/µl for fragment 1, and 50ng/µl for fragment 2.

We sent these PCR products individually for sequencing. The alignment result of this sequence is shown on Figure 1.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/3/31/T--TUDelft--venda_10_15_fig1.png)

*Sequence alignment of integration product (two red arrows) with the target EPO. Pink box represent the sgRNA binding site.*

Figure 1 shows the sequence alignment between the two fragments and the target EPO gene.From the sequence alignment, we can confirm that integration occured upstream the sgRNA binding site. The overlapping alignment between the two products suggested that integration does not always occur at the exact base pair position (~10bp variation), which might be the result of the flexible linker between dxCas9 and Tn5.

Next, we used pGEM T-Easy cloning method to clone these fragments on individual plasmid to be able to sequence verify them individually. By doing this we will be able to see the variations within that band individually. The protocol for pGEM T-Easy can be found on the protocol page. We used 2µl of 10x diluted sample of isolated fragment 1 or 1µl of isolated fragment 2 to be cloned into the plasmid. We were able to get white colonies and isolated their plasmid, but however, the sequence verification result did not come on time.

Close

* July
  + [Week: 16/07/2018 - 22/07/2018](#JulyInVivo3)
* August
  + [Week: 30/07/2018 - 05/08/2018](#AugustInVivo1)
  + [Week: 06/08/2018 - 12/08/2018](#AugustInVivo2)
  + [Week: 13/08/2018 - 19/08/2018](#AugustInVivo3)
  + [Week: 20/08/2018 - 26/08/2018](#AugustInVivo4)
  + [Week: 27/08/2018 - 02/09/2018](#AugustInVivo5)
* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberInVivo1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberInVivo2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberInVivo3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberInVivo4)

### July

[ ]
Week 3 16/07/2018 - 23/07/2018

Tuesday, 17/07/2018

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

The following high fidelity PCR reactions were carried out:

| Amplicon | Size (bp) | Fw primer | Rv primer | template | Elongation time (sec) | Annealing temperature (°C) |
| --- | --- | --- | --- | --- | --- | --- |
| LacZ-gRNA | 314 | IV003 | IV004 | Gblock | 60 | 60 |
| KanR CDS | 862 | IV005 | IV006 | pSB1K3 | 60 | 60 |

PCR mixtures were prepared, according to the High Fidelity PCR protocol. After the programme was finished, samples were kept at 4°C.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

PCR products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 50 µL Milli-Q.

Date
:   17/7/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured and verified, but not registered.

| Isolate | Concentration (ng/uL) |
| --- | --- |
| LacZ-gRNA | 85 |
| KanR cassette | 209 |

Close

### AUGUST

[ ]
Week 2 06/08/2018 - 12/08/2018

Friday, 10/08/2018

Date
:   10/8/2018

Operator
:   Kavish Kohabir

#### GBlock resuspension

GBlock *lacZ* gRNA (314bp) was resuspended according to the instructions provided by the supplier (IDT DNA).

Date
:   10/8/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify GBlock *lacZ* gRNA (314bp) and the Kanamycin resistance gene (KanR), high fidelity PCR was done according to the High Fidelity PCR protocol.

The following primer combination(s) were predicted to result in the following expected amplicon length(s)

| Template | Fw primer | Rv primer | expected size (bp) |
| --- | --- | --- | --- |
| *lacZ* Gblock | IV003 | IV004 | 314 |
| pSB1K3 | IV005 | IV006 | 862 |

The thermocycler programme was installed to have an extension time of 30sec and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Saturday, 11/08/2018

Date
:   11/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR samples from 10/8/2018 was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/27/T--TUDelft--20180811kAVISHlacZKANrjpg.png)

This gel shows the KanR gene with ME flanks (862bp) was successfully PCRed from pSB1K3 (lanes 5-7). Amplification of the Gblock gives a very faint band at the expected size (314bp) for *lacZ*  gRNA (lanes 2-4).

Date
:   11/8/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify the *lacZ* gRNA (314bp) and linearizing pACYC-dxCas9-LinkerTn5 (9388bp), high fidelity PCR was done. 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

| Template | Fw primer | Rv primer | expected size (bp) | elongation time |
| --- | --- | --- | --- | --- |
| *lacZ* gRNA Gblock | IV003 | IV004 | 314 | 15 sec |
| pACYC-dxCas9-LinkerTn5 | IV001 | IV002 | 9388 | 5 min |

The thermocycler programme was installed to have an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Sunday, 12/08/2018

Date
:   12/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR products from 11/8/2018 was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/8e/T--TUDelft--20180812KAVISHpcrLacZ_linearizedpacycdxcas9linkertn5.jpg)

This gel shows that pACYC-dxCas9-LinkerTn5 was successfully linearized (lane 8, 9 and 10), as there is a strong amplicon signal far above 5kbp, and we expected 9388bp. Amplification of the Gblock showed very faint bands at the expected size (314bp) (lane 2, 3 and 4).

Date
:   12/8/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

PCR samples from 10/8/2018 (*lacZ* gRNA and KanR) and 11/8/2018 (*lacZ* gRNA and linearized pACYC-dxCas9Tn5) were subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q (2 times 20).

Date
:   12/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Sample date | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- | --- |
| *lacZ* gRNA | 10/8/2018 | 180.6 | 1.9 | 2.0 |
| ME flanked KanR gene | 10/8/2018 | 220 | 1.8 | 2.1 |
| *lacZ* gRNA | 11/8/2018 | 155 | 1.8 | 2.0 |
| linearized pACYCdxCas9Tn5 | 11/8/2018 | 220 | 1.8 | 2.1 |

Date
:   12/8/2018

Operator
:   Kavish Kohabir

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble *lacZ* gRNA (insert) and linearized pACYC-dxCas9-LinkerTn5 (vector) to obtain pACYC-dxCas9-LinkerTn5-*lacZ*gRNA (9622bp).

| Component | Volume (uL) |
| --- | --- |
| Gibson Assembly Mastermix 2X | 10 |
| Vector | 9.4 (2 µg) |
| Insert | 0.6 (100 ng) |

The samples were incubated at 50°C for 20 minutes, after which they were kept at 4°C.

Date
:   12/8/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

Solid Luria Broth (LB) medium was prepared, complemented with chloramphenicol (Cam). Plates were stored at 4°C.

Close

[ ]
Week 3 13/08/2018 - 19/08/2018

Monday, 13/08/2018

Date
:   13/8/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | pACYC-dxCas9-LinkerTn5-*lacZ*gRNA (9622bp) | Gibson Assembly | 10µl | LB+Cam |
| *E.coli*DH5α | pACYC-dxCas9-LinkerTn5-*lacZ*gRNA (9622bp) | Gibson Assembly | 6µl | LB+Cam |
| *E.coli*DH5α | linearized pACYC-dxCas9-LinkerTn5 | *neg. control* | 2µl | LB+Cam |

200 µL LB medium was added as recovery medium.

Close

Tuesday, 14/08/2018

Date
:   14/8/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 13/8/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli*DH5α | 10µl pACYC-dxCas9-LinkerTn5-*lacZ*gRNA (9622bp) | LB+Cam | growth |
| *E.coli*DH5α | 6µl pACYC-dxCas9-LinkerTn5-*lacZ*gRNA (9622bp) | LB+Cam | growth |
| *E.coli*DH5α | linearized pACYC-dxCas9-LinkerTn5 | *LB+Cam* | less growth |

The negative control shows that there is still instact plasmid template, or self-ligated plasmid present in the transformation mixture provided. However, since the amount of colonies is less, there is a strong indicatin that the pACYC-dxCas9-LinkerTn5-*lacZ*gRNA might be constructed. It was decided to proceed with colony PCR.

Date
:   14/8/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify propagation of pACYC-dxCas9-LinkerTn5-*lacZ*gRNA after transformation (13/8/2018). This plasmid contains a fragment with dxCas9-LinkerTn5-*lacZ*gRNA, which can be checked with primers 018 and IV004.

| Component | Volume (uL) |
| --- | --- |
| Template DNA | picked colony |
| Forward primer | 018 |
| Reverse primer | IV004 |
| Expected amplicon size (bp) | 2148 |

14 tubes were prepared for colony PCR, containing picked colony as template DNA. The thermocycler programme was installed to have an extension time of 2.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   14/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/88/T--TUDelft--kavishcolonyPCR20180814annotated.jpg)

An amplicon of 2148bp was expected for this PCR reaction. For almost all colonies, we observe a band around this height. Not all bands seem to be at exactly the same height. Based on this gel, it was decided to inoculate colony 1, 2, 3 and 4

Date
:   14/8/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Colonies 1, 2, 3 and 4 from the plates from 13/8/2018 were used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with Chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Wednesday, 15/08/2018

Date
:   15/8/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of colonies 1, 2, 3, 4 (plates 13/8/2018) were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   15/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured and verified to be around 1.8~1.9.

| Isolate | Concentration (ng/uL) |
| --- | --- |
| colony 1 | 32 |
| colony 2 | 35 |
| colony 3 | 41 |
| colony 4 | 36 |

Date
:   15/8/2018

Operator
:   Kavish Kohabir

#### Sequence Verification from plasmid

For verification of insertion of *lacZ* gRNA into pACYCdxCas9Tn5, primers IV003 and IV004 were used for sequencing. Only plasmid isolates from colony 1 and 2 were sent for sequencing. The sequencing starts from the homology flanks, in the direction of the *lacZ* gRNA. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | Volume (uL) | Final concentration |
| --- | --- | --- |
| Plasmid DNA (500ng) | 7.5 | 50 ng/µL |
| 10µM primer | 2.5 | 25 pmol |

Within 7 days, the sequence was retrievable online and alignment with the *in silico* designed molecule showed 100% match of the 1050 bp downstream of the sequencing primer binding site. This was only the case for the plasmid isolate from colony 1. **This means the pACYC-dxCas9-LinkerTn5-*lacZ*gRNA construct is sequence verified.**

Date
:   15/8/2015

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Volume added (µL) | Medium on which was plated |
| --- | --- | --- | --- |
| *E.coli*DH5α | pSB1K3 | 5 | LB+Kan |
| *E.coli*DH5α | - (neg. control) | - | LB+Kan |

200 uL LB medium was added as recovery medium.

Date
:   15/8/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol in the morning, the plates were left for overday incubation in the stove. After ~12hr incubation, the following results were observed:

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli* DH5α | pSB1K3 | LB+Kan | colony growth || *E.coli* DH5α | - | LB+Kan | no growth |

Date
:   15/8/2015

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

1 colony from the LB+Kan was used for inoculation of 5mL liquid starter culture (Luria Broth, complemented with Kanamycin) following the Liquid Starter Culture Protocol.

Close

Thursday, 16/08/2018

Date
:   16/8/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of pSB1K3 (in DH5alpha) were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30μL of pre-warmed MilliQ.

Date
:   16/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were verified to be around 1.8~1.9.

| Isolate | Concentration (ng/uL) |
| --- | --- |
| pSB1K3 | 37 |

Date
:   16/8/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

Solid Luria Broth (LB) medium was prepared, complemented with Kanamycin (Kan), Chloramphenicol (Cam), IPTG and X-gal, following the following steps:

1. Warm 250mL LB agar.
2. Mix 50mg X-gal in 2.5mL DMSO.
3. Add X-gal solution to LB agar.
4. Add 2.5mL 100mM IPTG to LB agar.
5. Add 250µL Kan (1000x stock solution) & 250µL Cam (1000x stock solution).
6. Pour plates: cool down while protecting from light.
7. store at 4°C, protected from light.

Date
:   16/8/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Strain BL21DE3 used for inoculation of 10mL liquid starter culture (Luria Broth, **not complemented with antibiotics**) following the Liquid Starter Culture Protocol.

Close

Friday, 17/08/2018

Date
:   17/8/2018

Operator
:   Kavish Kohabir

#### Genomic DNA Isolation

Starter culture(s) of BL21DE3 were subjected to genomic DNA isolation, following the  [genomic DNA isolation protocol.](https://2018.igem.org/Team%3ATUDelft/Experiments)

Vigorous shaking (step 5) was done for 30 minutes, instead of 10 minutes.

Close

[ ]
Week 4 20/08/2018 - 26/08/2018

Monday, 20/08/2018

Date
:   20/8/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

Solid Luria Broth (LB) medium was prepared, complemented with Cam. 40 plates were poured and stored at 4°C.

Date
:   20/8/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | pACYC-dxCas9Tn5*lacZ*gRNA | ME flanked KanR | Medium on which was plated |
| --- | --- | --- | --- |
| *E. coli* BL21DE3 | 4µl (~120ng) | 10µl (~2µg) | + LB+Cam+Kan+IPTG+X-gal + LB+Cam |
| *E. coli* BL21DE3 | 4µl (~120ng) | - | + LB+Cam+Kan+IPTG+X-gal + LB+Cam |

200 µL LB medium was added as recovery medium.

Date
:   20/8/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Strain DH5alpha+pACYC-dxCas9Tn5*lacZ*gRNA were used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with Chloramphenicol) following the Liquid Starter Culture Protocol.

Date
:   20/08/2018

Operator
:   Venda Mangkusaputra

#### Restriction

Restriction cloning was done to assemble EPO and sgRNA for LacZ into biobrick backbone. The following table shows an overview of which fragments were cut with the appropriate restriction enzymes.

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Biobrick backbone (uL) | EPO (ul) | sgRNA for LacZ(ul) || 10x CutSmart buffer | 2 | 2 | 2 |
| Fragment | 9 | 6 | 6 |
| EcoRI | 0.4 | 0.4 | 0.4 |
| PstI | 0.4 | 0.4 | 0.4 |
| MilliQ | 8.2 | 11.2 | 11.2 |

The samples were incubated for 4 hours at 37 °C. DNA was isolated by following PCR clean up kit protocol.
DNA was quantified by nanodrop. The following concentrations were achieved:

+ Biobrick backbone=40ng/ul
+ EPO=25ng/ul
+ sgRNA for LacZ=25ng/ul

Date
:   20/08/2018

Operator
:   Venda Mangkusaputra

#### Ligation

In order to assemble biobrick inserts into the biobrick backbone, ligation was performed. The following pipetting scheme was followed

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | EPO (uL) | sgRNA (uL) || Ligase Buffer (10x) | 2 | 2 || T4 DNA Ligase | 1 | 1 || DNA vector (~100 ng) | 4 | 4 || DNA fragment (XXX ng) | 13 | 13 ||

The samples were incubated for 4 hours at room temperature.

Date
:   20/08/2018

Operator
:   Venda Mangkusaputra

#### Transformation of chemically competent cells

The following transformations for EPO and sgRNA for LacZ biobrick were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | EPO BB | Restriction-ligation | 6µl | LB-cam |
| *E.coli*DH5α | sgRNA for LacZ BB | Restriction-ligation | 6µl | LB-cam |

200 uL LB medium was added as recovery medium.

Close

Tuesday, 21/08/2018

Date
:   21/8/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 20/8/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

All colonies on X-gal plates were white, possibly suggesting that LacZ was disrupted and kanR is present in the cell. However, to exclude false positives, a crucial control is required: BL21DE3 on LB+X-Gal+IPTG, to reassure that the parental strain has intact *lacZ* in the first place.

Date
:   21/8/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of DH5alpha+pACYC-dxCas9Tn5*lacZ*gRNA were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   21/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/µL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pACYC-dxCas9Tn5*lacZ*gRNA | 159 | 1.85 | 1.94 |
| pACYC-dxCas9Tn5*lacZ*gRNA | 179 | 1.84 | 1.75 |
| pACYC-dxCas9Tn5*lacZ*gRNA | 152 | 1.84 | 1.86 |
|  |

Date
:   21/8/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- |
| *E.coli*BL21DE3 | pACYCdxCas9Tn5 + pSB1K3 | 4 each | LB+Cam+Kan+X-gal+IPTG |

200 µL LB medium was added as recovery medium.

Date
:   21/8/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Four colonies from the plate from 20/8/2018 were used for inoculation of 5mL liquid starter culture (Luria Broth, complemented with chlorampenicol and kanamycin) following the Liquid Starter Culture Protocol.

Close

Wednesday, 22/08/2018

Date
:   22/8/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 21/8/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strain | DNA | Medium | Result |
| --- | --- | --- | --- |
| *E.coli* BL21DE3 | pACYCdxCas9Tn5 + pSB1K3 | LB+Cam+Kan+X-gal+IPTG | 11 white colonies |

pSB1K3 is supposed to give a red colony phenotype. The pSB1K3 is suspected to be contaminated with other kanamycin (an)other resistant plasmid(s)

Date
:   22/8/2018

Operator
:   Kavish Kohabir

#### Genomic DNA Isolation

Starter culture(s) of inoculated colonies in LB+Cam+Kan were subjected to genomic DNA isolation, following the  [genomic DNA isolation protocol.](https://2018.igem.org/Team%3ATUDelft/Experiments)

Date
:   22/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| colony 1 | 39 | 1.9 | 1.9 |
| colony 2 | 46 | 1.9 | 1.3 |
| colony 3 | 40 | 1.9 | 1.2 |
| colony 4 | 52 | 1.8 | 1.5 |

Date
:   22/8/2018

Operator
:   Kavish Kohabir

#### Primer working stock preparation

The following primers were resuspended in sterile Milli-Q water as directed by the manufaturer, in order to make 100µM stock solutions.

The primers in these 100 µM stocks were heated to 60 °C for 20 minutes and centrifuged at maximum speed (~17,000 x g) for 2 minutes. 10µM primer working stock solutions were prepared by making 10x dilution aliquots with sterile Milli-Q, each in a total volume of 100µl.

Date
:   22/8/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

The following solid media were prepared:

+ Luria Broth (LB) + X-gal + IPTG
+ Luria Broth (LB) + X-gal + IPTG + Cam
+ Luria Broth (LB) + X-gal + IPTG + Cam + Kan

Final concentrations in these media are:

+ 0.1µmol IPTG per mL medium
+ 40 µg X-gal per mL medium

Date
:   22/8/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify integration of KanR after transformation from 20/8/2018. This can be checked with primers IV007 & IV008.

| Component |  |
| --- | --- |
| Template DNA | Isolated gDNA |
| Forward primer | IV007 |
| Reverse primer | IV008 |
| Expected amplicon size (bp) | 5680\* |

*\*5680bp only amplifies if the KanR integrated in the lacZ locus. If the locus was not disrupted, 4818bp amplicons are expected. as a positive control, BL21DE3 gDNA was used as template.*

5 tubes were prepared for colony PCR, containing isolated gDNA as template DNA. The thermocycler programme was installed to have an extension time of 6.5 minutes and an annealing temperature of 60°C. After the programme was finished, samples were kept at 4°C.

Date
:   22/08/2018

Operator
:   Venda Mangkusaputra

#### Colony PCR

Colony PCR was done to verify the success of EPO and sgRNA for LacZ biobrick. 3 different colonies from each plate were subjected to colony PCR. Two different primer sets were used. Biobrick primers 031 and 032 were used to verify the size of the insert. Primers 038 and 039 were used to verify the presence of EPO fragment within the biobrick backbone. Primers IV003 and IV004 were used to verify the presence of sgRNA for LacZ fragment within the biobrick backbone.

The PCR components and program can be found on the colony PCR protocol page. The PCR products were run on 2% TAE agarose gel shown on Figure 1. Upper lanes of Figure 1 shows amplification of insert with primers that anneal to the biobrick backbone flanking the insert (031 and 032). Lower lanes on Figure 1 shows amplification of insert with internal primers. All and all, the results confirmed insertion of the correct insert length.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/81/T--TUDelft--venda_08_22_fig1.png)

**Figure 2% TAE Agarose gel of colony PCR**. *Lanes are (1-3) colony 1-3 harboring EPO Biobrick (BB) amplified by biobrick primers 031 and 032, (4-6) colony 1-3 harboring sgRNA DNA template for LacZ Biobrick (BB) amplified by biobrick primers 031 and 032, (7) DNA ladder, (8-10) colony 1-3 harboring EPO Biobrick (BB) amplified by internal primers 038 and 039, (11-13) colony 1-3 harboring sgRNA DNA template for LacZ Biobrick (BB) amplified by internal primers IV003 and IV004, (14) DNA ladder.*

Close

Thursday, 23/08/2018

Date
:   23/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

gDNA PCR samples from 22/8/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/ff/T--TUDelft--20180823KAVISHgDNAcolonyPCR.jpg)

This gel shows various bands, but no integration (5680bp) nor intact *lacZ* (4818bp). This suggests the parental strain is contaminated with a *lacZ*-deficient strain. This goes along with the absence of any expected blue colony phenotype from the transformation done on 20/8/2018.

Date
:   23/8/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of 2 colonies with BioBrick (pSB1C3-*lacZ*gRNA) were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   23/8/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| colony 1 (*lacZ*gRNa) | 17 | 1.63 | 0.95 |
| colony 2 (*lacZ*gRNa) | 23 | 1.63 | 0.88 |
|

Date
:   23/8/2018

Operator
:   Kavish Kohabir

#### Sequence Verification

##### Sequence from plasmid

For verification of *lacZ* in pSB1C3, primers VF2 and VR were used for sequencing. The sequencing starts from the biobrick backbone in the direction of the insert. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | Volume (uL) | Final concentration |
| --- | --- | --- |
| Plasmid DNA (500ng) | 7.5 | >50 ng/uL |
| 10uM primer | 2.5 | 25 pmol |

Within 7 days, the sequence was retrievable online and alignment of colony 1 with the *in silico* designed molecule showed 100% match of the 1050 bp downstream of the sequencing primer binding site.

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Friday, 24/08/2018

Date
:   24/8/2018

Operator
:   Kavish Kohabir & Alexander

#### Chemically Competent Cells Preparation

Chemically competent cells were made of *E. coli* DH5alpha and *E. coli* BL21DE3, following the [Chemically Competent Cells Preparation protocol](https://2018.igem.org/Team%3ATUDelft/Experiments#chemcomptcellsprep-scroll).

Close

[ ]
Week 5 27/08/2018 - 02/09/2018

Monday, 27/08/2018

Date
:   27/8/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify the KanR cassette\* (~1kb), high fidelity PCR was done using pSB1K3 as template DNA and IV009 and IV010 as forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

*NOTE: previously (10/8/2018), the KanR **gene** was used for amplification. This is not the same as the **whole cassette**, which includes the promoter and terminator.*

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | expected size (bp) |
| --- | --- | --- | --- |
| ME-flanked KanR cassette (MEpKantME) | IV009 | IV010 | 1060 |
| KanR cassette (pKant) | IV011 | IV012 | 1014 |

The thermocycler programme was installed to have an extension time of 45sec and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   27/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

MEpKantME and pKant PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
The gel was completely empty, only the ladder was visible. It is not clear what exactly went wrong. Perhaps make more offers to the PCR gods?

Date
:   27/8/2018

Operator
:   Kavish Kohabir

#### Testing new stock of BL21DE3 cells

Previously (23/8/2018), it was observed that the BL21DE3 strain stock might be contaminated with other Kanamycin resistant cells. The following inoculations were carried out with both that old stock, and a fresh new stock, obtained from dr. Theo van Laar.

| BL21DE3 stock | Medium |
| --- | --- |
| old | LB+Cam |
| new | LB+Cam |
| old | LB+Kan |
| new | LB+Kan |
| old | LB+IPTG+X-gal\* |
| new | LB+IPTG+X-gal\* |

*\*LB+IPT+X-gal plates from 22/8/2018 were used.*

Close

Tuesday, 28/08/2018

Date
:   28/8/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify the KanR cassette\* (~1kb), high fidelity PCR was done using pSB1K3 as template DNA and IV009 and IV010 as forward and reverse primers respectively. 8 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

*NOTE: previously (10/8/2018), the KanR **gene** was used for amplification. This is not the same as the **whole cassette**, which includes the promoter and terminator.*

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | expected size (bp) | extension time |
| --- | --- | --- | --- | --- |
| ME-flanked KanR cassette (MEpKantME) | IV009 | IV010 | 1060 | 40sec |
| KanR cassette (pKant) | IV011 | IV012 | 1014 | 40sec |
| gDNA PCR | IV007 | IV008 | 5680\* | 6.5min |

\*5680bp should appear only in case of KanR integration in the *lacZ* locus. Otherwise, this should result in 4818bp bands (intact *lacZ*).

The thermocycler programme was installed to have an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   28/8/2018

Operator
:   Kavish Kohabir

#### Inoculation Results

After inoculating BL21DE3 controls on solid medium, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| BL21DE3 stock | Medium | Result |
| --- | --- | --- |
| old | LB+Cam | white colony growth |
| new | LB+Cam | no colony growth |
| old | LB+Kan | white colony growth |
| new | LB+Kan | no colony growth |
| old | LB+IPTG+X-gal\* | blue colony growth |
| new | LB+IPTG+X-gal\* | blue colony growth |

*\*LB+IPT+X-gal plates from 22/8/2018 were used.*

Based on these results, it was concluded that the old BL21DE3 batch already was contaminated with kanamycin and chloramphenicol resistance. The new batch does not show this, and therefore meets the requirements to continue *in vivo* screen work. Also, the plates poured on 22/8/2018 show good viability and chromogenic conversion of X-gal.

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Wednesday, 29/08/2018

Date
:   29/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR samples from 28/8/2018 were run on a 0.8% agarose gel in TAE buffer. 5 μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.
Again, the obtained gel was completely empty, only showing the reference ladders. It is not sure why this failed, but could have to do with too high annealing temperature. A gradient PCR could sort this out.

Date
:   29/8/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- |
| *E.coli*BL21DE3 | pACYC-dxCas9Tn5*lacZ*gRNA (9622bp) | 1µl (~150ng) | LB+Cam |

200 µL LB medium was added as recovery medium.

Date
:   29/8/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify the KanR cassette\* (~1kb), high fidelity PCR was done using pSB1K3 as template DNA and IV009 and IV010 as forward and reverse primers respectively. Also, another attempt with IV007 and IV008 on gDNA was done to get a positive control band at 4818bp. 25µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

All samples contained 1µL of DMSO. The thermocycler programme was installed to have an extension time of 1 minute/kb amplicon and an annealing temperature ranging from 55 to 67°C (55; 56.1; 57.8; 59.8; 62.5; 64.7; 66.2; 67). After the programme was finished, samples were kept at 4 °C.

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Thursday, 30/08/2018

Date
:   30/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR samples from 29/8/2018 and 30/8/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/94/T--TUDelft--20180830KanRandgDNAPCR.jpg)

Date
:   30/8/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify propagation of pACYC-dxCas9Tn5*lacZ*gRNA after transformation from 29/8/2018. This plasmid contains *lacZ* gRNA, which can be checked with primers 018 & IV004.

| Component | Volume (uL) |
| --- | --- |
| Template DNA | picked colony |
| Forward primer | 018 |
| Reverse primer | IV004 |
| Expected amplicon size (bp) | 2148 |

10 tubes were prepared for colony PCR, containing picked colony as template DNA. 1 positive control (already isolated pACYC-dxCas9Tn5*lacZ*gRNA) was included. The thermocycler programme was installed to have an extension time of 2.5min and an annealing temperature of 60°C. After the programme was finished, samples were kept at 4°C.

Date
:   30/8/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Colony PCR samples was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f1/T--TUDelft--20180830KAVISHcolonyPCRpACYCdxcas9tn5lacZgRNA.jpg)

This gel all tested colonies contain the *lacZ* gRNA next to Tn5 and dxCas9 (amplicon = 2148bp). it was decided to subject colony 1 to further analysis.

Date
:   30/8/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Colony 1 (BL21DE3+pACYC-dxCas9Tn5*lacZ*gRNA) of the plate from 29/8/2018 was used for inoculation of 60mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the Liquid Starter Culture Protocol.

Date
:   30/8/2018

Operator
:   Kavish Kohabir

#### Cell banking

Strain *E. coli* DH5alpha pSB1C3-*lacZ*gRNA (colony 1) was stocked and stored at -80oC following the cell banking protocol.

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Friday, 31/08/2018

Date
:   31/8/2018

Operator
:   Kavish Kohabir

#### High Fidelity Genomic PCR

To amplify the *lacZ* region (~4.8kb; no integration), high fidelity PCR was done using isolated BL21DE3 gDNA as template DNA and IV007 and IV008 as forward and reverse primers respectively. 25µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 3min and an annealing temperature range from 60°C to 65°C. After the programme was finished, samples were kept at 4 °C.

Date
:   31/8/2018

Operator
:   Kavish Kohabir

#### Cell banking

Strain(s) BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA was stocked and stored at -80oC following the cell banking protocol.

Date
:   31/8/2018

Operator
:   Kavish Kohabir

#### Electrocompetent Cell Preparation

60mL of *E. coli* BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA was used for making electrocompetent cell preparation protocol. Washing steps were only done with cold, sterile MilliQ. The final resuspension was done with 50% glycerol.

Date
:   31/8/2018

Operator
:   Kavish Kohabir

#### Genomic Colony PCR

Genomic colony PCR was done as an approach to verify whether primers IV007 & IV008 give a ~4.8kb amplicon.

8 tubes were prepared for colony PCR, containing supernatant of boiled picked blue colonies (24/8/2018) as template DNA. The thermocycler programme was installed to have an extension time of 6min and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

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Sunday, 02/09/2018

Date
:   2/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR samples from 31/8/2018 were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/ab/T--TUDelft--20180902KAVISHcolonyPCRbothboiledandfromcolonygoedverhaalmaaruiteindelijkheefthetnietgewerkt.jpg)

This gel shows that colony PCR from boiled colony as template does not work, not even for 1 of the 8 screened colonies. However, when using gDNA as template, the expected 4.8kb amplicon shows for annewaling temperatures lower than 63°C. From 61°C onwards, there is also another amplicon, as byproduct.

Close

### SEPTEMBER

[ ]
Week 1 03/09/2018 - 09/09/2018

Thursday, 06/09/2018

Date
:   6/9/2018

Operator
:   Kavish Kohabir

#### Electroporation

The following transformations were carried out according to the protocol for electroporation:

| Strains | Plasmid/fragment | Plate medium |
| --- | --- | --- |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | LB+Kan+Cam+X-gal+IPTG |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | LB+Kan+Cam+X-gal+IPTG |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | LB+Cam |

200 μl LB medium was added as recovery medium.

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Friday, 07/09/2018

Date
:   7/9/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 6/9/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strains | Plasmid/fragment | Plate medium | Result |
| --- | --- | --- | --- |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | LB+Kan+Cam+X-gal+IPTG | no growth |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | LB+Kan+Cam+X-gal+IPTG | no growth |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | LB+Cam | white colony growth |
| *E. coli* BL21DE3 | - | LB+Cam | no growth |

The transformation results show that the bacth of competent cells indeed already contains the plasmid pACYCdxCas9Tn5*lacZ*gRNA. Transformation with the pSB1K3 plasmid does not seem to confer kanamycin resistance. Perhaps, something is wrong with the pSB1K3 isolate.

Date
:   7/9/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

To determine the feasibility of *E. coli* BL21DE3 hosting 2 plasmids (pSB1K3 & pACYC-derived), the following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Volume added (uL) | Medium |
| --- | --- | --- | --- |
| *E.coli*BL21DE3 | pSB1K3 | 2 | LB+Kan |
| *E.coli*BL21 | pSB1K3+pACYC-DUET | 2 each | LB+Kan+Cam |
| *E.coli*BL21DE3 | - | - | LB+Kan |
| *E.coli*BL21 | - | - | LB+Kan+Cam |

200 µL LB medium was added as recovery medium. 50µL was plated.

Date
:   7/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify KanR cassette with (MEpKantME\*) and without (pKant\*) MEs (~1kb), high fidelity PCR was done using the following forward and reverse primers. 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

*\*pKant = promotor-Kan-terminator*

| Template | Fw primer | Rv primer | expected fragment | expected size (bp) |
| --- | --- | --- | --- | --- |
| pSB1K3 | IV013 | IV014 | MEpKantME | ~1kb |
| pSB1K3 | IV015 | IV016 | pKant | ~1kb |

The thermocycler programme was installed to have an extension time of 30sec and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Close

Saturday, 08/09/2018

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### Cell banking

DH5alpha+pSB1K3 and DH5alpha+pSB1C3 were stocked and stored at -80oC following the cell banking protocol.

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR products for MEpKantME and pKant were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/25/T--TUDelft--20180908kavishPCRforKanRandMEKanRME.jpg)

The gel shows multiple bands and indicates the plasmid sample may be contaminated with other DNA. This is the case, regardless of flanking with MEs or not.

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify KanR cassette with (MEpKantME\*) and without (pKant\*) MEs (~1kb), high fidelity PCR was done using the following forward and reverse primers. 50µL PCR mixtures were prepared, according to the High Fidelity PCR protocol. The same preparation was done as on 7/9/2018, but now with a gradient of annealing temperatures.

Primer combination(s) and expected fragment(s) are identical to as on 7/9/2018. Based on previous experiments, it suspected that the usual pSB1K3 plasmid sample is contaminated, so it was decided to work with a different vial (isolated in 2010, by iGEM Team TU Delft).

The thermocycler programme was installed to have an extension time of 45sec and an annealing temperature range from 65°C to 72°C. After the programme was finished, samples were kept at 4 °C.

Date
:   8/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

KanR cassette PCR fragments (with and without MEs) were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/4/40/T--TUDelft--20180908_2010pSB1K3templatewerktdusblijkbaarwelLOLOL.jpg)

This gel shows that the initial template used indeed did not contain pur pSB1K3. The 2010 isolate was a useful alternative to this, showing that the problematics was due to template contamination.

Close

Sunday, 09/09/2018

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Volume added (µL) | Medium on which was plated |
| --- | --- | --- | --- |
| *E.coli*DH5α | pSB1K3 | 1 (~350ng) | LB+Kan || *E.coli*DH5α | - | - | LB+Kan |

200 µL LB medium was added as recovery medium.

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### Inoculation of *in vivo* assay controls

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid | Medium |
| --- | --- | --- |
| *E.coli* BL21DE3 | pACYCdxCas9Tn5*lacZ*gRNA | LB+Kan |
| *E.coli* BL21DE3 | pACYCdxCas9Tn5*lacZ*gRNA + pSB1K3 | LB+Kan |

200 µL LB medium was added as recovery medium.

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify MEpKantME (~1kb, entails full expressional cassette for kanamycin resistance, flanked with MEs), high fidelity PCR was done using pSB1K3 as template DNA and IV013 and IV014 as forward and reverse primers respectively. PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 45sec and an annealing temperature of 65°C. After the programme was finished, samples were kept at 4 °C.

Date
:   9/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

MEpKantME PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL
was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f9/T--TUDelft--20180909KavishMEpKantME.jpg)

Close

[ ]
Week 2 10/09/2018 - 16/09/2018

Wednesday, 12/09/2018

Date
:   12/9/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Strain DH5alpha+pSB1K3 (red phenotype)
and BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA were used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with kanamycin for pSB1K3, and chloramphenicol for pACYC-derived vectors) following the Liquid Starter Culture Protocol.

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Thursday, 13/09/2018

Date
:   13/9/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of DH5alpha + pSB1K3 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   13/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| pSB1K3 | 125 | 1.9 | 2.0 |
| pSB1K3 | 130 | 1.9 | 2.1 |
|  |
|  |

Date
:   13/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify MEpKantME & pKant (~1kb), high fidelity PCR was done using freshly isolated pSB1K3 as template DNA. PCR mixtures were prepared, according to the High Fidelity PCR protocol.

Primer combination(s) and expected fragment(s)

| Sample | Fw primer | Rv primer | Template | expected size (bp) |
| --- | --- | --- | --- | --- |
| MEpKantME | IV013 | IV014 | pSB1K3 | ~1kb |
| pKant | IV015 | IV016 | pSB1K3 | ~1kb |

The thermocycler programme was installed to have an extension time of 45sec and an annealing temperature of 65°C. After the programme was finished, samples were kept at 4 °C.

Date
:   13/9/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Medium |
| --- | --- | --- |
| *E.coli*BL21DE3 | pACYCdxCas9Tn5*lacZ*gRNA | LB+Cam |
| *E.coli*BL21DE3 | pACYCTn5 | LB+Cam |
| *E.coli*BL21DE3 | pACYCdxCas9Tn5 | LB+Cam |

200uL LB medium was added as recovery medium.

Date
:   13/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

PCR samples for kanamycin expression cassettes were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/59/T--TUDelft--20180913KavishMEpKantMEandpKant.jpg)

This gel shows that, finally, the kanamycin cassettes (each ~1000bp) are appropriately amplified, without byproducts. This means the PCR protocol & template used on 13/9/2018 is correct.

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Friday, 14/09/2018

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify propagation of pACYCTn5 and pACYCdxCas9Tn5 after transformation from 13/9/2018. These plasmids contain the T7 expression system, which can be checked with primers 029 & 030.

| Component | Volume (uL) |
| --- | --- |
| Template DNA | picked colony |
| Forward primer | 029 |
| Reverse primer | 030 |
| Expected amplicon size | pACYCTn5: ~1.5kb  pACYCdxCas9Tn5: ~6.2kb |

Tubes were prepared for colony PCR, containing picked as template DNA. The thermocycler programme was installed to have an extension time of 6.5min and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/82/T--TUDelft--20180914KAVISHColonyPCR.jpg)

The gel shows that BL21DE3 was successfully transformed with pACYCdxCas9Tn5 (lane 2) and also with pACYCTn5 (lane 5). DH5alpha was not rendered with a pACYC-Gblock3&4 insert (lane 6 & 7), as the expected size would be 6kb.

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

The following strains were used for inoculation of 30mL liquid starter cultures:

+ BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA
+ BL21DE3 + pACYCdxCas9Tn5
+ BL21DE3 + pAYCTn5

Preparation of the LB media (Luria Broth, complemented with cloramphenicol) was done following the Liquid Starter Culture Protocol.
The cultures were grown until OD0.5, prior to making them electrocompetent.

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### Electrocompetent Cell Preparation

The following strains were made electrocompetent:

+ BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA
+ BL21DE3 + pACYCdxCas9Tn5
+ BL21DE3 + pAYCTn5

.

Washing steps were only done with cold, sterile MilliQ. The final resuspension was done with 50% glycerol.

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

MEpKantME & pKant PCR products from 13/9/2018 were subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q (2x20µl per column).

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| MEpKantME | 993 | 1.85 | 2.15 |
| MEpKant | 1153 | 1.87 | 2.29 |
| pKant | 823 | 1.78 | 2.06 |
|  |

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

The following solid media were prepared:

+ Luria Broth (LB) + X-gal + IPTG
+ Luria Broth (LB) + X-gal + IPTG + Cam
+ Luria Broth (LB) + X-gal + IPTG + Cam + Kan

Final concentrations in these media are:

+ 0.1µmol IPTG per mL medium
+ 40 µg X-gal per mL medium

Date
:   14/9/2018

Operator
:   Kavish Kohabir

#### DpnI Digestion

To eliminate any residual template DNA, a DpnI digestion was performed with the following reaction mixture:

| Component | Volume (uL) |
| --- | --- |
| 10x CutSmart buffer | 5 |
| PCR product | 40 |
| Enzyme (DpnI) | 1 |
| MilliQ | 4 |

The reaction was incubated for 4 hours at 37 °C.
Heat inactivation was performed for 30 minutes at 80 °C.

Close

Saturday, 15/09/2018

Date
:   15/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Kanamycin PCR samples were run on a 0.8% agarose gel in TAE buffer. 1μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/1/1f/T--TUDelft--20180915KAVISH_pkant-mepkantme.jpg)

Date
:   15/9/2018

Operator
:   Kavish Kohabir

#### DpnI Digest Clean-up

DpnI digested kanamycin PCR products were subjected to Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date
:   15/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| MEpKantMEDpnI | 1537.3 | 1.86 | 2.29 |
| pKantDpnI | 735 | 1.82 | 2.18 |
|  |
|  |

Date
:   15/9/2018

Operator
:   Kavish Kohabir

#### Electroporation

The following transformations were carried out according to the protocol for electroporation:

| Strain | Plasmid/fragment size (bp) | Medium\* |
| --- | --- | --- |
| *E. coli* DH5alpha | pACYC-DUET | 2 |
| *E. coli* BL21DE3 | - | 4 |
| *E. coli* BL21DE3 | - | 5 |
| *E. coli* BL21DE3 | - | 3 || *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | 1 & 2 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 1 & 4 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 1 & 4 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 1 & 4 |
| *E. coli* BL21DE3 + pACYCTn5 | - | 1 & 2 |
| *E. coli* BL21DE3 + pACYCTn5 | pKantDpnI | 1 & 4 |
| *E. coli* BL21DE3 + pACYCTn5 | MEpKantMEDpnI | 1 & 4 |
| *E. coli* BL21DE3 + pACYCTn5 | pSB1K3 | 1 & 4 |

*\*Some samples were plated on several media. The media are displayed with the corresponding numbers:*

1. LB+Xgal+IPTG+Cam+Kan
2. LB+Xgal+IPTG+Cam
3. LB+Xgal+IPTG
4. LB+Kan
5. LB+Cam

Of every linear fragment used, 200ng was electroporated. Of every plasmid, 50ng was electroporated. 200 μl LB medium was added as recovery medium.

Close

Sunday, 16/09/2018

Date
:   16/9/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 14/9/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

*\*Some samples were plated on several media. The media are displayed with the corresponding numbers:*

1. LB+Xgal+IPTG+Cam+Kan
2. LB+Xgal+IPTG+Cam
3. LB+Xgal+IPTG
4. LB+Kan
5. LB+Cam

| Strain | Plasmid/fragment size (bp) | Medium\* | Observation |
| --- | --- | --- | --- |
| *E. coli* DH5alpha | pACYC-DUET | 2 | white phenotype![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/66/T--TUDelft--20180915-DH5alpha-pACYC.jpg) |
| *E. coli* BL21DE3 | - | 4 | no growth |
| *E. coli* BL21DE3 | - | 5 | no growth |
| *E. coli* BL21DE3 | - | 3 | ![](https://static.igem.org/mediawiki/2018/8/81/T--TUDelft--20180915-A-LBXgalIPTG.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | 1 | ![](https://static.igem.org/mediawiki/2018/8/81/T--TUDelft--20180915Bcamkan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | 2 | ![](https://static.igem.org/mediawiki/2018/3/3f/T--TUDelft--20180915Bcamxgal.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 1 | no growth![](https://static.igem.org/mediawiki/2018/2/2a/T--TUDelft--20180915Ccamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 4 | no growth![](https://static.igem.org/mediawiki/2018/f/f2/T--TUDelft--20180915Ckan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 1 | no growth![](https://static.igem.org/mediawiki/2018/a/a1/T--TUDelft--20180915Dcamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 4 | no growth![](https://static.igem.org/mediawiki/2018/7/7c/T--TUDelft--20180915Dkan.jpeg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 1 | ![](https://static.igem.org/mediawiki/2018/f/fa/T--TUDelft--20180915Ecamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 4 | ![](https://static.igem.org/mediawiki/2018/8/8d/T--TUDelft--20180915Ekan.jpeg) |
| *E. coli* BL21DE3 + pACYCTn5 | - | 1 | ![](https://static.igem.org/mediawiki/2018/d/d2/T--TUDelft--20180915Fcamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCTn5 | - | 2 | ![](https://static.igem.org/mediawiki/2018/2/2e/T--TUDelft--20180915Fcamxgal.jpeg) |
| *E. coli* BL21DE3 + pACYCTn5 | pKantDpnI | 1 | no growth![](https://static.igem.org/mediawiki/2018/c/cc/T--TUDelft--20180915Gcamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCTn5 | pKantDpnI | 4 | no growth![](https://static.igem.org/mediawiki/2018/f/f9/T--TUDelft--20180915Gkan.jpg) |
| *E. coli* BL21DE3 + pACYCTn5 | MEpKantMEDpnI | 1 | no growth![](https://static.igem.org/mediawiki/2018/5/59/T--TUDelft--20180915Hcamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCTn5 | MEpKantMEDpnI | 4 | no growth![](https://static.igem.org/mediawiki/2018/c/ca/T--TUDelft--20180915Hkan.jpg) |
| *E. coli* BL21DE3 + pACYCTn5 | pSB1K3 | 1 | ![](https://static.igem.org/mediawiki/2018/0/0a/T--TUDelft--20180915Icamkanxgal.jpg) |
| *E. coli* BL21DE3 + pACYCTn5 | pSB1K3 | 4 | ![](https://static.igem.org/mediawiki/2018/5/56/T--TUDelft--20180915Ikan.jpg) |

Close

[ ]
Week 3 17/09/2018 - 23/09/2018

Monday, 17/09/2018

Date
:   17/9/2018

Operator
:   Kavish Kohabir

#### Solid Medium Preparation

The following solid media were prepared:

+ Luria Broth (LB) + X-gal + IPTG
+ Luria Broth (LB) + X-gal + IPTG + Cam
+ Luria Broth (LB) + X-gal + IPTG + Cam + Kan

Final concentrations in these media are:

+ 0.1µmol IPTG per mL medium
+ 40 µg X-gal per mL medium

Date
:   17/9/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

A colony of BL21DE3+pACYCdxCas9Tn5*lacZ*gRNA was used for inoculation of 60mL liquid starter culture (Luria Broth, complemented with chloramphenicol) following the Liquid Starter Culture Protocol.

The idea was to inoculate a white phenotype colony (BL21DE3 + pACYCTn5 + MEpKantME, plated on LB+Xgal+IPTG+Cam+Kan, 15/9/2018). But the phenotype has changed over two days to form a red colony. **This was the first indication of what was going to become a hard time. Red colony phenotype formation is latent and can develop over 5 days.**

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Wednesday, 19/09/2018

Date
:   19/9/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

The following strains were used for inoculation of 30mL liquid starter cultures:

+ BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA

Preparation of the LB media (Luria Broth, complemented with cloramphenicol) was done following the Liquid Starter Culture Protocol.
The cultures were grown until OD0.5, prior to making them electrocompetent. The starter culture included IPTG induction from OD0.2 onwards.

Date
:   19/9/2018

Operator
:   Kavish Kohabir

#### Electrocompetent Cell Preparation

The following strains were made electrocompetent:

+ BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA
+ BL21DE3 + pACYCdxCas9Tn5
+ BL21DE3 + pAYCTn5

.

Washing steps were only done with cold, sterile MilliQ. The final resuspension was done with 50% glycerol.

Date
:   19/9/2018

Operator
:   Kavish Kohabir

#### Electroporation

As a second attempt, with 800ng-1µg DNA instead of 200ng (compared to 15/9/2018), the following transformations were carried out according to the protocol for electroporation. *It was assumed that some of the controls are not necessesary to repeat, since they have repeatedly shown to be consistent.*

| Strain | Plasmid/fragment size (bp) | Medium\* |
| --- | --- | --- |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | 1 & 2 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 1, 2 & 4 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 1, 2 & 4 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 1, 2 & 4 |

*\*Some samples were plated on several media. The media are displayed with the corresponding numbers:*

1. LB+Xgal+IPTG+Cam+Kan
2. LB+Xgal+IPTG+Cam
3. LB+Xgal+IPTG
4. LB+Kan
5. LB+Cam

Of every linear fragment used, 200ng was electroporated. Of every plasmid, 50ng was electroporated. 200 μl LB medium was added as recovery medium with 0.1mM IPTG.

Close

Thursday, 20/09/2018

Date
:   20/9/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 19/9/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed.
*\*Some samples were plated on several media. The media are displayed with the corresponding numbers:*

1. LB+Xgal+IPTG+Cam+Kan
2. LB+Xgal+IPTG+Cam
3. LB+Xgal+IPTG
4. LB+Kan
5. LB+Cam

| Strain | Plasmid/fragment size (bp) | Medium\* | Observation |
| --- | --- | --- | --- |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | 1 | ![](https://static.igem.org/mediawiki/2018/8/88/T--TUDelft--20180919Bcamkan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | - | 2 | ![](https://static.igem.org/mediawiki/2018/f/f6/T--TUDelft--20180919Bcam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 1 | ![](https://static.igem.org/mediawiki/2018/7/7f/T--TUDelft--20180919Ccamkan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 2 | ![](https://static.igem.org/mediawiki/2018/0/0c/T--TUDelft--20180919Cam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pKantDpnI | 4 | ![](https://static.igem.org/mediawiki/2018/8/8a/T--TUDelft--20180919Ckan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 1 | ![](https://static.igem.org/mediawiki/2018/c/cb/T--TUDelft--20180919Dcamkan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 2 | ![](https://static.igem.org/mediawiki/2018/b/b6/T--TUDelft--20180919Dcam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | MEpKantMEDpnI | 4 | ![](https://static.igem.org/mediawiki/2018/2/2e/T--TUDelft--20180919Dkan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 1 | ![](https://static.igem.org/mediawiki/2018/8/8e/T--TUDelft--20180919Ecamkan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 2 | ![](https://static.igem.org/mediawiki/2018/1/1d/T--TUDelft--20180919Ecam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1K3 | 4 | ![](https://static.igem.org/mediawiki/2018/e/ed/T--TUDelft--20180919Ekan.jpg) |

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Friday, 21/09/2018

Date
:   21/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify kanamycin with ME flanks and biobrick flanks (~1kb) for placing it into a biobrick, high fidelity PCR was done using pSB1K3 as template DNA and IV017 & IV018 as forward and reverse primers respectively. PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 50sec and an annealing temperature of 65°C. After the programme was finished, samples were kept at 4 °C.

Date
:   21/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

Biobrick flanked MepKantME was run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/3/36/T--TUDelft--20180921kavishkanRbiobirckk.jpg)

Date
:   21/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

Biobrick flanked PCR products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date
:   21/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| Biobrick flanked MEpKantME | 620 | 2.25 | 1.9 |

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Sunday, 23/09/2018

Date
:   23/9/2018

Operator
:   Kavish Kohabir

#### Restriction

Restriction cloning was done for assembling MEpKantME in pSB1C3. The following table shows an overview of what fragment(s) was/were cut with what restriction enzyme(s).

|  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) | Volume (uL) || 10x CutSmart buffer | 2 | 2 |
| Fragment | 3.5µl (2µg)  Biobrick flanked MEpKantME | 15µl (2µg)  pSB1C3 |
| Enzyme | 0.4µl EcoRI | 0.4µl EcoRI |
| Enzyme | 0.4µl SpeI | 0.4µl XbaI |
| MilliQ | 13.7 | 2.2 |

The samples were incubated for 4 hour(s) at 37 °C, and were not heat inactivated.

Date
:   23/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

restricted PCR products were subjected to PCR Clean-Up, according to the protocol. Elution was done in 30 µL Milli-Q.

Date
:   23/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| BBMEpKantMEBBEcoRI/SpeI | 75 | 1.9 | 1.5 |
| BBMEpKantMEBBEcoRI/SpeI | 1.9 | 1.7 |

Date
:   23/9/2018

Operator
:   Kavish Kohabir

#### Ligation

In order to assemble cut fragments ligation was performed. A 20µl ligation reaction mixture was prepared with the following composition

|  |  |  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Component | Volume (uL) || Ligase Buffer (10x) | 2 || T4 DNA Ligase | 1 || DNA vector (~100 ng) | 4.6 || DNA fragment (~500 ng) | 6.5 || MilliQ | 5.9 |

The samples were incubated overnight at 4°C for ligation.

Close

[ ]
Week 4 24/09/2018 - 30/09/2018

Monday, 24/09/2018

Date
:   24/9/2018

Operator
:   Kavish Kohabir

#### Transformation of chemically competent cells

The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | MEpKantME in pSB1C3\* | restriction-ligation | 2 | LB+Cam |
| *E.coli*DH5α | MEpKantME in pSB1C3\* | restriction-ligation | 2 | LB+Cam+Kan |
| *E.coli*DH5α | MEpKantME in pSB1C3\* | restriction-ligation | 18 | LB+Cam |
| *E.coli*DH5α | MEpKantME in pSB1C3\* | restriction-ligation | 18 | LB+Cam+Kan |
| *E.coli*DH5α | - | - | - | LB+Cam |
| *E.coli*DH5α | - | - | - | LB+Cam+Kan |

\*default RFP is NOT cut out of the backbone.

200 uL LB medium was added as recovery medium.

Close

Tuesday, 25/09/2018

Date
:   25/9/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 24/9/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strain | Plasmid/insert | Cloning method | Medium | Result |
| --- | --- | --- | --- | --- |
| *E.coli*DH5α | MEpKantME in pSB1C3\* | restriction-ligation | LB+Cam | ![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a0/T--TUDelft--20180924BBMeKantcam.jpg) |
| *E.coli*DH5α | MEpKantME in pSB1C3\* | restriction-ligation | LB+Cam+Kan | ![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/da/T--TUDelft--20180924kancam.jpg) |
| *E.coli*DH5α | - | - | LB+Cam | ![Gel electrophoresis](https://static.igem.org/mediawiki/2018/4/4b/T--TUDelft--20180924dh5alphacam.jpg) |
| *E.coli*DH5α | - | - | LB+Cam+Kan | No growth![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a3/T--TUDelft--20180925kavishDH5alphakancam.jpg) |

Date
:   25/9/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify construction f the right plasmid, and propagation thereof. This plasmid contains ME-flanked kanamycin and RFP, placed in a pSB1C3 backbone, which can be checked with primers VF2 and VR.

| Component | Volume (uL) |
| --- | --- |
| Template DNA | picked colony |
| Forward primer | VF2 |
| Reverse primer | VR |
| Expected amplicon size (bp) | 2454\* |

*\*We expect 1383bp in case of no placement of MEpKantME in the vector.*

Tubes were prepared for colony PCR, containing picked colony as template DNA. The thermocycler programme was installed to have an extension time of 3 minutes and an annealing temperature of 60°C. After the programme was finished, samples were kept at 4°C.

Date
:   25/9/2018dd>

    Operator
    :   Kavish Kohabir

#### DNA Gel Electrophoresis

colony PCR samples were run on a 0.8% agarose gel in TAE buffer. 5μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/5a/T--TUDelft--20180925colonypcrMEKanRME-RFP.png)

Based on this gel, it was decided to continue with colony 4 & 6 for further aclaration.

Date
:   25/9/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

Colony 4 & 6 of the transformant plate (24/9/2018) were used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with chloramphenicol AND kanamycin) following the Liquid Starter Culture Protocol.

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Wednesday, 26/09/2018

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### Plasmid Isolation

Overnight starter cultures of colony 4 & 6 were subjected to plasmid isolation following the Plasmid Isolation Protocol. Final elution was done with 30 μL of pre-warmed MilliQ.

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were verified to be around 1.8~1.9.

|  |  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
| Isolate | Concentration (ng/uL) || pSB1C3-RFP-MEpKantME colony 4 | 264 |
| pSB1C3-RFP-MEpKantME colony 4 | 250 |
| pSB1C3-RFP-MEpKantME colony 6 | 252 |
| pSB1C3-RFP-MEpKantME colony 6 | 229 |

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### Sequence Verification

##### Option 1: Sequence from plasmid

For verification of MEpKantME being inserted into the pSB1C3, next to RFP, primers VF2, IV020 and VR were used for sequencing. The sequencing starts from the biobrick backone. The 10µL sequencing sample was prepared in a 1.5 mL tube as follows:

| Component | Volume (uL) | Final concentration |
| --- | --- | --- |
| Plasmid DNA (500ng) | 3 | 50 ng/uL |
| 10uM primer | 2.5 | 25 pmol |
| MilliQ | 4.5 | - |

Within 7 days, the sequence was retrievable online and alignment with the *in silico* designed molecule showed 100% match of the 1050 bp downstream of the sequencing primer binding site.

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### High Fidelity PCR

To amplify MEpKantME with long arms (3560bp), high fidelity PCR was done using pSB1C3-RFP-MEpKantME as template DNA and IV023 & VR as forward and reverse primers respectively. PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 3.5 minutes and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### DNA Gel Electrophoresis

The extended-arm MEpKantME fragments were run on a 0.8% agarose gel in TAE buffer. 3μL was pipetted into each lane. The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f7/T--TUDelft--20180926LONGFLANKEDFRAGMENTAHAHAAAHAAHHHAAHAHAHAHAA.jpg)

This gel shows that this amplification worked as designed. The Kanamyin cassette is now protected better against exonuclease degradation of the ME flanks. (approximately 1200bp spacer arms).

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

PCR products of long-armed MEpKantME were subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| long-armed MEpKantME (colony 4) | 1342 | 1.87 | 2.30 |
| long-armed MEpKantME (colony 6) | 1506 | 1.86 | 2.29 |
|  |
|  |

Date
:   26/9/2018

Operator
:   Kavish Kohabir

#### DpnI Digestion

To eliminate any residual template DNA, a DpnI digestion was performed with the following reaction mixture:

| Component | Volume (uL) |
| --- | --- |
| 10x CutSmart buffer | 5 |
| PCR product | 34 (~45-50µg) |
| Enzyme (DpnI) | 1 |
| MilliQ | 10 |

The reaction was incubated overnight at 37 °C. No heat inactivation was performed.

Close

Thursday, 27/09/2018

Date
:   27/9/2018

Operator
:   Kavish Kohabir

#### PCR clean-up

50 μL of restriction product was subjected to PCR Clean-Up, according to the protocol. Elution was done in 40 µL Milli-Q.

Date
:   27/9/2018

Operator
:   Kavish Kohabir

#### Nanodrop DNA Quantification

The following isolates were quantified according to the Nanodrop DNA Quantification protocol. The concentration and purity of the DNA (260/280 and 260/230 ratios) were measured.

| Isolate | Concentration (ng/uL) | 260/280 ratio | 260/230 ratio |
| --- | --- | --- | --- |
| long-armed MEpKantMEDpnI (colony 4) | 496 | 1.86 | 2.24 |
| long-armed MEpKantMEDpnI (colony 6) | 588 | 1.85 | 2.22 |
|  |
|  |

Date
:   27/9/2018

Operator
:   Kavish Kohabir

#### Electroporation

The following transformations were carried out according to the protocol for electroporation:

| Strains | Plasmid/fragment size (bp) | Amount added | Plate medium |
| --- | --- | --- | --- |
| *E. coli* BL21DE3 | pSB1C3-MEpKantME-RFP | 50ng | 1 & 2 |
| *E. coli* BL21DE3 | long-armed MEpKantMEDpnI | 1µg | 1 & 2 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1C3-MEpKantME-RFP | 50ng | 1, 2 & 3 |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | long-armed MEpKantMEDpnI | 1µg | 1, 2 & 3 |

*\*Some samples were plated on several media. The media are displayed with the corresponding numbers:*

1. LB+Xgal+IPTG+Cam+Kan
2. LB+Xgal+IPTG+Cam
3. LB+Kan

200μl LB medium was added as recovery medium.

Close

Friday, 28/09/2018

Date
:   28/9/2018

Operator
:   Kavish Kohabir

#### Transformation Results

After carrying out the transformation protocol on 27/9/2018, the plates were left for overnight incubation. After ~14-18hr incubation, the following results were observed:

| Strains | Plasmid/fragment size (bp) | Plate medium | Observation |
| --- | --- | --- | --- |
| *E. coli* BL21DE3 | pSB1C3-MEpKantME-RFP | 1 | ![](https://static.igem.org/mediawiki/2018/6/6b/T--TUDelft--20180928pBL21DE3pSB1C3MEpKantME-RFP_XgalIPTGCamKan.jpg) |
| *E. coli* BL21DE3 | pSB1C3-MEpKantME-RFP | 2 | ![](https://static.igem.org/mediawiki/2018/3/3c/T--TUDelft--20180928pBL21DE3pSB1C3MEpKantME-RFP_XgalIPTGCam.jpg) |
| *E. coli* BL21DE3 | long-armed MEpKantMEDpnI | 1 | ![](https://static.igem.org/mediawiki/2018/1/1b/T--TUDelft--20180928pBL21DE3longarmedMEpKant_XgalIPTGCamKan.jpg) |
| *E. coli* BL21DE3 | long-armed MEpKantMEDpnI | 2 | ![](https://static.igem.org/mediawiki/2018/8/8b/T--TUDelft--20180928pBL21DE3longarmedMEpKant_XgalIPTGCam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1C3-MEpKantME-RFP | 1 | ![](https://static.igem.org/mediawiki/2018/f/ff/T--TUDelft--20180928BL21DE3pACYCdxCas9Tn5lacZgRNApSB1C3MEpKanME-RFP_XgalIPTGCamKan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1C3-MEpKantME-RFP | 2 | ![](https://static.igem.org/mediawiki/2018/e/e1/T--TUDelft--20180928BL21DE3pACYCdxCas9Tn5lacZgRNApSB1C3MEpKanME-RFP_XgalIPTGCam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | pSB1C3-MEpKantME-RFP | 3 | ![](https://static.igem.org/mediawiki/2018/6/6e/T--TUDelft--20180928BL21DE3pACYCdxCas9Tn5lacZgRNApSB1C3MEpKanME-RFP_Kan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | long-armed MEpKantMEDpnI | 1 | ![](https://static.igem.org/mediawiki/2018/e/e7/T--TUDelft--20180928BL21DE3pACYCdxCas9Tn5lacZgRNAlongflanksMEpKanME-RFP_XgalIPTCamKan.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | long-armed MEpKantMEDpnI | 2 | ![](https://static.igem.org/mediawiki/2018/8/86/T--TUDelft--20180928BL21DE3pACYCdxCas9Tn5lacZgRNAlongflanksMEpKanME-RFP_XgalIPTCam.jpg) |
| *E. coli* BL21DE3 + pACYCdxCas9Tn5*lacZ*gRNA | long-armed MEpKantMEDpnI | 3 | ![](https://static.igem.org/mediawiki/2018/a/a3/T--TUDelft--20180928BL21DE3pACYCdxCas9Tn5lacZgRNAlongflanksMEpKanME-RFP_Kan.jpg) |

*\*Some samples were plated on several media. The media are displayed with the corresponding numbers:*

1. LB+Xgal+IPTG+Cam+Kan
2. LB+Xgal+IPTG+Cam
3. LB+Kan

Some of the colonies had white phenotype when observing them after 14-18h incubation. However, judging from previous observations, it is not excluded that RFP might come to expression at a later stage. Nevertheless, these colonies were inoculated for further identification.

Date
:   28/9/2018

Operator
:   Kavish Kohabir

#### Liquid Starter Culture Preparation

White colonies from LB+Cam+Kan+IPTG+Xgal plates were used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with chloramphenicol & kanamycin) following the Liquid Starter Culture Protocol.

Date
:   28/9/2018

Operator
:   Kavish Kohabir

#### Colony PCR

Colony PCR was done to verify whether *lacZ* was really disrupted. The following rimer setup was used:

![](https://static.igem.org/mediawiki/2018/4/49/T--TUDelft--2018_invivoprimers.png)

With this setup, the following amplicon sizes are expected:

| Primers | No integration | Integration |
| --- | --- | --- |
| IV007 & IV019 | no band | ?\* |
| IV020 & IV008 | no band | ?\* |
| IV007 & IV022 | 2180bp | 3218bp |
| IV021 & IV008 | 2658bp | 3696bp |

\*This amplicon size depends on the integration site of the donor DNA. The maximum expected size is 5kb.

23 colonies were used for doing 4 PCR reactions each + a control (isolated parental BL21DE3 gDNA for each PCR; 92 reactions total. Resuspende colony served as template DNA for each reaction. The thermocycler programme was installed to have an extension time of 5min and an annealing temperature of 60 °C. After the programme was finished, samples were kept at 4°C.

![](https://static.igem.org/mediawiki/2018/0/01/T--TUDelft--20180928MEGAMASSAHUGEIMMENSECOLONYPCRHEUJJJJJJ1.JPG)
![](https://static.igem.org/mediawiki/2018/d/df/T--TUDelft--20180928MEGAMASSAHUGEIMMENSECOLONYPCRHEUJJJJJJ2.JPG)
![](https://static.igem.org/mediawiki/2018/b/ba/T--TUDelft--20180928MEGAMASSAHUGEIMMENSECOLONYPCRHEUJJJJJJ3.JPG)

Close

* June
  + [Week: 25/06/2018 - 01/06/2018](#JunePurification4)
* July
  + [Week: 02/07/2018 - 08/07/2018](#JulyPurification1)
  + [Week: 09/07/2018 - 15/07/2018](#JulyPurification2)
  + [Week: 16/07/2018 - 22/07/2018](#JulyPurification3)
  + [Week: 23/07/2018 - 29/07/2018](#JulyPurification4)
* August
  + [Week: 30/07/2018 - 05/08/2018](#AugustPurification1)
  + [Week: 06/08/2018 - 12/08/2018](#AugustPurification2)
  + [Week: 13/08/2018 - 19/08/2018](#AugustPurification3)
  + [Week: 20/08/2018 - 27/08/2018](#AugustPurification4)
  + [Week: 28/08/2018 - 02/09/2018](#AugustPurification5)
* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberPurification1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberPurification2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberPurification3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberPurification4)
* October
  + [Week: 01/10/2018 - 07/10/2018](#OctoberPurification1)
  + [Week: 08/10/2018 - xx/10/2018](#OctoberPurification2)

### June

[ ]
Week 4 25/06/2018 - 01/07/2018

Friday, 29/06/2018

Date
:   29/06/2018

Operator
:   Monique de Leeuw

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5 protein. A colony with Tn5\_3 strain was used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Saturday, 30/06/2018

Date
:   30/06/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 protein continued. Inoculated 1L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 12:00am | 0.09 |
| 1:00pm | 0.2 |
| 2:00pm | 0.49 |

After the culture OD600 reached 0.49, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Sunday, 01/07/2018

Date
:   01/07/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. 5.71g of cells of Tn5 protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 80uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| imidazole | 2M | 400uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/56/T--TUDelft--2018_Figure1_01072018.png)

**Figure expression of Tn5:**  *12% SDS PAGE of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane clarified lysate, lane 3 cell debris.
.*

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. 33.5mL clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 20mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/bd/T--TUDelft--2018_Figure2_01072018.png)

**Figure Nickel Affinity Chromatography.**   *12% SDS of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane clarified lysate, lane 3 cell debris, lane 4 flow through, lane 5 wash1, lane 6 wash2, lane 7 Elution 1, lane 8 Elution 2, lane 9 Elution 3, lane 10 Elution 4.*

No Tn5 was observed in the lysate or the elution fraction. At first, we thought an error occurred during expression, however after thorough analysis we identified our mistake. There was a miss communication between stain construction and bioprocess as a result the plasmid was in the incorrect host organism. The Tn5 plasmid was still in DH5 alpha. Going forward, all transformation into expression host will be covered by the bioprocess team, to avoid this mistake in the future.

Close

### July

[ ]
Week 1 02/07/2018 - 08/07/2018

Monday, 02/07/2018

Date
:   02/07/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The Tn5\_3 plasmid was transfomred into BL21AI for Tn5 protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | Tn5.3 (5210bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Tuesday, 03/07/2018

Date
:   03/07/2018

Operator
:   Monique de Leeuw

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5 protein. A colony with Tn5\_3 strain was used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Wednesday, 04/07/2018

Date
:   04/07/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 protein continued. Inoculated 1L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 10:30am | 0.12 |
| 11:30am | 0.25 |
| 12:30am | 0.56 |

After the culture OD600 reached 0.56, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Thursday, 05/07/2018

Date
:   05/07/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. 5.87g of cells of Tn5 protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 82uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| imidazole | 2M | 411uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f5/T--TUDelft--2018_Figure1A_05072018.png)

**Figure expression of Tn5:**  *12% SDS PAGE of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate.
.*

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. 33.5mL clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 20mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/0/04/T--TUDelft--2018_Figure2A_05072018.png)

**Figure Nickel Affinity Chromatography.**   *12% SDS of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 flow through, lane 5 wash1, lane 6 Elution 1, lane 7 Elution 2, lane 8 Elution 3, lane 9 Elution 4 , lane 10 elution 5*

A significant amount of Tn5 was observed in the nickel chromatography elution fractions (lane 6-9), however the Tn5 protein was also observed in the flowthrough and wash fractions (lane 4,5). It is recommended to optimize the nickel chromatography to reduce these losses, however a bigger consern is a double band for Tn5 is observed. One at the expected weight, and one slightly higher. It was unclear why the Tn5 would run higher. As a following step, elution fraction 1 (lane 6) was run under different denaturing conditions to fully denature the protein.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the degradation of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/8b/T--TUDelft--2018_Figure3_16082018.png)

**Figure Different sample gel preparation conditions.**   *12% SDS PAGE of Tn5 (expected size 53.3kDa) Lane 1 molecular ladder (kDa), lane 2 950C 30min, lane 3 950C 15min, lane 4 750C 7min, lane 5 No boiling.*

A double band for Tn5 observed for all conditions, both bands from the 12% gel were excised and sent for mass spectrometry. The mass spectrometry confirmed that both bands were full length Tn5 with both the N and C terminal intact. This concluded that there was not any protease digestion and that the results are potentially due to a gel artifact. As a result, the purification will continue.

Close

[ ]
Week 2 09/07/2018 - 15/07/2018

Monday, 09/07/2018

Date
:   09/07/2018

Operator
:   Janine Nijenhuis

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5. A colony with Tn5 strain was used for inoculation of 40mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Date
:   07/09/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Heparin Chromatography*

The Tn5 protein was further purified with Heparin chromatography. The 1mL HiTrap Heparin HP column was connected to the AKTA pure in the cold room at 4 ℃. 2.5mL of nickel chromatography elution fraction 1-5 from Tn5 protein was injected into the injection loop and the chromatography was followed according to the Purification Protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the heparin chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 20% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/8e/T--TUDelft--2018_Figure1_06072018.png)

**Figure Heparin Chromatography.**   *: 20% SDS PAGE of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane 2 starting material, lane 3 flow through, lane 4 wash, lane 5 elution 1, lane 6 elution 2, lane 7 elution 3, lane 8 elution 4, lane 9 elution 5, lane 10 elution 6, lane 11 elution 7, lane 12 elution 8, lane 13 elution 9, lane 14 elution 10, lane 15 elution 11.*

The heparin chromatography was successful. No losses were observed in the flowthrough or wash (ane 3-4) of the run, resulting in high recoveries. The first 4 elution fractions (lane 5-8) contained some low molecular weight contaminating proteins and therefore only elution fraction 6 to 11 (lane 10 to 15) were pooled.

Close

Tuesday, 10/07/2018

Date
:   10/07/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 continued. Inoculated 800 mL liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.6.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 09:23 | 0.01 |
| 10:20 | 0.07 |
| 11:18 | 0.25 |
| 12:01 | 0.61 |

After the culture OD600 reached 0.61, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Wednesday, 11/07/2018

Date
:   11/07/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. The cells of Tn5 protein were resuspended in 30 mL Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 120uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1 tablet |
| imidazole | 2M | 75uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 20mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 20% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/54/T--TUDelft--2018_NBJanine20180713.png)

**Figure Nickel Affinity Chromatography.**   *20% SDS PAGE of Tn5 (expected size 53.3kDa) Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 cell debris, lane 5 not bound lysate, lane 6 flow through, lane 7 wash, lane 8 elution 1, lane 9 elution 2, lane 10 elution 3, lane 11 elution 4, lane 12 elution 5, lane 13 elution 6.*

The nickel chromatography was not completely successful, but there is some purification of Tn5. The Tn5 did not completely bind to the nickel resin and was observed in the flowthrough and wash (lane 5-7). It is recommended to further optimize the nickel chromatography to further reduce losses, and reduce contaminating proteins due to non-specific binding to the nickel resin (lane 8-10). However for our purposes, the losses were tolerated. Elution fractions 1 to 3 were pooled (lane 8-10).

Close

Thursday, 12/07/2018

Date
:   12/07/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The dxcas9\_19 plasmid was transfomred into BL21AI for dxCas9 protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | dxcas9\_19 (7967bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Sunday, 15/07/2018

Date
:   15/07/2018

Operator
:   Monique de Leeuw

#### Objective

dxCas9 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9 protein. A colony with dxcas9-linker-Tn5\_9 strain was used for inoculation of 20mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

[ ]
Week 3 16/07/2018 - 22/07/2018

Monday, 16/07/2018

Date
:   16/07/2018

Operator
:   Janine Nijenhuis

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5. A colony with Tn5 strain was used for inoculation of 40mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Date
:   16/07/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9 protein continued. Inoculated 1L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 11:00am | 0.14 |
| 12:10am | 0.34 |
| 1:10am | 0.54 |

After the culture OD600 reached 0.54, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Tuesday, 17/07/2018

Date
:   17/07/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 continued. Inoculated 800 mL liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.6.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 12:20 | 0.23 |
| 13:05 | 0.25 |
| 14:10 | 0.64 |

After the culture OD600 reached 0.64, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Date
:   24/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9 protein started. Harvest the cells according to the purification protocol. 9.4g of cells of dxCas9 protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 131.6uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| imidazole | 2M | 658uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis]()

**Figure dxCas9 Expression:**  *12% SDS PAGE of dxCas9 (expected size 158.3kDa). Lane 1 molecular ladder (kDa), Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate.*

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. 51.2mL clarified lysate of dxCas9 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 20mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/a/a2/T--TUDelft--2018_Figure4_17072018.png)

**Figure Nickel Affinity Chromatography.**   *12% SDS PAGE of dxCas9 (expected size 158.3kDa) Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 flow through 1, lane 5 flow through 2, lane 6 flow through 3, lane 7 wash 1, lane 8 elution 1, lane 9 elution 2, lane 10 elution 3, lane 11 elution 4, lane 12 elution 5, lane 13 elution 6, lane 14 elution 1.*

The nickel chromatography was not successful. The dxCas9 did not bind to the nickel resin and was observed in the flowthrough and wash (lane 4-7). It is recommended to optimize the nickel chromatography by reducing the amount of imidazole in the binding buffer to 5mM for the next experiment.

Close

Wednesday, 18/07/2018

Date
:   18/07/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. The cells of Tn5 protein were resuspended in 30 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 60uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1 tablet |
| imidazole | 2M | 75uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 20mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain. Sadly no picture was taken. However, the purification was succesfull.

Close

### August

[ ]
Week 2 06/08/2018 - 12/08/2018

Monday, 06/08/2018

Date
:   06/08/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The dxcas9\_19 plasmid was transfomred into BL21AI for dxCas9 protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | dxcas9\_19 (7967bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Tuesday, 07/08/2018

Date
:   07/08/2018

Operator
:   Monique de Leeuw

#### Objective

dxCas9 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9 protein. A colony with dxcas9-linker-Tn5\_9 strain was used for inoculation of 20mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Wednesday, 08/08/2018

Date
:   08/08/2018

Operator
:   Janine Nijenhuis

#### Objective

dxCas9-Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9-Tn5. A colony with dxCas9-Tn5\_9 strain was used for inoculation of 40mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Date
:   08/08/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9 protein continued. Inoculated 1L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 10:30am | 0.20 |
| 11:30am | 0.38 |
| 12:00am | 0.52 |

After the culture OD600 reached 0.52, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Thursday, 09/08/2018

Date
:   09/08/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9-Tn5 continued. Inoculated 2L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.6.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 12:50 | 0.23 |
| 13:30 | 0.25 |
| 14:10 | 0.47 |
| 14:20 | 0.58 |

After the culture OD600 reached 0.58, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Date
:   09/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9 protein started. Harvest the cells according to the purification protocol. 4.4g of cells of dxCas9 protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 61.6uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| imidazole | 2M | 77uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/4/48/T--TUDelft--2018_Figure1_17072018.png)

**Figure dxCas9 Expression:**  *12% SDS PAGE of dxCas9 (expected size 158.3kDa). Lane 1 molecular ladder (kDa), lane 2 pre-induced, lane 3 whole lysate, lane 4 clarified lysate, lane 5 cell debris.*

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. 29mL clarified lysate of dxCas9 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 5mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/b/b3/T--TUDelft--2018_Figure2_17072018.png)

**Figure Nickel Affinity Chromatography.**   *12% SDS PAGE of dxCas9 (expected size 158.3kDa) Lane 1 molecular ladder (kDa), lane 2 pre-induction, lane 3 whole lysate, lane 4 clarified lysate, lane 5 cell debris, lane 6 flow through, lane 7 wash 1, lane 8 wash 2, lane 9 elution 1, lane 10 elution 2, lane 11 elution 3, lane 12 elution 4, lane 13 elution 5, lane 14 elution 6.*

The nickel chromatography was not completely successful, but improved significantly after an the concentration of imidazole in the binding buffer was reduced to 5mM. The dxCas9 did not completely bind to the nickel resin and was observed in the flowthrough and wash (lane 6-8). It is recommended to further optimize the nickel chromatography to further reduce losses, and reduce contaminating proteins due to non-specific binding to the nickel resin (lane 9-10). However for our purposes, the losses were tolerated, and only elution fractions 3 to 6 were pooled (lane 11-14). Additionally, a double band for dxCas9 is observed. The purification will be continued however, we will look deeper into this observation. Is this double band due to degradation, or some other interaction with the dxcas9? We are working with technicians on performing mass spectrometry.

Close

Friday, 10/07/2018

Date
:   10/08/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9-Tn5 protein started. Harvest the cells according to the purification protocol. The cells of dxCas9-Tn5 protein were resuspended in 30 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 60uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1 tablet |
| imidazole | 2M | 75uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of dxCas9-Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 5mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the dxCas9-Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain. Sadly no picture was taken. The results showed that the fusion protein would not bind to the column material. It was decided to do another purification, but with a lower concentration of Imidazole.

Date
:   10/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Heparin Chromatography*

The dxCas9 protein was further purified with Heparin chromatography. The 1mL HiTrap Heparin HP column was connected to the AKTA pure in the cold room at 4 ℃. 2mL of nickel chromatography elution fraction 3-6 from dxCas9 protein was injected into the injection loop and the chromatography was followed according to the Purification Protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the heparin chromatography of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/6f/T--TUDelft--2018_Figure1_18072018.png)

**Figure Heparin Chromatography.**   *12% SDS PAGE of dxCas9 (expected size 158.3kDa). Lane 1 molecular ladder (kDa), lane 2 starting material, lane 3 flow through, lane 4 wash, lane 5 elution 1, lane 6 elution 2, lane 7 elution 3, lane 8 elution 4, lane 9 elution 5, lane 10 elution 6, lane 11 elution 7, lane 12 elution 8, lane 13 elution 9, lane 14 elution 10, lane 15 elution 11.*

The heparin chromatography was successful. The protein recoveries were high, with no losses in the flowthrough and wash (lane 3-4). The first 4 elution fractions (lane 5-8) contained some low molecular weight contaminating proteins, as a result only elution fraction 5 to 10 (lane 9 to 14) were pooled. Again the double band for dxCas9 was observed on the 12% SDS PAGE, which we have seen with all purification.

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Saturday, 11/08/2018

Date
:   11/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Dialysis*

Finally the dxCas9 protein is dialysed and concentrated. 3mL of sample (Heparin chromatography elution fraction 5-10 of dxCas9 protein) were dialysed according to the purification protocol with dialysis buffer (20 mM Tris-HCl, 100 mM KCl, 5% glycerol, 1mM beta-Mercaptoethanol, pH 7.5). After dialysis, the sample of dxCas9 protein was stored at -20℃, according to the purification protocol.

In order to analyse the concentrated dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/4/47/T--TUDelft--2018_Figure1_19072018.png)

**Figure Final protein:**  *8% SDS PAGE of dxCas9 (expected size 158.3kDa). Lane 1 molecular ladder (kDa), lane 2 final dxCas9 post dialysis.*

Note the dxCas9 is only one band on the 8% PAGE. The purification will be continued however, we will look deeper into this observation. What causes this double band? Is there degradation, or some other interaction with the dxcas9? Also what happened during the final dialysis step to remove the double band? Trace amounts of contaminating background is observed, however dxCas9 is significantly more in quantity and the background was tolerated for functionality testing. The final concentration of the solution was determined according to the BCA protocol on our wiki and resulted in a concentration of 0.46mg/mL or 2.88µM. The

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Sunday, 12/08/2018

Date
:   12/08/2018

Operator
:   Janine Nijenhuis

#### Objective

dxCas9-Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9-Tn5. Two colonies with dxCas9-Tn5\_9 strain were used for inoculation of two 20mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

[ ]
Week 3 13/08/2018 - 19/08/2018

Monday, 13/08/2018

Date
:   13/08/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9-Tn5 continued. Inoculated 2L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.6.

**Table :** Growth represented by OD600 over time

| Time | Col 1 OD600 | Col 2 OD600 |
| 12:00 | 0.06 | 0.05 |
| 12:50 | 0.08 | 0.08 |
| 13:50 | 0.28 | 0.30 |
| 14:30 | 0.56 | 0.56 |

After both cultures OD600 reached 0.56, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Date
:   13/08/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The dxcas9\_19 plasmid was transfomred into BL21AI for dxCas9 protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | dxcas9\_19 (7967bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Tuesday, 14/08/2018

Date
:   14/08/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9-Tn5 protein started. Harvest the cells of the cultures of col 1 and col 2 according to the purification protocol. The cells of both cultures with dxCas9-Tn5 protein were resuspended in 24 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added for both cultures:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 60uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1 tablet |
| imidazole | 2M | 75uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

Two times 1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of dxCas9-Tn5 protein of each colony was added to one of the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resins were washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 1mM DTT, pH8) and 0.5mM Imidazole for Col 1 and 0mM Imidazole for Col 2, and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the dxCas9-Tn5 protein of both colonies, two precast Tris-Glycine polyacrylamide gel were run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f9/T--TUDelft--2018_NBJanine14082018.png)

**Figure Nickel Affinity Chromatography Col 1.**   *8% SDS PAGE of dxCas9-Tn5 (expected size 214.7kDa). Lane 1 high range molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 cell debris, lane 5 flow through, lane 6 wash, lane 7 elution 1, lane 8 elution 2, lane 9 elution 3, lane 10 elution 4, lane 11 elution 5, lane 12 elution 6.*

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/5d/T--TUDelft--2018_NBJanine14082018_2.png)

**Figure Nickel Affinity Chromatography Col 2.**   *8% SDS PAGE of dxCas9-Tn5 (expected size 214.7kDa). Lane 1 high range molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 cell debris, lane 5 flow through, lane 6 wash, lane 7 elution 1, lane 8 elution 2, lane 9 elution 3, lane 10 elution 4, lane 11 elution 5, lane 12 elution 6, lane 13 cell debris.*

As the two figures show, even with decreasing the concentration of Imidazole, no purification of dxCas9-Tn5 happened. The fusion protein broke apart. This can already be seen in the samples before purification (figure col 1 lane 2-5, figure col 2 lane 2-5). The conclusion is that the fusion protein probably breaks at the linke, due to two bands at approximatly the same height as dxCas9 and Tn5. Another purification will be done, now with a gradient elution.

Date
:   14/08/2018

Operator
:   Monique de Leeuw

#### Objective

dxCas9 was expressed and purified to produce samples for mass spectrometry and other analysis to identify why there are two bands for the dxCas9 protein.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9 protein. A colony with dxcas9-linker-Tn5\_9 strain was used for inoculation of 20mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Wednesday, 15/08/2018

Date
:   15/08/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9 protein continued. Inoculated 12L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 10:30am | 0.16 |
| 11:30am | 0.34 |
| 12:00am | 0.56 |

After the culture OD600 reached 0.56, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Thursday, 16/08/2018

Date
:   16/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9 protein started. Harvest the cells according to the purification protocol. 8.4g of cells of dxCas9 protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 117.6uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| imidazole | 2M | 147uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/5/51/T--TUDelft--2018_Figure1_16082018.png)

**Figure dxCas9 Expression:**  *12% SDS PAGE of dxCas9 (expected size 158.3kDa). Lane 1 molecular ladder (kDa), lane 2 pre-induction, lane 3 whole lysate, lane 4 clarified lysate.*

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. 49mL clarified lysate of dxCas9 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 5mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/cf/T--TUDelft--2018_Figure2_16082018.png)

**Figure Nickel Affinity Chromatography.**   *12% SDS PAGE of dxCas9 (expected size 158.3kDa) Lane 1 molecular ladder (kDa), lane 2 pre-induction, lane 3 whole lysate, lane 4 clarified lysate, lane 5 flow through, lane 6 elution 1, lane 7 elution 2, lane 8 elution 3.*

The nickel chromatography was reproducible, and again a double band for dxCas9 is observed. One at the expected weight, and one slightly higher. It was unclear why the dxCas9 would run higher. As a following step, elution fraction 1 (lane 6) was run on a 6% SDS PAGE gel under different denaturing conditions for better resolution.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the degradation of the dxCas9 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 6% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/8b/T--TUDelft--2018_Figure3_16082018.png)

**Figure Different sample gel preparation conditions.**   *12% SDS PAGE of dxCas9 (expected size 158.3kDa) Lane 1 molecular ladder (kDa), lane 2 95 deg C 60min, lane 3 95 deg C 30min, lane 4 95 deg C 15min, lane 5 95 deg C 5min, lane 75 deg C 7min, lane 7 No boiling, lane 8 molecular ladder (kDa).*

On the 6% tris-glycine SDS PAGE a single band for dxCas9 was observed for all conditions, suggesting that the double band might be some artifact of the 12% SDS PAGE. To further confirm these results, both bands from the 12% gel were excised and sent for mass spectrometry. The mass spectrometry confirmed that both bands were full length dxCas9 with both the N and C terminal intact. This concluded that there was not any protease digestion and that the results are potentially due to a gel artifact. As a result, the purification will continue.

Close

Sunday, 19/08/2018

Date
:   19/08/2018

Operator
:   Janine Nijenhuis

#### Objective

dxCas9-Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9-Tn5. A colony with dxCas9-Tn5\_9 strain was used for inoculation of 40mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

[ ]
Week 4 20/08/2018 - 26/08/2018

Monday, 20/08/2018

Date
:   20/08/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9-Tn5 continued. Inoculated 2L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.3.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 11:00 | 0.06 |
| 12:20 | 0.10 |
| 13:25 | 0.35 |

After OD600 reached 0.35, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Tuesday, 21/08/2018

Date
:   21/08/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9-Tn5 protein started. Harvest the cells according to the purification protocol. The cells with dxCas9-Tn5 protein were resuspended in 30 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added for both cultures:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 60uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1 tablet |
| imidazole | 2M | 75uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of dxCas9-Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resins were washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 0.5mM Imidazole, 1mM DTT, pH8), and eluded with with 9\* 500μL (6CV) of elution buffer with an increasing concentration of Imidazole (starting from 0.5mM to 250mM) (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the dxCas9-Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/cb/T--TUDelft--2018_NBJanine20180821.png)

**Figure Nickel Affinity Chromatography.**   *8% SDS PAGE of dxCas9-Tn5 (expected size 214.7kDa). Lane 1 high range molecular ladder (kDa), lane 2 whole lysate, lane 3 cell debris, lane 4 clarified lysate, lane 5 flow through, lane 6 wash, lane 7 elution 1, lane 8 elution 2, lane 9 elution 3, lane 10 elution 4, lane 11 elution 5, lane 12 elution 6, lane 13 elution 7, lane 14 elution 8, lane 15 elution 9.*

As the figure shows, even with a increasing concentration of Imidazole during elution, the fusion protein breaks down. This can be seen in elution fractions 3-8 (lane 9-14). The conclusion is that another method needs to be thought of to purify the fusion protein.

Date
:   21/08/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The dxcas9-linker-Tn5\_9 plasmid was transfomred into BL21AI for fusion protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | dxcas9-linker-Tn5\_9 (9432bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Wednesday, 22/08/2018

Date
:   22/08/2018

Operator
:   Monique de Leeuw

#### Objective

Fusion was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of fusion protein. A colony with dxcas9\_linker-Tn5\_9 strain was used for inoculation of 20mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Thursday, 23/08/2018

Date
:   23/08/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the fusion protein continued. Inoculated 2L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 10:50am | 0.28 |
| 11:25am | 0.34 |
| 12:00am | 0.58 |

After the culture OD600 reached 0.58, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Friday, 24/08/2018

Date
:   24/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the fusion protein started. Harvest the cells according to the purification protocol. 7.6g of cells of fusion protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 7.5).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 106uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| PMSF | 0.2M | 266uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/e8/T--TUDelft--2018_Figure1_24082018.png)

**Figure Fusion Expression:**  *12% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 cell debris, lane 5 clarified lysate.*

#### Protein Purification - *Heparin Chromatography*

The fusion protein was further purified with Heparin chromatography. The 1mL HiTrap Heparin HP column was connected to the AKTA pure in the cold room at 4 ℃. 42.4mL of crude lysate from fusion protein was injected into the injection loop and the chromatography was followed according to the Purification Protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the heparin chromatography of the fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/9a/T--TUDelft--2018_Figure2_24082018.png)

**Figure Heparin Chromatography.**   *8% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 elution 1, lane 3 elution 2, lane 4 elution 3, lane 5 elution 4, lane 6 elution 5, lane 7 elution 6, lane 8 elution 7, lane 9 elution 8, lane 10 elution 9, lane 11 elution 10, lane 12 elution 11, lane 13 elution 12.*

Degradation was suspected for our fusion protein. Results seem to suggest that the fusion protein is being cleaved at the linker, resulting in both the dxCas9 and the fusion protein being present in our sample. The heparin chromatography was relatively successful at separating the two populations. The dxCas9 has a lower affinity to the heparin resin as a result it elutes at lower concentrations of salt (lane 2-10). As the concentration of salt increases less dxCas9 and more fusion elutes (lane 6-10). At high levels of salt the mostly fusion elutes (lane 11-13). Only these last three elution fractions are pooled (lane 11-13). Thus losses are tolerated, to obtain a relatively pure fusion fraction which can be further purified by MonoQ.

Close

Saturday, 25/08/2018

Date
:   25/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification *- MonoQ Chromatography*

The fusion protein was further purified with MonoQ chromatography. The 1mL MonoQ 5/50 GL column was connected to the AKTA pure in the cold room at 4 ℃. 6mL of sample (4x diluted heparin chromatography elution fraction 10-12 of fusion protein) was injected into the injection loop and the chromatography was followed according to the Purification Protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse monoQ of the fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/6b/T--TUDelft--2018_Figure1_25082018.png)

**Figure MonoQ chromatography:**  *8% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 starting material, lane 3 flow through, lane 4 wash, lane 5 elution 1, lane 6 elution 2, lane 7 elution 3, lane 8 elution 4, lane 9 elution 5, lane 10 elution 6, lane 11 elution 7, lane 12 elution 8, lane 13 elution 9, lane 14 elution 10, lane 15 elution 11.*

The elution peak is broad (lane 6-14), however, the fractions are relatively pure and the dxCas9 contamination was mostly eliminated. These 9 fractions were pooled and further purified.

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Sunday, 26/08/2018

Date
:   26/08/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Dialysis*

Finally the fusion protein is dialysed and concentrated. 4.5mL of sample (MonoQ chromatography elution fraction 2-10 of fusion protein) were dialysed according to the purification protocol with dialysis buffer (20 mM Tris-HCl, 100 mM KCl, 5% glycerol, 1mM beta-Mercaptoethanol, pH 7.5).

#### Protein Purification - *Concentration*

After dialysis, 4.5mL of sample of fusion protein was concentrated to a final volume of 0.6mL and stored at -20℃, according to the purification protocol.

In order to analyse the concentrated fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/82/T--TUDelft--2018_Figure1_26082018.png)

**Figure Concentration:**  *8% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 Crude lysate, lane 3 final fusion post concentration 9uL, lane 4 final fusion post concentration 4.5uL, lane 5 final fusion post concentration 2.25uL.*

Trace amounts of contaminating background was observed, however, the fusion was significantly more in quantity and the background was tolerated for functionality testing. The final concentration of the solution was determined according to the BCA protocol and resulted in a concentration of 0.57mg/mL (2.64µM).

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[ ]
Week 5 27/08/2018 - 02/09/2018

Monday, 27/08/2018

Date
:   27/08/2018

Operator
:   Janine Nijenhuis

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5. A colony with Tn5 strain was used for inoculation of 40mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Tuesday, 28/08/2018

Date
:   28/08/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 continued. Inoculated 1L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.3.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 12:55 | 0.04 |
| 14:25 | 0.11 |
| 15:30 | 0.48 |

After OD600 reached 0.48, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Wednesday, 29/08/2018

Date
:   29/08/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. The cells with Tn5 protein were resuspended in 30 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added for both cultures:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 60uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1 tablet |
| imidazole | 2M | 75uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resins were washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 20mM imidazole, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 250mM imidazole, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/d0/T--TUDelft--2018_NBJanine20180830.png)

**Figure Nickel Affinity Chromatography.**   *8% SDS PAGE of Tn5 (expected size 53.3kDa). Lane 1 high range molecular ladder (kDa), lane 2 post induction, lane 3 whole lysate, lane 4 cell debris, lane 5 clarified lysate, lane 6 flow through, lane 7 wash, lane 8 elution 1, lane 9 elution 2, lane 10 elution 3, lane 11 elution 4, lane 12 elution 5, lane 13 elution 6.*

The nickel chromatography was not completely successful, but there is some purification of Tn5. The Tn5 did not completely bind to the nickel resin and was observed in the flowthrough (lane 6). It is recommended to further optimize the nickel chromatography to further reduce losses, and reduce contaminating proteins due to non-specific binding to the nickel resin (lane 8). However for our purposes, the losses were tolerated.

Close

### September

[ ]
Week 1 03/09/2018 - 09/09/2018

Monday, 03/09/2018

Date
:   03/09/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The Tn5\_3 plasmid was transfomred into BL21AI for Tn5 protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | Tn5.3 (5210bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Tuesday, 04/09/2018

Date
:   04/09/2018

Operator
:   Monique de Leeuw

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5 protein. A colony with Tn5\_3 strain was used for inoculation of 10mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Wednesday, 05/09/2018

Date
:   05/09/2018

Operator
:   Janine Nijenhuis

#### Objective

Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of Tn5. A colony with Tn5 strain was used for inoculation of 100mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Date
:   05/09/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 protein continued. Inoculated 1L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 10:30am | 0.22 |
| 11:30am | 0.35 |
| 12:00am | 0.52 |

After the culture OD600 reached 0.52, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Thursday, 06/09/2018

Date
:   06/09/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the Tn5 continued. Inoculated 3L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.3.

**Table :** Growth represented by OD600 over time

| Time | Flask 1 OD600 | Flask 2 OD600 | Flask 3 OD600 |
| 12:50 | 0.03 | 0.05 | 0.05 |
| 14:25 | 0.05 | 0.07 | 0.08 |
| 15:20 | 0.14 | 0.18 | 0.17 |
| 15:40 | 0.27 | 0.33 | 0.26 |
| 16:00 | 0.37 | 0.40 | 0.39 |

After the three cultures reached an OD600 of respectively 0.37, 0.4 and 0.39, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Date
:   06/09/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. 6.17g of cells of Tn5 protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 86uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| imidazole | 2M | 432uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/3/3d/T--TUDelft--2018_Figure1_05072018.png)

**Figure expression of Tn5:**  *12% SDS PAGE of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane 2 pre-Induction, lane 3 whole lysate, lane 4 clarified lysate.
.*

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. 33.5mL clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resin was washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 20mM Imidazole, 10% glycerol, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 250 mM Imidazole, 10% glycerol, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/6/69/T--TUDelft--2018_Figure2_05072018.png)

**Figure Nickel Affinity Chromatography.**   *12% SDS of Tn5 (expected size 53.3kDa). Lane 1 molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 cell debris, lane 5 flow through 1, lane 6 flow through 2, lane 7 wash 1, lane 8 wash 2, lane 9 wash 3, lane 10 elution 1, lane 11 elution 2, lane 12 elution 3, lane 13 elution 4, lane 14 elution 5, lane 15 elution 6.*

A significant amount of Tn5 was observed in the nickel chromatography elution fractions (lane 10-14), however the Tn5 protein was also observed in the flowthrough and wash elution fractions (lane 5-9). It is recommended to optimize the nickel chromatography to reduce these losses. For our purposes, the Tn5 losses were tolerated because the amounts that we obtained were sufficient to proceed to functionality testing of the Tn5. To increase the Tn5 amount, the first five elution fractions were pooled (lane 10-14).

Close

Friday, 07/09/2018

Date
:   07/09/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. The three cultures were combined to one. The French Press was however out of order, so the cell pellet is stored at -20 ℃.

Close

[ ]
Week 2 10/09/2018 - 16/09/2018

Wednesday, 12/09/2018

Date
:   12/09/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the Tn5 protein started. Harvest the cells according to the purification protocol. The cells with Tn5 protein were resuspended in 72 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 8).

After resuspension the following additives were added for both cultures:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| beta-mercaptoethanol | 0.5M | 5.04uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 2 tablet |
| imidazole | 2M | 180uL |

The cells were lysed and clarified according to the the purification protocol.

#### Protein Purification - *Nickel Affinty Chromatography*

1mL of 50% His-Select Ni resin, was washed according to the Purification protocol. Clarified lysate of Tn5 protein was added to the washed His-Select Ni resin and incubated with gentle mixing for 60minutes at 4 ℃. The His-Select Ni resins were washed with washing buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 20mM imidazole, 1mM DTT, pH8), and eluded with with 6\* 500μL (6CV) of elution buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, 250mM imidazole, 1mM DTT, pH8) at 4 ℃, according to the Purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the nickel affinty chromatography of the Tn5 protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/3/35/T--TUDelft--2018_NBJanine20180912.png)

**Figure Nickel Affinity Chromatography.**   *8% SDS PAGE of Tn5 (expected size 53.3kDa). Lane 1 high range molecular ladder (kDa), lane 2 whole lysate, lane 3 clarified lysate, lane 4 cell debris, lane 5 flow through, lane 6 wash, lane 7 elution 1, lane 8 elution 2, lane 9 elution 3, lane 10 elution 4, lane 11 elution 5, lane 12 elution 6.*

The nickel chromatography was not completely successful, but there is some purification of Tn5. The Tn5 did not completely bind to the nickel resin and was observed in the flowthrough and wash (lane 6-7). It is recommended to further optimize the nickel chromatography to further reduce losses, and reduce contaminating proteins due to non-specific binding to the nickel resin (lane 9-10). However for our purposes, the losses were tolerated.

Close

[ ]
Week 4 24/09/2018 - 30/09/2018

Monday, 24/09/2018

Date
:   24/09/2018

Operator
:   Janine Nijenhuis

#### Objective

dxCas9-Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9-Tn5. A colony with dxCas9-Tn5\_9 strain was used for inoculation of 100mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Tuesday, 25/09/2018

Date
:   25/09/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9-Tn5 continued. Inoculated 5L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.3.

**Table :** Growth represented by OD600 over time

| Time | Flask 1 OD600 | Flask 2 OD600 | Flask 3 OD600 |
| 11:30 | 0.03 | 0.08 | 0.04 |
| 13:50 | 0.30 | 0.22 | 0.22 |
| 14:10 | X | 0.37 | 0.34 |

After the three cultures reached an OD600 of respectively 0.30, 0.37 and 0.34, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Wednesday, 26/09/2018

Date
:   26/09/2018

Operator
:   Janine Nijenhuis

#### Protein Purification - *Cell lysis*

The downstream processing of the dxCas9-Tn5 protein started. Harvest the cells according to the purification protocol. The cells with dxCas9-Tn5 protein were resuspended in a total volume of 150 mL of Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 7.5).

After resuspension the following additives were added for both cultures:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| beta-mercaptoethanol | 14.3M | 10.5uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 3 tablets |
| imidazole | 2M | 375uL |
| PMSF | 0.2M | 3mL |

The cells were lysed and clarified according to the the purification protocol. However, something went wrong, and instead of a clear suspension, only foam came out of the homogenizer. Because it looked like denatured proteins, it is decided to start again with the purification, because it cannot be said for sure the fusion protein will work.

Close

Thursday, 27/09/2018

Date
:   27/09/2018

Operator
:   Janine Nijenhuis

#### Objective

dxCas9-Tn5 was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of dxCas9-Tn5. A colony with dxCas9-Tn5\_9 strain was used for inoculation of 100mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Friday, 28/09/2018

Date
:   28/09/2018

Operator
:   Janine Nijenhuis

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the dxCas9-Tn5 continued. Inoculated 3L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 0.3.

**Table :** Growth represented by OD600 over time

| Flask number OD600 | 13:50 | 15:30 | 16:25 |
| 1 | 0.11 | 0.18 | 0.57 |
| 2 | 0.09 | 0.11 | 0.38 |
| 3 | 0.07 | 0.16 | 0.55 |
| 4 | 0.07 | 0.16 | 0.56 |
| 5 | 0.05 | 0.15 | 0.54 |
| 6 | 0.06 | 0.16 | 0.54 |
| 7 | 0.06 | 0.17 | 0.54 |

After the seven cultures all reached an OD600 higher than 0.3, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

### October

[ ]
Week 1 01/10/2018 - 07/10/2018

Monday, 01/10/2018

Date
:   01/10/2018

Operator
:   Monique de Leeuw

#### Transformation of chemically competent cells

The dxcas9-linker-Tn5\_9 plasmid was transfomred into BL21AI for fusion protein production. The following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*BL21AI | dxcas9-linker-Tn5\_9 (9432bp) | Heat shock | 1 | LB chloramphenicol |

200 uL LB medium was added as recovery medium.

Close

Tuesday, 02/10/2018

Date
:   02/10/2018

Operator
:   Monique de Leeuw

#### Objective

Fusion was expressed and purified to produce protein for *in vitro* functionality evaluation.

#### Protein expression - *Seed Culture*

A seed culture was made to start Upstream Processing (USP) of fusion protein. A colony with dxcas9\_linker-Tn5\_9 strain was used for inoculation of 20mL liquid starter culture (Luria Broth, complemented with 34 μg/mL chloramphenicol) following the Liquid Starter Culture Protocol.

Close

Wednesday, 03/10/2018

Date
:   03/10/2018

Operator
:   Monique de Leeuw

#### Protein Expression - *Inoculation & Induction*

The upstream processing of the fusion protein continued. Inoculated 2L liquid media (Luria Broth, complemented with 34 μg/mL chloramphenicol) with the seed culture and grown to an OD600 of 2.

**Table :** Growth represented by OD600 over time

| Time | OD600 |
| 10:50am | 0.08 |
| 11:25am | 0.32 |
| 12:00am | 0.58 |

After the culture OD600 reached 0.58, the Protein Expression protocol was followed. After the expression is completed, the cells were transferred to Downstream Processing (DSP).

Close

Thursday, 04/10/2018

Date
:   04/10/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Cell lysis*

The downstream processing of the fusion protein started. Harvest the cells according to the purification protocol. 7.2g of cells of fusion protein were resuspended in Lysis buffer (20 mM Tris-HCl, 250 mM NaCl, 10% glycerol, pH 7.5).

After resuspension the following additives were added:

| Additive | Stock Concentration | Volume added |
| --- | --- | --- |
| DTT | 0.5M | 101uL |
| EDTA-free Complete protease inhibitor tablets | n/a | 1tablets |
| PMSF | 0.2M | 252uL |

The cells were lysed and clarified according to the the purification protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the expression of the fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 12% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f9/T--TUDelft--2018_Figure1_04102018.png)

**Figure Fusion Expression:**  *12% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 Whole lysate, lane 2 clarified, lane 3 flow through 1, lane 4 flow through 2 , lane 5 molecular ladder (kDa).*

#### Protein Purification - *Heparin Chromatography*

The fusion protein was further purified with Heparin chromatography. The 1mL HiTrap Heparin HP column was connected to the AKTA pure in the cold room at 4 ℃. 45.2mL of crude lysate from fusion protein was injected into the injection loop and the chromatography was followed according to the Purification Protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse the heparin chromatography of the fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol.The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/0/02/T--TUDelft--2018_Figure2_04102018.png)

**Figure Heparin Chromatography #1.**   *8% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 Whole lysate, lane 2 clarified, lane 3 flow through 1, lane 4 flow through 2 , lane 5 molecular ladder (kDa), lane 6 wash, lane 7 elution 1, lane 8 elution 2, lane 9 elution 3, lane 10 elution 4, lane 11 elution 5, lane 12 elution 6, lane 13 elution 7, lane 14 elution 8, lane 15 elution 9.*
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/d8/T--TUDelft--2018_Figure1_05102018.png)

**Figure Heparin Chromatography #2.**   *8% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 molecular weight (kDa), lane 2 elution 10, lane 3 elution 11, lane 4 elution 12 , lane 5 elution 13, lane 6 elution14, lane 7 elution 15, lane 8 elution 16, lane 9 elution 17, lane 10 elution 18, lane 11 elution 19, lane 12 elution 20, lane 13 elution 21, lane 14 elution 22, lane 15 elution 23.*

8-15

Degradation of our fusion protein is still significant. Results seem to suggest that the fusion protein is being cleaved at the linker, resulting in both the dxCas9 and the fusion protein being present in our sample. The heparin chromatography was relatively successful at separating the two populations. The dxCas9 has a lower affinity to the heparin resin as a result it elutes at lower concentrations of salt. As the concentration of salt increases less dxCas9 and more fusion elutes (gel1, lane 9-15 and gel2, lane 1-7). At high levels of salt the mostly fusion elutes (gel2, lane 8-15). Only these last eight elution fractions are pooled. Thus losses are tolerated, to obtain a relatively pure fusion fraction which can be further purified by MonoQ.

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Friday, 05/10/2018

Date
:   05/10/2018

Operator
:   Monique de Leeuw

#### Protein Purification *- MonoQ Chromatography*

The fusion protein was further purified with MonoQ chromatography. The 1mL MonoQ 5/50 GL column was connected to the AKTA pure in the cold room at 4 ℃. 16mL of sample (4x diluted heparin chromatography elution fraction 16-23 of fusion protein) was injected into the injection loop and the chromatography was followed according to the Purification Protocol.

#### Tris-Glycine Polyacrylamide Gel

In order to analyse monoQ of the fusion protein, a precast Tris-Glycine polyacrylamide gel was run with a 6% stacking gel and 8% separation gel according to the SDS PAGE Tris-Glycine Polyacrylamide Gel protocol. The polyacrylamide gel ran for 90min at 50volt. After the was completed, the gel was stained with SimplyBlue SafeStain and imaged using the GelDoc system. The result of the gel is shown below:
![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/c1/T--TUDelft--2018_Figure1_06102018.png)

**Figure MonoQ chromatography:**  *8% SDS PAGE of fusion (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 starting material, lane 3 flow through, lane 4 wash, lane 5 elution 1, lane 6 elution 2, lane 7 elution 3, lane 8 elution 4, lane 9 elution 5, lane 10 elution 6, lane 11 elution 7, lane 12 elution 8, lane 13 elution 9, lane 14 elution 10, lane 15 elution 11.*

The elution peak is broad (lane 5-13), however, the fractions are relatively pure and the dxCas9 contamination was mostly eliminated. These 9 fractions were pooled and further purified.

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Saturday, 06/10/2018

Date
:   06/10/2018

Operator
:   Monique de Leeuw

#### Protein Purification - *Dialysis*

Finally the fusion protein is dialysed and concentrated. 4.5mL of sample (MonoQ chromatography elution fraction 2-10 of fusion protein) were dialysed according to the purification protocol with dialysis buffer (20 mM Tris-HCl, 100 mM KCl, 5% glycerol, 1mM beta-Mercaptoethanol, pH 7.5).

#### Protein Purification - *Concentration*

After dialysis, 4.5mL of sample of fusion protein was concentrated to a final volume of 0.6mL and stored at -20℃, according to the purification protocol. Trace amounts of contaminating background was observed, however, the fusion was significantly more in quantity and the background was tolerated for functionality testing. The final concentration of the solution was determined according to the BCA protocol and resulted in a concentration of 0.56mg/mL.

Close

* September
  + [Week: 03/09/2018 - 09/09/2018](#SeptemberSequencing1)
  + [Week: 10/09/2018 - 16/09/2018](#SeptemberSequencing2)
  + [Week: 17/09/2018 - 23/09/2018](#SeptemberSequencing3)
  + [Week: 24/09/2018 - 30/09/2018](#SeptemberSequencing4)
* October
  + [Week: 01/10/2018 - 07/10/2018](#OctoberSequencing1)
  + [Week: 08/10/2018 - 14/10/2018](#OctoberSequencing2)

### September

[ ]
Week 2 10/09/2018 - 16/09/2018

Wednesday, 12/09/2018

Date
:   12/09/2018

Operator
:   Timothy Páez

#### High Fidelity PCR

With the objective to generate *EPO* biobrick with a Kanamycin fragment as intron, high fidelity PCR was done to amplify EPO biobrick from junction 2 between introns. Primers 103 and 104 were used to PCR amplify the backbone. At the same time, primers 105 and 106 were used to PCR amplify a fragment from Kanamycin from the plasmid pSB1K3. Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

50 µL PCR mixtures were prepared, according to the High Fidelity PCR protocol.

The thermocycler programme was installed to have an extension time of 1.5 minutes and an annealing temperature of 65 °C. After the programme was finished, samples were kept at 4 °C.

Date
:   12/09/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

PCR products of *EPO* plasmid and kanamycin PCR were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane with 1 µL of loading buffer.
- 4 µL of 500 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/e/ec/T--TUDelft--12_09_EPO_PCR.png)

Figure 1. Gel electrophoresis from EPO plasmid and Kanamycin fragment PCR. Lanes are (1-2) Amplification of EPO backbone, (3) negative control, (L) 500 bp ladder, (4-5) amplification of Kanamycin fragment and (6) negative control.

Date
:   12/09/2018

Operator
:   Timothy Páez

#### Gibson Assembly

According to the displayed reaction mixture composition, Gibson Assembly was done to assemble Kanamycin fragment and Epo backbone to obtain EPO+Kan intron plasmid (3.2 kbp).

| Component | Volume (uL) |
| --- | --- |
| Gibson Assembly Mastermix 2X | 10 |
| Vector | EPO biobrick (100 ng) |
| Fragment | Kanamycin fragment (75 ng) |

The samples were incubated at 50°C for 60 minutes, after which they were kept on ice for subsequent transformation.

Date
:   12/09/2018

Operator
:   Timothy Páez

#### Transformation of chemically competent cells

Using the Gibson assembly reaction, the following transformations were carried out according to the protocol for transformation of chemically competent cells:

| Strain | Plasmid & size (bp) | Cloning method | Volume added (uL) | Medium on which was plated |
| --- | --- | --- | --- | --- |
| *E.coli*DH5αlpha | EPO backbone (2600 bp) | Restriction Ligation | 10 µL | LB with Chloroamphenicol |

400 µL LB medium was added as recovery medium.

Close

Friday, 14/10/2018

Date
:   19/09/2018

Operator
:   Timothy Páez

#### Colony PCR

Colony PCR was done to verify propagation of *Escherichia coli* DH5 alpha cells after transformation with Gibson assembly product from 12\_09\_2018 containing EPO+Kan intron plasmid. This insertion can be checked with primers 103 and 104 (*EPO* amplification), Primer sequences can be found at:  [Primer sequences](https://2018.igem.org/Team%3ATUDelft/Primers).

Date
:   14/09/2018

Operator
:   Timothy Páez

#### DNA Gel Electrophoresis

PCR products of *EPO+kan* intron plasmid and kanamycin PCR were run on a 0.8 % agarose gel in TAE buffer.
- 5 μL of PCR product was pipetted into each lane with no loading buffer.
- 4 µL of 200 bp DNA ladder
- 80 V for 30 minutes
- The gel was imaged using a gel documentation system with UV-light.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/c/ca/T--TUDelft--EPO_KAN_PCR_TIMMY.png)

Figure 1. Gel electrophoresis from EPO+Kan intron plasmid PCR. Lanes are (1-4) Amplification of EPO backbone with kanamycin fragment intron, (L) 200 bp ladder and (5-6) negative control of only EPO plasmid.

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### October

[ ]
Week 2 08/10/2018 - 14/10/2018

Monday, 08/10/2018

Date
:   08/10/2018

Operator
:   Timothy Páez

#### Library preparation for MinION sequencing

##### Sequencing of sample: Lambda DNA

Lambda DNA suplemented in the Rapid Sequencing kit from ONT MinION (RAD004) was library preped and sequenced following the protocol indicated for MinION sequencing. Protocols can be accessed here:  [Protocols](https://2018.igem.org/Team%3ATUDelft/Experiments).

###### Sequencing stats

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/d/d5/T--TUDelft--Timmy_FirstSeq_1.png)

Figure 1. Read length histogram for Lambda DNA sequencing run during 6 hours with MinION in Flow Cell FloMin106 with RAD004 kit for library preparation.

###### Data processing

After sequencing run, the following analysis were performed:
- Albacore software for base calling.
- *De novo* assembly of reads with Canu for 40 kb genome size.

The assembly resulted in 1 contig that was aligned to a known reference sequence with > 99% identity.

Close

Wednesday, 10/10/2018

Date
:   08/10/2018

Operator
:   Timothy Páez

#### Library preparation for MinION sequencing

##### Sequencing of sample: Mix of DNA molecules at equimolar concentrations

A solution with different types of DNA molecules was prepared before library preparation. This solution contained equimolar concentrations of different molecules, defined below:
- EPO PCR amplification: 600 bp.
- EPO + Kanamycin amplification: 1200 bp.
- EPO Biobrick plasmid: 2600 bp
- pACYC plasmid: 4000 bp
- EPO+Amp plasmid:2800bp
This solution was library prepared library preped following the protocol indicated for MinION sequencing. Protocols can be accessed here:  [Protocols](https://2018.igem.org/Team%3ATUDelft/Experiments). After the fragmentation step 1, a new DNA molecule was added to asess weather unadapted DNA is sequenced or not. This sequence was:
- dxCas9 PCR amplification: 4000 bp

###### Sequencing stats

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/8/8e/T--TUDelft--Timmy_2RUN_EPO.png)

Figure 1. Read length histogram for Lambda DNA sequencing run during 6 hours with MinION in Flow Cell FloMin106 with RAD004 kit for library preparation.

###### Data processing

After sequencing run, the following analysis were performed:
- Albacore software for base calling.
- Samptools multiple alignment to reference molecules
- Samtools sort alignment, stats, view, and export as fasta mapped files.

###### Sequencing stats

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/f/f3/T--TUDelft--Sequencing_1.png)

Figure 1. Read length histogram for Lambda DNA sequencing run during 6 hours with MinION in Flow Cell FloMin106 with RAD004 kit for library preparation.

Close

Sunday, 14/10/2018

Date
:   14/10/2018

Operator
:   Timothy Páez

#### Fusion protein functionality testing: integration of sequencing adapters on EPO backbone and Tn5 DNA

We aimed to perform the integration of adapters with our fusion protein on EPO biobrick circular DNA plasmid.

The components of the reagents can be found on the protocol. The sequence of the adapter used in this assay were sequencing adapters specified at  [Sequences](https://2018.igem.org/Team%3ATUDelft/Primers) page.

**Fusion protein:adapter DNA complex formation**
Tn5 or fusion protein was loaded with adapter DNA shown above by following the pipetting scheme shown on Table 2. All samples and reagents were kept on ice during preparation.

**Table 2:** Pipetting scheme for transposome formation.

|  | Reaction with fusion protein (µl) | Reaction no fusion protein (µl) || Buffer | 1 | 0.6 |
| Fusion (~3µM) | 8 | 0 |
| Tn5 (~8µM) | 0 | 0 |
| Adapter (25µM) | 1 | 0.6 |
| Water | 0 | 4.8 |
| Total | 10 | 6 |

The reagents were pipetted in the following order: Water, 10x functionality buffer, dxCas9-Tn5, and adapter. The samples were incubated at 37°C for 1 hour.

To add appropriate amount of sgRNA, the pipetting scheme on Table 3 was followed. sgRNA J2.1 signifies version 1 of sgRNA targeting juntion 2 of EPO gene:

**Table 3:** Pipetting scheme for sgRNA loading.

|  | Reaction with sgRNA J2.1(µl) | Reaction no sgRNA(µl) || Transposome | 4 | 4 |
| sgRNA (100ng/µl) | 0.8 | 0 |
| MgCl2 Buffer | 0.2 | 0.2 |
| Water | 0 | 0.8 |

The samples were incubated at 37°C for 10 minutes.
Target DNA (EPO cDNA) was added to make final concentration of ~5ng/µl. One negative control was done where water instead of EPO cDNA was added.

The samples were incubated at 37°C. At incubation time = 1h. 2.5 µl of sample were taken from each reaction and was subjected to PCR. PCR mix and cycle can be found on protocol.

The PCR products were analysed on 5% native TBE polyacrylamide gel. The resulting gel is either stained with ethidium bromide and imaged with GelDoc system, or directly imaged with Typhoon Imaging System.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/9/9e/T--TUDelft--Sequencing_adapters_on_EPO_plasmid.png)

**Figure 1. 5% TBE native PAGE stained by EtBr and imaged by GelDoc system.Integration reaction after 1 hour.**  *Lanes are (L) DNA ladder (100-1000bp), (1) amplified product with fw EPO (2) amplified product with rv EPO, (3) amplified product with fw EPO without sgRNA, (4) amplified product with rv EPO without sgRNA, (5) amplified product with fw EPO without fusion protein, (6) amplified product with rv EPO without fusion protein, (7) amplified product with fw EPO without adapter DNA, (8) amplified product with rv EPO without adapter DNA*

Figure 1 did not show integration of adapters on plasmid DNA. The reaction was repeated with plasmid linearization before the integration.

![Gel electrophoresis](https://static.igem.org/mediawiki/2018/2/28/T--TUDelft--SequencingAdapters.png)

Integration on linearized plasmid worked.

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# Team:TUDelft/Experiments

ADOPE

[![](https://static.igem.org/mediawiki/2018/0/09/T--TUDelft--2018_adope_logo_white.png)](https://2018.igem.org/Team%3ATUDelft)

* Project
  + [Description](https://2018.igem.org/Team%3ATUDelft/Description)
  + [Design](https://2018.igem.org/Team%3ATUDelft/Design)
  + [Results](https://2018.igem.org/Team%3ATUDelft/Results)
  + [Demonstration](https://2018.igem.org/Team%3ATUDelft/Demonstrate)
* Wetlab
  + [Experiments (Protocols)](https://2018.igem.org/Team%3ATUDelft/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ATUDelft/Notebook)
  + [InterLab](https://2018.igem.org/Team%3ATUDelft/InterLab)
  + [Safety](https://2018.igem.org/Team%3ATUDelft/Safety)
  + [Measurement](https://2018.igem.org/Team%3ATUDelft/Measurement)
* Parts
  + [Parts Overview](https://2018.igem.org/Team%3ATUDelft/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3ATUDelft/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ATUDelft/Composite_Parts)
  + [Part Collection](https://2018.igem.org/Team%3ATUDelft/Part_Collection)
  + [Sequences](https://2018.igem.org/Team%3ATUDelft/Primers)
* Modeling
  + [Model](https://2018.igem.org/Team%3ATUDelft/Model)
  + [Software Tool](https://2018.igem.org/Team%3ATUDelft/Software)
  + [Improvement](https://2018.igem.org/Team%3ATUDelft/Improve)
* Human Practices
  + [Integrated Human Practices](https://2018.igem.org/Team%3ATUDelft/Human_Practices)
  + [Education and Engagement](https://2018.igem.org/Team%3ATUDelft/Public_Engagement)
  + [Entrepreneurship](https://2018.igem.org/Team%3ATUDelft/Entrepreneurship)
  + [Applied Design](https://2018.igem.org/Team%3ATUDelft/Applied_Design)
* [EurAsian Meetup](https://2018.igem.org/Team%3ATUDelft/EurAsianMeetup)
* Team
  + [Team Members](https://2018.igem.org/Team%3ATUDelft/Team)
  + [Collaborations](https://2018.igem.org/Team%3ATUDelft/Collaborations)
  + [Contributions](https://2018.igem.org/Team%3ATUDelft/Contributions)
  + [Team Building](https://2018.igem.org/Team%3ATUDelft/Team_Building)
* Acknowledgements
  + [Attributions](https://2018.igem.org/Team%3ATUDelft/Attributions)
  + [Sponsors](https://2018.igem.org/Team%3ATUDelft/Sponsors)
* + [Judging form](https://igem.org/2018_Judging_Form?team=TUDelft)
  + [Medal Requirements](https://2018.igem.org/Team%3ATUDelft/Medals)
  + [Awards](https://2018.igem.org/Team%3ATUDelft/Awards)

![Wetlab Protocols](https://static.igem.org/mediawiki/2018/e/e1/T--TUDelft--wetlabprothead.png)

Antibiotic Stock Solution

Preparation of a 1000X antibiotic stock solution:

1. Weigh X grams of the specific antibiotic of interest.
2. Resolve in 95% ethanol (Chloramphenicol) or MilliQ (other antibiotics).

BCA Protein Quantification

This protocol is based on the [Pierce BCA protein assay kit](https://www.thermofisher.com/order/catalog/product/23225)by Thermo Scientific protocol.

1. Prepare a set of protein standards using one 2mg/mL Albumin Standard (BSA) ampule according to the table below:
   *NOTE: Use the same diluent as the samples. The expected working range = 20-2000µg/mL.*

| Vial | Volume of MilliQ (µL) | Source of BSA | Volume of source BSA (µL) | Final BSA concentration (µg/µL) |
| --- | --- | --- | --- | --- |
| A | 0 | Stock | 300 | 2000 |
| B | 125 | Stock | 375 | 1500 |
| C | 325 | Stock | 325 | 1000 |
| D | 175 | Vial B | 175 | 750 |
| E | 325 | Vial C | 325 | 500 |
| F | 325 | Vial E | 325 | 250 |
| G | 325 | Vial F | 325 | 125 |
| H | 400 | Vial G | 100 | 25 |
| I | 400 | n/a | 0 | 0 |

2. Determine the amount of total volume of working reagent (WR) required by using the following formula:
   Total volume WR = (# standards + # unknowns) × (# replicates) × (200 µL)
   *NOTE: With pipetting you always take a bit extra (e.g. 10%), so not exactly the amount needed for x samples*
3. Prepare the BCA working reagent by mixing 50 parts of BCA Reagent A with 1 part of BCA Reagent B (50:1, Reagent A:B).
   *NOTE: The WR is stable for several days when stored in a closed container at room temperature.*
4. Pipette 25µL of each standard or unknown sample replicate into a microplate well.
5. Add 200µL of the WR to each well and mix plate thoroughly.
6. Cover plate and incubate at 37 °C for 30 minutes.
7. Cool plate to room temperature.
8. Measure the absorbance at or near 562nm on a plate reader.

Blunt End Ligation

1. Thaw the ligase buffer on ice, to prevent damaging the ATP
2. For phosphorylation, prepare a sample a sample as follows:

   | Component | Volume (µL) |
   | --- | --- |
   | 10x Ligase buffer | 2.5 |
   | T4 DNA Ligase | 1 |
   | ATP | 2.5 |
   | T4 PNK enzyme | 1 |
   | DNA vector | X (~250 ng) |
   | DNA fragment | Y\* |
   | MilliQ | Up to 25 µL |

   *\* NOTE: The desired vector:insert ratio will be 1:3. Use a ligation calculator to calculate the amounts of vector DNA and insert DNA to be added.*
3. Incubate for at one hour at 37 °C.
4. Cancel phosphorylation process by incubating for 30 minutes at 65 °C.
5. Add 1 µL of Ligase T4 and incubate for at least four hours at 4 °C.

Blood Sample Preparation

1. Centrifuge the whole blood (9mL EDTA anticoagulated tube) at 1200 x g for 10 minutes.
2. Pipet of the plasma carefully (approximately 4 to 4.5mL) and put in a separate tube. When you only want the cfDNA and not the white blood cells be sure to not disturb the buffycoat with the pipet.
3. Centrifuge the plasma at 24000 x g for 20 minutes.
4. Take of the plasma without disturbing the pallet and aliquot into 1.5mL tubes, 1mL per tube.
5. Store at -20 °C.
6. For every follow up steps frozen aliquots can be taken from the freezer and thawed at room temperature. When thawed, the samples should be directly processed. Avoid thawing and freezing cycles, since this will cause degradation of cfDNA.

F. Thurik (2016). Cell-free placental DNA beyond Down syndrome.

Cell Banking

*NOTE: All work is performed under asceptic conditions.*

1. For one cryostock, take a 1.5mL sample from an [overnight liquid culture](#liquidstarterculture-scroll).
2. Centrifuge the 2mL tubes at 2000rpm for 10 min.
3. Decant the supernatant without disturbing the pellet.
4. Add fresh sterile LB medium to the pellet, 1/3 volume of the starting volume of the culture.
5. Completely resuspended the pellet by vortexing the tube.
6. Add sterile 80% glycerol solution, the same volume as fresh LB in step 4.
7. Mix by vortexing.
8. Make a 1mL aliquot in cryotubes and label it with the cell type, plasmid type, protein type, operator and date.
9. Store the vials at -80 ºC and update the inventory.

Chemically Competent Cells Preparation

This protocol s over 3 days of execution time, starting from a -80 °C mother stock. Throughout the protocol, it is recommended to work under aseptic conditions in order to prevent contamination risks.

#### Day 1

1. Keep the -80 °C strain stock of interest on ice.
2. Streak the strain on [solid selective medium](#solidmedium-scroll) and incubate overnight at 37 °C while shaking.

#### Day 2

3. Prepare a 10mL [liquid starter culture](#liquidstarterculture-scroll) with one of the colonies that grew on the selective plate. Let the culture grow overnight at 37 °C, shaking at 180rpm.
4. Sterilise solutions of 100 mM CaCl2 and of 100 mM CaCl2 + 15 % Glycerol in advance.
   NOTE: Volumes depend on the total culture volume to be prepared in step 5. For example, if 1L is used in step 7, 300mL of 100mM CaCl2 and 10mL of 100mM CaCl2 + 15% glycerol will be used.

#### Day 3

5. Inoculate 1:100 of overnight culture in the desired volume of LB with antibiotic when required (eg. 10mL of culture in 1L of LB).
6. Incubate in a shaker at 37 °C, 180rpm to OD600nm ~0.4-0.6 (measure OD600nm every 30 minutes).
7. Harvests the cells by centrifugation at 4000 x g for 5 minutes in centrifuge tubes, decant supernatant.
8. Resuspend cells by gently pipetting 1/5 (of the volume of LB from step 5) of ice-cold 100 mM CaCl2 and incubate on ice for 20 minutes.
9. Pellet the cells by centrifugation at 4000 x g for 5 minutes in centrifuge tubes, decant supernatant.
10. Resuspend cells by gently pipetting 1/10 (of the volume of LB from step 5) of ice-cold 100 mM CaCl2 and incubate on ice for 60 minutes.
11. Pellet the cells by centrifugation at 4000 x g for 5 minutes in centrifuge tubes, decant supernatant.
12. Resuspend cells by gently pipetting 1/100 (of the volume of LB from step 5) of ice-cold 100 mM CaCl2 + 15% glycerol and keep on ice.
13. Chemical competent cells can either immediately be used for [heat shock transformation](#chemcomptcellstrans-scroll) or stored in aliquots of 50µL in microcentrifuge tubes at -80 °C.

Chemically Competent Cells Transformation

1. Get as many aliquots of [competent cells](#chemcomptcellsprep-scroll) (50µL) from the -80 °C freezer as transformations to be done and put them on ice for 10-15 min.
   *NOTE: Do not forget positive and negative controls (no DNA). If commercial competent cells (highly efficient) are used, an aliquot of 50µL can be split in two equal volumes of 25 µL and used for two transformations.*
2. Add DNA of interest to the 50µL of competent cells.
   1. Gibson Assembly mixture: 5µL
   2. Ligation mixture: 5µL
   3. Plasmids: 10 ng, volume depends on the plasmid concentration- Incubate on ice for 10-20 minutes.
   - Heat shock at 42 °C for exactly 45 seconds.
   - Add 200µL of hand-warm LB-medium.
   - Incubate at 37 °C with shaking (250 rpm) for 1 hour.
   - Plate the cells on [LB-agar plates](#solidmedium-scroll) with the correct antibiotic to select for the introduced plasmid . 50-75µL can be plated on one plate. The remaining cell culture can be pelleted and resuspended in some residual supernatant prior to plating.
     *NOTE: When adding more than 200µL LB medium during step 5, plate out 100µL on selective medium. Then, briefly centrifuge the remaining cell culture and plate out the cell pellet on selective medium.*
   - Incubate plates at 37 °C overnight.
     *NOTE: Alternatively, you can incubate over the weekend, leaving the plate on the bench.*

Colony PCR

1. Under aseptic conditions, pick a colony, resuspend it in 10µL of MilliQ water.
   *NOTE: A picked colony cannot be used again; it is recommended to restreak on a 'back-up'-plate with the same pipet tips and incubate it overnight at 37 °C.*
2. Incubate the resuspended colony at 90 °C for 10 min. Spin the suspension down and use the supernatant as template DNA for the PCR.
   *NOTE: Instead of separate boiling prior to PCR, this step can be incorporated in the PCR program. The initial denaturation step at 98 °C should then be prolonged to 5 minutes.*
3. Make sure every PCR reaction is composed as follows:

   | Component | Volume (µL) | Final concentration |
   | --- | --- | --- |
   | Gotaq 5X buffer\* | 10 | 1X |
   | 10mM dNTPs | 1 | 200µM |
   | Primer forward (10µM) | 1 | 200nM |
   | Primer reverse (10µM) | 1 | 200nM |
   | Boiled colony supernatant | 5 |  |
   | Gotaq polymerase (5U/µL) | 0.2 | 20U/mL |
   | MilliQ | 31.8 |  |

   *\* NOTE: Use Gotaq Buffer Green when it is required to run a verification gel afterwards.*- Add 5µL of supernatant of colony mixture to each PCR tube.
   - Close all tubes thoroughly and place them in a thermocycler with the following protocol:

     | Step | Time (s) | Temperature (°C) |
     | --- | --- | --- |
     | Initial denaturation | 150 | 98 |
     | Denaturation | 60 | 94 |
     | Annealing | 60 | Tann\* |
     | Extension | 60 sec per kb DNA | 72 |
     | Final extension | 600 | 72 |
     | Hold | ∞ | 4 |

     *\*NOTE: The annealing temperature (Tann) is dependent on the melting temperature (Tm) of the primers used. It is recommended to have Tann = Tm - 5 °C.*
   - The PCR product(s) can be checked on a agarose gel. In order to do so, cast a gel and prepare the samples according to the [DNA electrophoresis](#DNAgelelectrophoresis-scroll) protocol.

Dextrin-Capped Gold Nanoparticles (d-AuNPs) Generation

The generation of dextrin-capped gold nanoparticles generation was performed according to the protocol described by [Anderson *et al.* 2011](#references-scroll).

1. Prepare the following solutions:
   1. 20mM gold chloride salt (HAuCl4) in MilliQ. Store at 4 ºC.
   2. 25g/L dextrin in MilliQ.
   3. 10% (w/v) sodium carbonate (Na2CO3) in MilliQ.
   4. dH2O pH=9, adjusted with sodium hydroxide (NaOH).- Add 25mL of the dextrin solution (25g/L) to a sterile 250mL flask.
   - Add 5mL of the Gold Chloride salt solution (20mM).
   - Adjust pH of the solution to pH=9 with 10% (w/v) Na2CO3 by checking the pH with pH indicator strips.
   - Complete the reaction mixture by adding dH2O pH=9 up to a total reaction volume of 50mL.
   - Incubate the flask at 50 ºC in the dark with continuous shaking (250rpm) for 3 hours.
   - Measure the absorbance spectrum of the sample every 20 minutes.
     *NOTE: Change in color to red is the final indication of the ion Au+3 reduction to Au0.*
   - When the reaction mixture shows a clear shift in color to red, stop the reaction and store the dextrin-capped gold nanoparticle batch at room temperature in a closed glass container in the dark.

###### References:

1. [Anderson, M.J. *et al.*, 2011. One step alkaline synthesis of biocompatible gold
   nanoparticles using dextrin as capping agent. Journal of Nanoparticles Research, 13, pp2843–2851](https://link.springer.com/article/10.1007/s11051-010-0172-3)

Dextrin-Capped Gold Nanoparticles (d-AuNPs) Testing

Evaluating of the functionality of dextrin-capped gold nanoparticles is performed in a transparent 96 well plate with flat bottom. There are several analyses being conducted, which are:

* Salt stability
* ssDNA induced stability.
* ssDNA and target DNA stability.

The following table indicates different solutions needed for each type of analysis:

| Analysis | d-AuNPs (µL) | ssDNAp (µL) | NaCl solution (X mM) (µL) | dsDNA target (µL) | MilliQ / Hybridization buffer (µL) |
| --- | --- | --- | --- | --- | --- |
| Salt stability | 20 | 0 | 20 | 0 | 20 |
| ssDNA induced stability | 20 | 4 | 20 | 0 | 16 |
| ssDNA and target dsDNA induced stability | 20 | 4 | 20 | 100 | 6 |

For each specific functionality testing reaction, perform the following steps directly in the wells of the 96 wells plate, unless indicated otherwise.

#### Salt Stability

1. Add 20µL of MilliQ.
2. Add 20µL of NaCl solutions (varying concentrations).
3. Use one well as salt blank by adding 20µL MilliQ instead of NaCl solution.
4. Add 20µL of d-AuNPs.
5. Mix gently by pipetting and incubate at 21 ºC during 10 minutes.
6. Quantify the visible absorption spectrum of the solutions or measure absorbance at 520nm and 620nm.

#### ssDNAp Induced Stability

1. Add 4µL of ssDNAp (1µM).
2. Use one wells as ssDNAp blank by adding 4 µL MilliQ instead of ssDNAp.
3. Add 16µL of MilliQ.
4. Add 20µL of NaCl solutions (varying concentrations).
5. Use one well as salt blank by adding 20µL MilliQ instead of NaCl solution.
6. Add 20µL of d-AuNPs.
7. Mix gently by pipetting and incubate at 21 ºC during 10 minutes.
8. Quantify the visible absorption spectrum of the solutions or measure absorbance at 520nm and 620nm.

#### ssDNAp and dsDNA Target Induced Stability

Prepare the following reaction in a PCR tube:

1. Add 4µL of ssDNAp (1µM).
2. Use one well as ssDNAp blank by adding 4µL MilliQ instead of ssDNAp.
3. Add 10µL of dsDNA target (~1nM).
4. Add 6µL of Hybridization buffer.
5. Mix gently by pipetting and add tubes on thermal cycler with the following program:

   | Step | Time (s) | Temperature (°C) |
   | --- | --- | --- |
   | Denaturation | 300 | 95 |
   | Annealing | 60 | 57 |
   | Relaxation | 600 | 20 |

   - Add 20µL of the reaction mixture into the wells of 96 well plate
   - Add 20µL of NaCl solutions (varying concentrations or defined concentration based on previous results).
   - Use one well as salt blank by adding 20µL MilliQ instead of NaCl solution.
   - Add 20µL of d-AuNPs.
   - Mix gently by pipetting and incubate at 21 ºC during 10 minutes.
   - Quantify the visible absorption spectrum of the solutions or measure absorbance at 520nm and 620nm.

DNA Gel Electrophoresis

Gel Electrophoresis and DNA staining makes use of mutagenic chemicals like EtBr, SYBR Safe or any other DNA staining. Wear protection (gloves) when carrying out this protocol and work in an assigned area for this work to prevent contamination of the rest of the lab.

1. For a 0.8% (w/v) gel in TAE, weigh agarose powder for a 0.8% (w/v) gel.
   *E.g. weigh 1.6g and add to 200mL TAE buffer for a 0.8% gel.*
2. Add weighed agarose in TAE buffer (1X) and warm the solution (in a microwave) until it is completely dissolved. Let the solution cool down to hand warm temperature.
   *NOTE: Make sure the lid is not completely closed to avoid possible exploding of the glass bottle.*
3. Pour gel in gel tray and mix well with [SYBR Safe.](https://www.thermofisher.com/nl/en/home/life-science/dna-rna-purification-analysis/nucleic-acid-gel-electrophoresis/dna-stains/sybr-safe.html) For a small gel (~ 40mL solution) add 1µL of SYBR Safe; for a large gel (~80mL) add 2µL of SYBR Safe. Add a comb to create wells for the samples. Allow the agarose to solidify (approximately 20 minutes).
   *NOTE: For better mixing, add SYBR Safe to bottle instead of tray.*
4. Transfer the gel to the electrophoresis cell, remove the combs and cover the gel in TAE buffer (1X).
   *NOTE: Mind the direction of DNA migration when placing the gel in the cell.*
5. Prepare the electrophoresis samples by adding Nucleic Acid Loading Buffer conform the manufacturer’s instructions.
6. Load the molecular weight marker (DNA ladder) in the first well according to manufacturer’s instructions (generally 3-5µL) and load 5-10µL of the dyed samples in the other wells.
   *NOTE: Do not contaminate the loading buffer and ladder with SYBR Safe! Do not touch it while wearing a glove.*
7. Connect the cables of the gel tray following the colour code and run at 80-110V for 40-60 min.
   *NOTE: Mind the direction of DNA migration when placing the lid on the cell.
   NOTE: Time and voltage depend on the density of the gel and the length of the lane in the gel.*

DNA Purification from Agarose Gel

This protocol is based on the [Wizard® SV Gel and PCR Clean-Up System](https://nld.promega.com/products/dna-purification-quantitation/dna-fragment-purification/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281) of Promega Corporation.

1. Excise a proper band containing the DNA from gel and place the excised gel slice in a 1.5 mL Eppendorf tube.
2. Weigh the excised gel slice.
3. Add 10µL Membrane Binding Solution per 10 mg of gel slice. Vortex and incubate at 50-65 °C until gel slice is completely dissolved.
4. Insert the SV Minicolumn into the Collection Tube and label both of them according to the labelling of your samples.
5. Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate the Minicolumn at room temperature for 1 minute.
6. Centrifuge the SV Minicolumn at maximum speed for 1 minute.
7. Discard the flow through and reinsert the SV Minicolumn into the Collection Tube.
8. Add 700µL of Membrane Wash Solution.
   *NOTE: Upon prior use, dilute the solution with 95% ethanol following the manufacturer's’ instructions.*
9. Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
10. Discard the flow through and reinsert the SV Minicolumn into the Collection Tube.
11. Repeat the washing step with 500µL of Membrane Wash Solution and centrifuge for 5 minutes at maximum speed.
12. Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the Minicolumn assembly lid open to allow full evaporation of ethanol
    *NOTE: Leaving the column at room temperature ameliorates evaporation of residual ethanol traces.*
13. Transfer the SV Minicolumn to a clean labelled 1.5mL Eppendorf tube.
14. Add 50µL of pre-warmed MilliQ directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
    *NOTE: Use 30µL when higher final concentrations of DNA are required or when small quantities of DNA are suspected.*
15. Incubate the SV Minicolumn at room temperature for 5 minutes.
16. Centrifuge at maximum speed for 1 minute.
17. Discard the SV Minicolumn, cap the tube containing the eluted DNA, measure the DNA concentration and keep the DNA at 4 °C (for immediate use) or -20 °C (for storage).

DpnI Digestion

Our DpnI digestions were performed with [New England Biolabs DpnI (20.000 units/mL)](https://www.neb.com/products/r0176-dpni#Product%20Information), which is compatible with CutSmart Buffer by the same manufacturer.

1. Prepare a sample in a 0.5mL microcentrifuge tube as follows:

   | Component | Volume (µL) |
   | --- | --- |
   | 10x CutSmart buffer (NEB) | 4 |
   | Purified PCR product | 30 |
   | Restriction Enzyme DpnI | 1 |
   | MilliQ | 5 |

   - Incubate for 1.5 hours at 37 °C.
   - Heat inactivate the enzyme by incubating at 80 °C for 20 minutes.

dxCas9 Protein Functionality Testing: Mobility Shift

This protocol describes how the functionality of the gRNA:dxCas9:DNA complex or the gRNA:dxCas9-Tn5:DNA complex is evaluated to bind to the target DNA using a mobility shift assay. The protocol consists of two parts: binding of dxCas9(-Tn5) to the gRNA and binding of the gRNA:dxCas9(-Tn5) to the target DNA

#### gRNA:dxCas9(-Tn5) complex formation

*NOTE: all samples and reagents are kept on ice during preparation.*

1. Load the purified dxCas9 or dxCas9-Tn5 protein with gRNA provided by [Arbor Biotechnologies](https://arbor.bio/) by combining the following components:

1. 1-100nM dxCas9 or dxCas9-Tn5
2. 1.6-160nM gRNA
*NOTE: the gRNA and dxCas9 or dxCas9-Tn5 is mixed in a 1:1.6 molar ratio.* 3. 10X functionality buffer (20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol, 10mM MgCl2 pH 7.5)
4. MilliQ (add up to a final volume of 10µL)

2. Pipette the reagents in the following order in a 0.5mL tube: MilliQ, 10X functionality buffer, dxCas9 or dxCas9-Tn5 protein and gRNA.
3. Incubate at 37 °C for 10 minutes.

#### gRNA:dxCas9(-Tn5):Target DNA complex formation

1. Add 1nM Target DNA (our case EPO cDNA) to the gRNA:dxCas9 or gRNA:dxCas9-Tn5 complex.
2. Incubate at 37 °C for 1 hour.
3. Analyse the samples on a [5% native TBE polyacrylamide gel](#nativeTBEpolyacrylamidegel-scroll) to observe the mobility shift.

dxCas9 Protein Functionality Testing: Trypsin Resistance

This protocol describes how the trypsin resistance of the gRNA-loaded dxCas9 and dxCas9-Tn5 proteins were evaluated due to a protein conformational change. The protocol consists of two parts: binding of dxCas9(-Tn5) to the gRNA and the trypsin resistance assay

#### gRNA:dxCas9(-Tn5) complex formation

*NOTE: all samples and reagents are kept on ice during preparation.*

1. Load the purified dxCas9 or dxCas9-Tn5 protein with gRNA provided by [Arbor Biotechnologies](https://arbor.bio/) by adding the following components:

1. 1-100nM dxCas9 or dxCas9-Tn5
2. 1.6-160nM gRNA
*NOTE: the gRNA and dxCas9 or dxCas9-Tn5 is mixed in a 1:1.6 molar ratio.* 3. 10X functionality buffer (20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol, 10mM MgCl2 pH 7.5)
4. MilliQ (add up to a final volume of 10µL)

2. Pipette the reagents in the following order in a 0.5mL tube: MilliQ, 10X functionality buffer, dxCas9 or dxCas9-Tn5 protein and gRNA.
3. Incubate at 37 °C for 10 minutes.

#### Trypsin Resistance Assay

1. Add 0.1-10nM Trypsin to the gRNA:dxCas9 or gRNA:dxCas9-Tn5 complex.
2. Incubate at 23 °C for 30 minutes.
3. Analyse the samples on a [8% SDS Tris-Glycine polyacrylamide gel](#SDSPAGEelectrophoresis-scroll) to observe the trypsin resistance.

Electrocompetent Cells Preparation

This protocol spans over 3 days of execution time, starting from a -80 °C mother stock. Throughout the protocol, it is recommended to work under aseptic conditions in order to prevent contamination risks.

#### Day 1

1. Sterilize the required 50mL MilliQ and 250µL glycerol in advance.
2. Keep the -80 °C strain stock of interest on ice.
3. Streak the strain on [solid selective medium](#solidmedium-scroll) and incubate overnight at 37 °C.

#### Day 2

4. Prepare a 10mL [liquid starter culture](#liquidstarterculture-scroll) with one of the colonies that grew on the selective plate. Let the culture grow overnight at 37 °C, shaking at 180rpm.

#### Day 3

5. Use 0.5mL of the starter culture to inoculate 35mL selective liquid medium.
6. Grow at 37°C while shaking 250 rpm till an OD600 of ~0.5.
7. Centrifuge for 10 minutes at 4 °C at 3900rpm.
8. Discard the supernatant and resuspend pellet in 20mL cold MilliQ.
9. Centrifuge for 10 minutes at 4 °C at 3900rpm.
10. Discard the supernatant and resuspend pellet in 20mL cold MilliQ.
11. Centrifuge for 10 minutes at 4 °C at 3900rpm.
12. Discard supernatant and resuspend in 200µL 50% glycerol.
13. Prepare aliquots of 50µL.
14. Either transform the [electrocompetent cells](#electrocomptcellstrans-scroll) straight away or store the electrocompetent cells at -80 °C.

Electrocompetent Cells Transformation

*NOTE: This protocol allows for a single transformation.*

1. Thaw a 50µL aliquot of [electrocompetent cells](#electrocomptcellsprep-scroll) on ice.
2. Add ~200 ng DNA to the cells and keep on ice for 20 minutes.
   *NOTE: in case of purified plasmid DNA, 1ng DNA is enough.*
3. Transfer all the content to an electro-shock cuvette.
4. Electro-shock the cells with the Electro Cell Manipulator at 2.5kV.
5. Immediately add 0.2-1mL of recovery medium (eg. SOC-medium). Resuspend and transfer to a 1.5mL tube.
6. Incubate at 37 °C at 250rpm for 1 hour.
7. Plate the cells on [solid medium](#solidmedium-scroll) with appropriate antibiotics and incubate overnight at 37 °C.

G-Block Resuspension

This protocol is based on the instructions for G-Block resuspension given by  [Integrated DNA Technologies (IDT)](https://eu.idtdna.com/pages).

1. Centrifuge the tube containing the gBlock for 3−5 seconds (>3,000 x g) to pellet the material to the bottom of the tube.
2. Add an appropriate volume of sterile MilliQ to the tube for a desired final concentration. The required volume of MilliQ can be read from the table below (and the label on the IDT tube that contains your fragment):

   | Final concentration (ng/µL) | µL MilliQ to add to 250ng | µL MilliQ to add to 250ng | µL MilliQ to add to 250ng |
   | --- | --- | --- | --- |
   | 10 | 25 | 50 | 100 |
   | 20 | not recommended | 25 | 50 |
   | 50 | not recommended | 10 | 20 |
3. Incubate at 50 °C for 20 minutes.
4. Briefly vortex and centrifuge.
5. Store the resuspended gBlock at -20 °C.

Genomic DNA Isolation

*This protocol is based on MO BIO Laboratories Inc. UltraClean® Microbial DNA Isolation Kit. For more information on the composition of the MD solutions, and for safety precautions, we recommend consulting MO BIO Laboratories’  [kit manual.](https://www.google.com/url?q=https://mobio.com/media/wysiwyg/pdfs/protocols/12224.pdf&sa=D&ust=1539523909568000&usg=AFQjCNH3FVpt7FGSE7QuzdhzmSrmp_oB1A).*

1. Add 1.8 ml of microbial (bacteria, yeast) culture to a 2mL Collection Tube (provided) and centrifuge at 10,000 x g for 30 seconds at room temperature. Decant the supernatant and spin the tubes at 10,000 x g for 30 seconds at room temperature and completely remove the media supernatant with a pipette tip. NOTE: Based on the type of microbial culture, it may be necessary to centrifuge longer than 30 seconds.
2. Resuspend the cell pellet in 300µl of MicroBead Solution and gently vortex to mix. Transfer resuspended cells to MicroBead Tube.
3. Check Solution MD1. If Solution MD1 is precipitated, heat the solution at 60 °C until the precipitate has dissolved. Add 50µl of Solution MD1 to the MicroBead Tube.
4. Optional: To increase yields, to minimize DNA shearing, or for difficult cells, see Alternative lysis methods in the “Hints & Troubleshooting Guide” section before continuing.
5. Secure MicroBead Tubes horizontally using the MO BIO Vortex Adapter tube holder for the vortex (MO BIO Catalog# 13000-V1) or secure tubes horizontally on a flat-bed vortex pad with tape. Vortex at maximum speed for 10 minutes. (See “Hints & Troubleshooting Guide” for less DNA shearing).
6. Make sure the 2mL MicroBead Tubes rotate freely in the centrifuge without rubbing. Centrifuge the tubes at 10,000 x g for 30 seconds at room temperature.
   *CAUTION: Be sure not to exceed 10,000 x g or tubes may break.*
7. Transfer the supernatant to a clean 2mL Collection Tube (provided). Expect 300-350µl of supernatant.
8. Add 100 µl of Solution MD2, to the supernatant. Vortex for 5 seconds. Then incubate at 4 °C for 5 minutes.
9. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g.
10. Avoiding the pellet, transfer the entire volume of supernatant to a clean 2mL Collection Tube (provided). Expect approximately 450µL in volume.
11. Shake to mix Solution MD3 before use. Add 900 µl of Solution MD3 to the supernatant and vortex for 5 seconds.
12. Load about 700µL into the Spin Filter and centrifuge at 10,000 x g for 30 seconds at room temperature. Discard the flow through, add the remaining supernatant to the Spin Filter, and centrifuge at 10,000 x g for 30 seconds at room temperature. NOTE: A total of 2 to 3 loads for each sample processed are required. Discard all flow through liquid.
13. Add 300µL of Solution MD4 and centrifuge at room temperature for 30 seconds at 10,000 x g.
14. Discard the flow through.
15. Centrifuge at room temperature for 1 minute at 10,000 x g.
16. Being careful not to splash liquid on the spin filter basket, place Spin Filter in a new 2mL Collection Tube (provided)
17. Add 50µL of Solution MD5 to the center of the white filter membrane.
18. Centrifuge at room temperature for 30 seconds at 10,000 x g.
19. Discard Spin Filter. The DNA in the tube is now ready for any downstream application. No further steps are required. Store the DNA at -20 °C until further use.

Gibson Assembly

This Gibson Assembly protocol is based on the [protocol provided by New England Biolabs](https://international.neb.com/products/e2611-gibson-assembly-master-mix#Product%20Information).

1. Thaw 10µL of 2X Gibson Assembly mastermix (New England Biolabs) on ice.
2. Add backbone and insert; depending on the assembly, assembly pieces can be added in a predetermined ratio (recommended is ratio 1:3 mol vector over mol insert, so not a 1:3 ratio based on weight). Do not exceed the total volume of 10µL.
   *NOTE: An online ligation calculator can subsequently be used to calculate the amount of the assembly pieces that is required.*
3. If applicable, fill up the reaction volume to 20µL with MilliQ.

   | Component | Volume (µL) |
   | --- | --- |
   | Gibson Assembly Master Mix 2X (NEB) | 10 |
   | Vector | X (? ng) |
   | Insert fragment | Y (? ng) |
   | MilliQ | 10-X-Y |

   *NOTE: The vector should have compatible overhangs with the insert(s)*
4. Incubate the assembly reaction at 50 °C for 60 minutes and place on ice for subsequent [chemical transformation](#chemcomptcellstrans-scroll) or [electroporation](#electrocomptcellstrans-scroll). Otherwise, store at -20 °C.

High Fidelity PCR

1. For each PCR in 50µL reaction volume, make sure the composition is as follows:

   | Component | Volume (µL) | Final concentration |
   | --- | --- | --- |
   | 5X Phusion HF buffer | 10 | 1X |
   | 10mM dNTPs | 1 | 200µM |
   | Primer forward (10µM) | 2.5 | 200nM |
   | Primer reverse (10µM) | 2.5 | 200nM |
   | DNA template | ~10ng to 250ng\* |  |
   | Phusion polymerase | 0.5 | 20U/mL |
   | MilliQ | up to 50µL |  |

   *\* NOTE: The 10ng - 250ng of template DNA are approximate, choose a volume that works fine for all your samples.
   NOTE: High fidelity PCR with Phusion polymerase can be optimized per case by adding 3% of DMSO.*
2. Close all tubes thoroughly and place them in a thermocycler with the following protocol:

   | Step | Temperature (°C) | Time (s) |
   | --- | --- | --- |
   | Initial denaturation | 98 | 30 |
   | Denaturation | 98 | 10 |
   | Annealing | Tann\* | 15 |
   | Extension | 72 | 15-30 sec per kb DNA |
   | Final extension | 72 | 300 |
   | Hold | 4 | ∞ |

   *\* NOTE: The annealing temperature (Tann) is dependent on the melting temperature (Tm) of the primers used. It is recommended to have Tann = Tm - 5 °C.*
3. The PCR product(s) can be checked on agarose gel. In order to do so, cast a gel and prepare the samples according to the [DNA electrophoresis](#DNAgelelectrophoresis-scroll) protocol.

Ligation

1. Thaw the ligase buffer on ice, to prevent damaging the ATP.
2. Prepare a sample as follows:

   | Component | Volume (µL) |
   | --- | --- |
   | 10X Ligase buffer | 2 |
   | T4 DNA Ligase | 1 |
   | DNA vector | X (~100ng) |
   | DNA fragment | Y\* |
   | MilliQ | 17-X-Y |

   *\* NOTE: The desired vector:insert ratio will be 1:3. Use a ligation calculator to calculate the amounts of vector DNA and insert DNA to be added.*
3. Incubate for at least one hour at 4 °C. Optimally, incubate overnight at 4 °C.

Liquid Medium Preparation

1. Dissolve Luria Broth powder in water according to instructions by manufacturer.
2. Heat sterilize (121 °C) the medium in the autoclave.
3. After cooling down, add the required antibiotic under aseptic conditions.
   *NOTE: With our antibiotic stock solutions of 1000x we used 1µL of antibiotic solution per mL of LB-agar.*
4. Store the medium at 4 °C if complemented with antibiotics.

Liquid Starter Culture (10mL)

1. Label as many 15mL Falcon tubes/Erlenmeyer flasks as the number of colonies you want to grow.
2. Under aseptic conditions, distribute 10mL of liquid medium (LB or SOC) for each starter culture.
   *NOTE: Supply with relevant antibiotics.*
3. Under aseptic conditions, pick a colony with the inoculation loop and swirl it in the starter culture to inoculate.
   *NOTE: A picked colony cannot be used again; it is recommended to restreak on a 'back-up'-plate and incubate it overnight at 37 °C.*
4. Grow the liquid cultures at 37 °C overnight, shaking at 250rpm.

MinION Flow Cell Washing

This protocol describes the steps to be taken to wash a flow cell after performing a sequencing run.

1. Thaw components of the Wash kit (WKE\_1012) at room temperature.
2. After the sequencing run has stopped, add 150µL of Buffer A to the priming port (without opening the SpotOn port).
3. Incubate for 10 minutes at room temperature.

For using immediately:

4. Add 500 µL of buffer B slowly on the priming port.
5. Slowly take out the waste solution from the waste chamber.
6. The flow cell is ready for priming and adding a new DNA library.

For storage:

7. Add 500 µL of storage buffer slowly on the priming port.
8. Slowly take out the waste solution from the waste chamber.
9. Store flow cell at 4 ºC.

Nanodrop DNA quantification

This protocol intends to quantify DNA amount, as well as the purity, in DNA purification or extraction samples, through spectroscopy. Make sure the Nanodrop machinery is installed to measure at wavelengths for dsDNA (260nm) and impurities at 230nm and 280nm.

1. Clean the measurement surface with a piece of tissue and ethanol.
2. Use 1µL of sterile MilliQ as a blank.
3. Clean the measurement surface with a piece of tissue.
4. Use 1µL of a sample to measure its concentration.
   *NOTE: It is best to measure the same sample in triplo and use the average value. If you have multiple samples, clean the measurement surface in between measurements.*
5. When done measuring, wipe the measurement surface with a tissue and MilliQ.

Nanodrop Protein quantification

This protocol intends to quantify protein amount, as well as the purity, in purification or extraction samples, through spectroscopy. Make sure the Nanodrop machinery is installed to measure at wavelengths for 280nm and 260nm.

1. Clean the measurement surface with a piece of tissue and ethanol.
2. Use 2µL of sterile MilliQ as a blank.
3. Clean the measurement surface with a piece of tissue.
4. Use 2µL of a sample to measure its concentration.
   *NOTE: It is best to measure the same sample in triplo and use the average value. If you have multiple samples, clean the measurement surface in between measurements.*
5. When done measuring, wipe the measurement surface with a tissue and MilliQ.

Nanodrop RNA quantification

This protocol intends to quantify RNA amount, as well as the purity, in purification or extraction samples, through spectroscopy. Make sure the Nanodrop machinery is installed to measure at wavelengths for ssRNA (260nm) and impurities at 230nm and 280nm.

1. Clean the measurement surface with a piece of tissue and ethanol.
2. Use 1µL of sterile MilliQ as a blank.
3. Clean the measurement surface with a piece of tissue.
4. Use 1µL of a sample to measure its concentration.
   *NOTE: It is best to measure the same sample in triplo and use the average value. If you have multiple samples, clean the measurement surface in between measurements.*
5. When done measuring, wipe the measurement surface with a tissue and MilliQ.

Native TBE Polyacrylamide Gel: Ethidium Bromide Staining

A native PAGE electrophoresis is used to separate DNA based on their size by using an electric current. This protocol describes how to prepare a native PAGE gels, how to prepare samples and how to run the electrophoresis.
*NOTE: In this protocol we use a 5% separation gel (this is for DNA samples between 50 bp and 1,000 bp).*

#### Preparing the native PAGE gel

1. Prepare the following solutions for 2 gels (total 12mL):

   | Component | Volume |
   | --- | --- |
   | 40% Acrylamide | 1.5mL |
   | 10X TBE buffer | 1.2mL |
   | 10% APS | 120µL |
   | TEMED | 12µL |
   | MilliQ | 9.168mL |

   *NOTE: Polymerization will start when 10% APS and TEMED are added.*
2. Assemble the casting station and pour the separation gel between the glass plates.
3. Immediately place the gel comb on top of the gel.
4. Allow the gel to solidify, then remove the plates from the casting station.
5. The gels are ready for usage, or can be stored at 4 °C for later usage.

#### Preparing samples

6. Add 2μL protein 6X Loading Dye to 10μL of sample.

#### Running PAGE gel

7. Run the gel at 150V and 25mA.
8. The gel is finished when the purple line is at the end of the gel.

#### Processing PAGE gel: Ethidium bromide staining

9. Turn off the power pack before opening the gel box.
10. Remove the gel from the cassette and place the gel in a clean staining tray of the appropriate size filled with a layer of MilliQ or TBE buffer.
11. Gently allow the gel to slide from the glass plate into the water.
12. Add 1µL of Ethidium bromide into the water.
13. Allow the gel to shake on a moving platform for 10 minutes.
14. Take a picture with the Gel doc system/Typhoon.

Native TBE Polyacrylamide Gel: Silver Staining

A native PAGE electrophoresis is used to separate DNA based on their size by using an electric current. This protocol describes how to prepare a native PAGE gels, how to prepare samples and how to run the electrophoresis.
*NOTE: In this protocol we use a 5% separation gel (this is for DNA samples between 50 bp and 1,000 bp).*

#### Preparing te native PAGE gel

1. Prepare the following solutions for 2 gels (total 12mL):

   | Component | Volume |
   | --- | --- |
   | 40% Acrylamide | 1.5mL |
   | 10X TBE buffer | 1.2mL |
   | 10% APS | 120µL |
   | TEMED | 12µL |
   | MilliQ | 9.168mL |

   *NOTE: Polymerization will start when 10% APS and TEMED are added.*
2. Assemble the casting station and pour the separation gel between the glass plates.
3. Immediately place the gel comb on top of the gel.
4. Allow the gel to solidify, then remove the plates from the casting station.
5. The gels are ready for usage, or can be stored at 4 °C for later usage.

#### Preparing samples

6. Add 2μL protein 6X Loading Dye to 10μL of sample.

#### Running PAGE gel

7. Run the gel at 150V and 25mA.
8. The gel is finished when the purple line is at the end of the gel.

#### Processing PAGE gel: Silver staining

9. Turn off the power pack before opening the gel box.
10. Remove the gel from the cassette and place the gel in a clean staining tray of the appropriate size filled with a layer of MilliQ or TBE buffer.
11. Gently allow the gel to slide from the glass plate into the water.
12. Discard the water.
13. Prepare the following solutions for silver staining with SilverQuest Silver staining kit.

    | Solution | Components |
    | --- | --- |
    | Fixing solution | 10mL Acetic Acid  40mL Ethanol  50mL MilliQ |
    | Sensitizing solution | 30mL Ethanol  10mL Sensitizer  60mL MilliQ |
    | Staining solution | 1mL Silver Stainer  100mL MilliQ |
    | Developing solution | 10mL Developer  1 drop Developer Enhancer  90mL MilliQ |

    *NOTE: All incubations are performed on a rotary shaker rotating at a speed of 1 revolution/second at room temperature.*
14. Cover the gel with 100mL fixing solution for one hour with gentle rotation.
    *NOTE: The gel can be stored in the fixative overnight if there is not enough time to complete the staining protocol.*
15. Decant the fixative solution and wash the gel in 30% ethanol for 10 minutes.
16. Decant the ethanol and wash the gel in 100mL of Sensitizing solution for 10 minutes.
17. Decant the Sensitizing solution and wash the gel in 100mL of 30% ethanol for 10 minutes.
18. Decant the 30% ethanol and wash the gel in 100mL of ultrapure water for 10 minutes.
19. Decant the water and indicate the gel in 100mL of Staining solution for 15 minutes.
20. Decant the Staining solution and wash the gel with 100mL of ultrapure water for 20–60 seconds.
    *Note: Washing the gel for more than a minute will remove silver ions from the gel resulting in decreased sensitivity.*
21. Incubate the gel in 100mL of Developing solution for 4–8 minutes until bands start to appear and the desired band intensity is reached.
22. After the appropriate staining intensity is achieved, immediately add 10 mL of Stopper directly to the gel still immersed in Developing solution.
23. Gently agitate the gel for 10 minutes.
    *Note: The color changes from pink to colorless indicating that the development has stopped.*
24. Decant the Stopper solution and wash the gel with 100 mL of ultrapure water for 10 minutes.
25. Take a picture with the Gel doc system.

PCR Clean-Up

This protocol is based on the [Wizard® SV Gel and PCR Clean-Up System](https://nld.promega.com/products/dna-purification-quantitation/dna-fragment-purification/wizard-sv-gel-and-pcr-clean-up-system/?catNum=A9281)  of Promega Corporation.

1. Add an equal volume of Membrane Binding Solution to the volume of PCR product.
2. Insert the SV Minicolumn into Collection Tube and label both of them according to the labelling of your samples.
3. Transfer the dissolved gel mixture to the Minicolumn assembly. Incubate the Minicolumn at room temperature for 1 minute.
4. Centrifuge the SV Minicolumn at maximum speed for 1 minute.
5. Discard the flow through and reinsert the SV Minicolumn into the Collection Tube.
6. Add 700µL of Membrane Wash Solution.
   *NOTE: Upon prior use, dilute the solution with 95% ethanol following the manufacturer's’ instructions.*
7. Centrifuge the SV Minicolumn assembly at maximum speed for 1 minute.
8. Discard the flow through and reinsert the SV Minicolumn into the Collection Tube.
9. Repeat the washing step with 500µL of Membrane Wash Solution and centrifuge for 5 minutes at maximum speed.
10. Once the Collection Tube is empty, centrifuge the Minicolumn assembly at maximum speed for 1 minute with the microcentrifuge lid open to allow ethanol full evaporation.
    *NOTE: Leaving the column at room temperature ameliorates evaporation of residual ethanol traces.*
11. Transfer the SV Minicolumn to a clean labelled 1.5mL Eppendorf tube.
12. Add 50µL of pre-warmed MilliQ directly to the centre of the SV Minicolumn, without touching the membrane with the pipette tip.
    *NOTE: Use 30µL when higher final concentrations of DNA are required or when small quantities of DNA are suspected.*
13. Incubate the SV Minicolumn at room temperature for 5 minutes.
14. Centrifuge at maximum speed for 1 minute.
15. Discard the SV Minicolumn, cap the tube containing the eluted DNA and keep the DNA at 4 °C (for immediate use) or -20 °C (for storage).

p-GEM T-Easy Cloning

The protocol is based on Promega’s [pGEM T-Easy Vector System](https://nld.promega.com/products/pcr/pcr-cloning/pgem-t-easy-vector-systems/?catNum=A1360) technical manual.
pGEM T-Easy vector system can be used to clone PCR amplification products by Taq polymerase on separate plasmids. The following steps are done to complete the protocol:
Briefly centrifuge the pGEM.-T Easy Vector and insert DNA tubes.

1. Set up ligation reactions in the following order:

* 5µl 2x Rapid Ligation Buffer, T4 DNA Ligase
* 1µl pGEM-T Easy vector
* Xµl PCR product (to obtain 3:1 insert:vector molar ratio)
* 1µl T4 DNA Ligase
* Xµl MilliQ to 10µl

2. Incubate overnight at 4C.
3. Transform ligates into competent cells according to the [chemical competent cell transformation protocol](#chemcomptcellstrans-scroll).
4. Blue/white colony screening to chose for colonies with inserts.
5. To calculate the amount of PCR product needed, the following formula can be used:
   ![equation](https://static.igem.org/mediawiki/2018/a/a6/T--TUDelft--equationprotocol.png)

Plasmid Isolation

This protocol is based on the protocol supplied with the [Promega PureYield™ Plasmid Miniprep Kit](https://nld.promega.com/resources/protocols/technical-bulletins/101/pureyield-plasmid-miniprep-system-protocol/) of Promega Corporation.

1. Centrifuge 1.5-3mL of a [liquid (starter) culture](#liquidstarterculture-scroll) for 1 minute in a 1.5mL microcentrifuge tube at maximum speed.
2. Discard the supernatant and resuspend in 600µL of MilliQ.
3. Add 100µL of Cell Lysis Buffer, and mix by inverting the tube.
4. Add 350µL of cold (4–8 °C) Neutralization Solution, and mix thoroughly by inverting.
5. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
6. Transfer the supernatant (~900µl) to a PureYield™ Minicolumn without disturbing the cell debris pellet.
7. Place the minicolumn into a Collection Tube and centrifuge at maximum speed in a microcentrifuge for 15 seconds.
8. Discard the flow through, and place the minicolumn into the same Collection Tube.
9. Add 200µl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 15 seconds.
10. Add 400µl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at maximum speed in a microcentrifuge for 30 seconds.
11. Transfer the minicolumn to a clean, labelled 1.5 ml Eppendorf tube, then add 30µl of sterile MilliQ directly to the minicolumn matrix. Incubate for 1 minute at room temperature.
12. Centrifuge for 15 seconds to elute the plasmid DNA. Discart the column and label the tube. The isolate can be stored at -20 °C or directly used.

Primer Working Stock

This protocol is based on the instructions for primer resuspension given by  [Integrated DNA Technologies (IDT).](https://eu.idtdna.com/pages)

1. Centrifuge the tube containing the primer for 3−5 seconds (>3,000 x g) to pellet the material to the bottom of the tube.
2. Dissolve the DNA material in sterile MilliQ according to the supplier IDT. This creates a 100µM stock solution.
3. Heat the primer stock solution to 65 °C for 20 minutes.
4. Centrifuge the primer stock solution at maximum speed (~17,000 x g) for 2 minutes.
5. Prepare a 10X diluted work solution (10µM) by dilution with sterile MilliQ.
6. Store both Stock and Work solutions at -20 °C.

Protein Expression

All work was performed within a sterile field created by a bunsen burner flame.

#### Cell Culture

1. Inoculate a single colony from an agar plate containing transformed cells in a [10 mL starter culture](#liquidstarterculture-scroll) with appropriate antibiotic selection marker.
2. Let the seed culture grow overnight at 37 °C with 180 rpm rotation.
3. Measure the OD600 to confirm growth (an OD600 of around 2 is to be expected).
4. Prepare 1L of media with the required antibiotics.
5. Inoculate the media with 10 mL of the seed culture (1:100 ratio).
6. Let the culture grow until an OD600 of 0.5.
7. When the required OD is reached, put the culture on ice for 30 minutes.
8. After the 30 minutes, induce the expression of the protein by adding 1M IPTG to a final concentration of 1mM and 20% arabinose to a final concentration 0.2% arabinose.
9. Grow the cells for 16 hours ot 18 °C and 180rpm rotation.

#### Cell Harvest

10. Harvest the liquid culture by centrifugation at 5200g and 4 °C for 15 minutes.
11. Discard the supernatant and weigh the pellet. Resuspend the pellet with 6mL PBS/g cells.
12. Centrifuge the cells 15 minutes at 5200 g and 4 °C, and again discard supernatant.
13. The cells are now directly passed off to downstream processing.

Protein Purification dxCas9

This protocol describes the optimized downstream processing of dxCas9.

#### Cell Lysis

1. Retrieve a washed cell pellet from upstream processing according to the [protein expression](#proteinexpressionandlysis-scroll) protocol.
   *NOTE: All following steps in this purification protocol are done at 4 °C.*
2. Resuspend the pellet in lysis buffer (20mM Tris-HCL, 250mM NaCl, 10% v/v glycerol, 1mM DTT, 5mM imidazole, pH 8.0) and one protease inhibitor tablet per 50mL lysis buffer. Make sure pellet is fully resuspended.
3. Lyse the cells using a high-pressure homogenizer (French Press) (2 rounds at 1 kbar).

#### Clarification

4. Clarify the lysate via centrifugation for 45 min at 16,000 g.
   *NOTE: The dxCas9 is now dissolved in the supernatant and the pellet contains cell debris.*

#### Nickel Affinity Chromatography

5. Perform Nickel affinity chromatography with a gravity column.
   *NOTE: 1mL of 50% His-Select Ni resin per 15mL clarified lysate for high expression proteins.*
6. Resuspend 1mL of 50% Hisselect Nickel Affinity gel in 10mL (20CV) water, spinning down at 3220g for 1 min and discard the supernatant. Repeat this three times.
7. Equilibrate the washed resin with 10mL (20CV) of equilibration buffer (20mM Tris-HCl, 250mM NaCl, 5mM Imidazole, 10% v/v glycerol, 1mM DTT, pH8), spinning down at 3220g for 1 min and discard the supernatant.
8. Add the Hisselect column material to the clarified lysate and incubate for one hour with gentle mixing.
9. Spin down the mixture, for 1 min at 2000 g.
   *NOTE: This mixture contains dxCas9 bound to Hisselect Nickel resin.*
10. Discard the supernatant by pipetting and load the resin onto a gravity column.
11. Wash the resin five times in 2mL (20CV) of washing buffer (20mM Tris-HCl, 250mM NaCl, 5mM Imidazole, 10% v/v glycerol, 1mM DTT, pH8) and collect the flowthrough.
12. Elute the dxCas9 from the resin with six fractions of 500μL of elution buffer (20mM Tris-HCl, 250mM NaCl, 1mM DTT and 250mM imidazole, pH 8.0), and collect in differently labeled tubes.
13. Measure the A280 of all six elution fractions at the Nanodrop, to roughly estimate the protein concentrations.
14. Load the all the samples including the elution fractions onto an [SDS 8% Tris-Glycine PAGE gel](#SDSPAGEelectrophoresis-scroll) to confirm presence of the protein.
15. Pool the fraction that contains the dxCas9.

#### Heparin Chromatography

16. Perform heparin chromatography on the AKTA pure with a 1 mL HiTrap Heparin HP column.
17. Set the following settings:
    * Flow rate: 0.5mL/min
    * Maximum column pressure: 0.5MPa- Perform the following method on the AKTA.

      | Chromatography step | Buffer | Column Volume (CV) | Volume (mL) | Fractionation |
      | --- | --- | --- | --- | --- |
      | Column washing | Water | 20 | 20 | 0 Fractions |
      | Column equilibrating | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM DTT, pH 7.5 | 20 | 20 | 0 Fractions |
      | Sample loading | Pooled dxCas9 fractions | 1-20 | 1-20 | 1 Fraction in the appropriate tube |
      | Washing | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM DTT, pH 7.5 | 20 | 20 | 1 Fraction in a 50mL Falcon tube |
      | Eluting | 20mM Tris-HCl, 1.5M NaCl, 10% v/v glycerol, 1mM DTT, pH 7.5 | 30 | 30 | 60 Fractions in a 1mL tube |
      | Column Washing | Water | 20 | 20 | 0 Fractions |
      | Column Storage | 20% ethanol | 20 | 20 | 0 Fractions |
    - Based on the A280 seen on the chromatogram select desired elution fractions that contains protein.
    - Load the all the samples including the desired elution fractions onto an [SDS 8% Tris-Glycine PAGE gel](#SDSPAGEelectrophoresis-scroll) to confirm presence of the protein.

    #### Dialysis

    - Pool the fraction that contain the dxCas9.
    - Prepare 2L of dialysis buffer (20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol and pH 7.5).
    - Inject the pooled fractions (0.5-3mL) into a pierce 10,000 Molecular weight cut off cassette.
    - Buffer exchange by dialysis of the pooled fractions for 1 hour in 1L dialysis buffer.
    - Replace the dialysis buffer with fresh buffer and repeat dialysis for 1 hour.
    - Remove the dxCas9 solution out of the dialysis cassette and measure the concentration with the [Pierce BCA protein assay kit](#BCAproteinquantification-scroll) by Thermo Scientific.
    - Aliquote the dxCas9 solution in 25µL aliquots and freeze at -20℃ for functionality testing assays.

Protein Purification Tn5

This protocol describes the optimized downstream processing of Tn5.

#### Cell Lysis

1. Retrieve a washed cell pellet from upstream processing according to the [protein expression](#proteinexpressionandlysis-scroll) protocol.
   *NOTE: All following steps in this purification protocol are done at 4 °C.*
2. Resuspend the pellet in lysis buffer (20mM Tris-HCL, 250mM NaCl, 10% v/v glycerol, 1mM DTT, 5mM imidazole, pH 8.0) and one protease inhibitor tablet per 50mL lysis buffer. Make sure pellet is fully resuspended.
3. Lyse the cells using a high-pressure homogenizer (French Press) (2 rounds at 1 kbar).

#### Clarification

4. Clarify the lysate via centrifugation for 45 min at 16,000 g.
   *NOTE: The Tn5 is now dissolved in the supernatant and the pellet contains cell debris.*

#### Nickel Affinity Chromatography

5. Perform Nickel affinity chromatography with a gravity column.
   *NOTE: 1mL of 50% His-Select Ni resin per 15mL clarified lysate for high expression proteins.*
6. Resuspend 1mL of 50% Hisselect Nickel Affinity gel in 10mL (20CV) water, spinning down at 3220 g for 1 min and discard the supernatant. Repeat this three times.
7. Equilibrate the washed resin with 10mL (20CV) of equilibration buffer (20mM Tris-HCl, 250mM NaCl, 5mM Imidazole, 10% v/v glycerol, 1mM DTT, pH8), spinning down at 3220 g for 1 min and discard the supernatant.
8. Add the Hisselect column material to the clarified lysate and incubate for one hour with gentle mixing.
9. Spin down the mixture, for 1 min at 2000 g.
   *NOTE: This mixture contains Tn5 bound to Hisselect Nickel resin.*
10. Discard the supernatant by pipetting and load the resin onto a gravity column.
11. Wash the resin five times in 2 mL (20CV) of washing buffer (20mM Tris-HCl, 250mM NaCl, 5mM Imidazole, 10% v/v glycerol, 1mM DTT, pH8) and collect the flowthrough.
12. Elute the Tn5 from the resin with six fractions of 500μL of elution buffer (20mM Tris-HCl, 250mM NaCl, 1mM DTT and 250mM imidazole, pH 8.0), and collect in differently labeled tubes.
13. Measure the A280 of all six elution fractions at the Nanodrop, to roughly estimate the protein concentrations.
14. Load the all the samples including the elution fractions onto an [SDS 12% Tris-Glycine PAGE gel](#SDSPAGEelectrophoresis-scroll) to confirm presence of the protein.
15. Pool the fractions that contain the Tn5.

#### Heparin Chromatography

16. Perform heparin chromatography on the AKTA pure with a 1 mL HiTrap Heparin HP column.
17. Set the following settings:
    * Flow rate: 0.5mL/min
    * Maximum column pressure: 0.5 MPa- Perform the following method on the AKTA.

      | Chromatography step | Buffer | Column Volume (CV) | Volume (mL) | Fractionation |
      | --- | --- | --- | --- | --- |
      | Column washing | Water | 20 | 20 | 0 Fractions |
      | Column equilibrating | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM DTT, pH 7.5 | 20 | 20 | 0 Fractions |
      | Sample loading | Pooled Tn5 fractions | 1-20 | 1-20 | 1 Fraction in the appropriate tube |
      | Washing | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM DTT, pH 7.5 | 20 | 20 | 1 Fraction in a 50mL Falcon tube |
      | Eluting | 20mM Tris-HCl, 1.5M NaCl, 10% v/v glycerol, 1mM DTT, pH 7.5 | 30 | 30 | 60 Fractions in a 1 mL tube |
      | Column Washing | Water | 20 | 20 | 0 Fractions |
      | Column Storage | 20% ethanol | 20 | 20 | 0 Fractions |
    - Based on the A280 seen on the chromatogram select desired elution fractions that contains protein.
    - Load the all the samples including the desired elution fractions onto an [SDS 12% Tris-Glycine PAGE gel](#SDSPAGEelectrophoresis-scroll) to confirm presence of the protein.

    #### Dialysis

    - Pool the fraction that contain the Tn5.
    - Prepare 2L of dialysis buffer (20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol and pH 7.5).
    - Inject the pooled fractions (0.5-3mL) into a pierce 10,000 Molecular weight cut off cassette.
    - Buffer exchange by dialysis of the pooled fractions for 1 hour in 1L dialysis buffer.
    - Replace the dialysis buffer with fresh buffer and repeat dialysis for 1 hour.
    - Remove the Tn5 solution out of the dialysis cassette.

    #### Protein Concentration

    - Wash a Amicon ultra-15mL centrifugal filter units with an 10,000 molecular weight cutoff with MilliQ water by centrifuging at 4,000 × g for approximately 10–20 minutes.
    - Add the Tn5 sample to the filter and centrifuged at 4,000 × g for approximately 10–20 minutes.
    - Measure the Tn5 protein concentration with the [Pierce BCA protein assay kit](#BCAproteinquantification-scroll) by Thermo Scientific.
    - Aliquote the Tn5 solution in 25µL aliquots and freeze at -20℃ for functionality testing assays.

Protein Purification dxCas9-Tn5

This protocol describes how the downstream processing of dxCas9-Tn5 was performed.

#### Cell Lysis

1. Retrieve a washed cell pellet from upstream processing according to the [protein expression](#proteinexpressionandlysis-scroll) protocol.
   *NOTE: All following steps in this purification protocol are done at 4 °C.*
2. Resuspend the pellet in lysis buffer (20mM Tris-HCL, 250mM NaCl, 1mM DTT, 10% v/v glycerol, 1mM PMSF, pH 7.5) and one protease inhibitor tablet per 50mL lysis buffer. Make sure pellet is fully resuspended.
3. Lyse the cells using a high-pressure homogenizer (French Press) (2 rounds at 1 kbar).

#### Clarification

4. Clarify the lysate via centrifugation for 45 min at 16,000 g.
   *NOTE: The dxCas9-Tn5 is now dissolved in the supernatant and the pellet contains cell debris.*
5. Filter the clarified lysate with a 0.45µm filter.

   #### Heparin Chromatography

   - Perform heparin chromatography on the AKTA pure with a 1mL HiTrap Heparin HP column.
   - Set the following settings:
     * Flow rate: 0.5mL/min
     * Maximum column pressure: 0.5 MPa- Perform the following method on the AKTA.

       | Chromatography step | Buffer | Column Volume (CV) | Volume (mL) | Fractionation |
       | --- | --- | --- | --- | --- |
       | Column washing | Water | 20 | 20 | 0 Fractions |
       | Column equilibrating | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM PMSF, pH 7.5 | 20 | 20 | 0 Fractions |
       | Sample loading | Crude dxCas9-Tn5 extract | 1-50 | 1-50 | 1 Fraction in the appropriate tube |
       | Washing | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM PMSF, 1mM DTT, pH 7.5 | 20 | 20 | 1 Fraction in a 50 mL Falcon tube |
       | Eluting | 20mM Tris-HCl, 1.5M NaCl, 10% v/v glycerol, 1mM PMSF, 1mM DTT, pH 7.5 | 30 | 30 | 60 Fractions in a 1mL tube |
       | Column Washing | Water | 20 | 20 | 0 Fractions |
       | Column Storage | 20% ethanol | 20 | 20 | 0 Fractions |
     - Based on the A280 seen on the chromatogram select desired elution fractions that contains protein.
     - Load the all the samples including the desired elution fractions onto an [SDS 8% Tris-Glycine PAGE gel](#SDSPAGEelectrophoresis-scroll) to confirm presence of the protein.

     #### MonoQ Chromatography

     - Perform anionic exchange chromatography on the AKTA pure with a 1mL MonoQ 5/50 GL column on the crude extract.- Set the following settings:
         * Flow rate: 0.5mL/min
         * Maximum column pressure: 20 MPa- Follow the following method on the AKTA

           | Chromatography step | Buffer | Column Volume (CV) | Volume (mL) | Fractionation |
           | --- | --- | --- | --- | --- |
           | Column washing | Water | 20 | 20 | 0 Fractions |
           | Column equilibrating | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM PMSF, 1mM DTT, pH 8.5 | 20 | 20 | 0 Fractions |
           | Sample loading | Pooled fractions diluted 4 times with 20mM Tris-HCl, 0mM NaCl, 10% v/v glycerol, 1mM PMSF, 1mM DTT, pH 8.5 | 1-20 | 1-20 | 1 Fraction in the appropriate tube |
           | Washing | 20mM Tris-HCl, 250mM NaCl, 10% v/v glycerol, 1mM PMSF, 1mM DTT, pH 8.5 | 20 | 20 | 1 Fraction in a 50 mL Falcon tube |
           | Eluting | 20mM Tris-HCl, 1.5M NaCl, 10% v/v glycerol, 1mM PMSF, 1mM DTT, pH 8.5 | 30 | 30 | 60 Fractions in a 1 mL tube |
           | Column Washing | Water | 20 | 20 | 0 Fractions |
           | Column Storage | 20% ethanol | 20 | 20 | 0 Fractions |
         - Based on the A280 seen on the chromatogram select desired elution fractions that contains protein.
         - Load the all the samples including the desired elution fractions onto an [SDS 8% Tris-Glycine PAGE gel](#SDSPAGEelectrophoresis-scroll) to confirm presence of the protein.

         #### Dialysis

         - Pool the fraction that contain the dxCas9-L-Tn5.
         - Prepare 2L of dialysis buffer (20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol, 1mM PMSF and pH 7.5).
         - Inject the pooled fractions (0.5-3mL) into a pierce 10,000 Molecular weight cut off cassette.
         - Buffer exchange by dialysis of the pooled fractions for 1 hour in 1L dialysis buffer.
         - Replace the dialysis buffer with fresh buffer and repeat dialysis for 1 hour.
         - Remove the dxCas9-Tn5 solution out of the dialysis cassette.

         #### Protein Concentration

         - Wash a Amicon ultra-15mL centrifugal filter units with an 10,000 molecular weight cutoff with MilliQ water by centrifuging at 4,000 × g for approximately 10–20 minutes.
         - Add the dxCas9-Tn5 sample to the filter and centrifuged at 4,000 × g for approximately 10–20 minutes.
         - Measure the dxCas9-Tn5 protein concentration with the [Pierce BCA protein assay kit](#BCAproteinquantification-scroll) by Thermo Scientific.
         - Aliquote the dxCas9-Tn5 solution in 25µL aliquots and freeze at -20℃ for functionality testing assays.

Qubit High Sensitivity dsDNA quantification

This protocol is based on the manual for the  [Qubit dsDNA High Sensitivity Assay Kit](https://www.thermofisher.com/order/catalog/product/Q32854) provided by Thermofischer Scientific.

1. Set up the required amount of tubes for your samples and add two for the calibration standards.
2. Label the lids. Do not label the sides of the tubes, since this will interfere with the measurements.
3. Prepare the Qubit working solution.
   n = amount of sample + two standards
4. Dilute n µL of dsDNA HS Reagent 1:200 in n \* 199µL dsDNA HS Buffer. Use a clean plastic tube each time you prepare a working solution. Do not use glass containers.
5. Add 190µL of the working solution to the tubes for the two standards.
6. Add 10µL of the standards to the according tubes.Mix by vortexing 2-3 seconds and be careful not to create bubbles.
7. Add 180-199µL of working solution to the sample tubes, dependent on the amount of sample to be measured. The amount of sample used for the measurement can range from 1 to 20µL. Dependent on this amount the added volume of working solution should be adapted, so that the final volume is always 200µL.
8. Add each sample to the according assay tube. Mix by vortexing 2-3 seconds.
9. Allow all tubes to incubate at room temperature for 2 minutes.
10. Read the standards and samples following the Qubit instrument instructions.

Restriction

1. Decide on which restriction enzyme(s) to cut with. Check [online (NEB)](https://international.neb.com/tools-and-resources/usage-guidelines/nebuffer-performance-chart-with-restriction-enzymes) what buffer the enzyme(s) work(s) in . For most of the restriction enzymes, the CutSmart buffer 10X can be used.
2. Prepare a 20µL sample as follows:

| Component | Volume (µL) |
| --- | --- |
| 10X CutSmart buffer (NEB) | 2 |
| Fragment (~1-2μg) | X |
| Restriction Enzyme(s) | 1 per enzyme |
| MilliQ | 18 - #RE added - X) |

3. Incubate for 4 hours at 37 °C.- Inactivate the restriction enzyme(s) by heating to 65 °C for 10 minutes.
     *NOTE: This last step can be skipped if the sample is evaluated on gel electrophoresis immediately.
     NOTE: Some enzymes are not inactivated by increasing the temperature to 65 °C.
     NOTE: [DNA Clean Up](#PCRcleanup-scroll) is recommended for subsequent cloning strategies.*

RNA Clean Up

This protocol is based on the [QIAGEN RNeasy MinElute Cleanup Kit](https://www.qiagen.com/us/shop/sample-technologies/rna/rna-clean-up/rneasy-minelute-cleanup-kit/#orderinginformation) suited for volumes smaller than 100µL.

1. Adjust the transcripts volume to 100µl with RNase-free water.
2. Add 350µL Buffer RLT and mix.
3. Add 250µL of absolute ethanol.
4. Transfer the sample to an RNeasy MinElute spin column placed in a 2mL collection tube. Close the lid and centrifuge for 15 seconds at minimum speed 8000x g.
5. Discard the flow-through and place the spin column in a new 2mL collection tube.
6. Add 500µL Buffer RPE to the spin column. Close the lid and centrifuge for 15 seconds at minimum speed 8000x g.
7. Discard the flow-through and place the spin column back to the same 2mL collection tube.
8. Add 500µL 80% ethanol to the spin column. Close the lid and centrifuge for 2 minutes at minimum speed 8000x g.
9. Discard the flow-through and place the spin column in a new 2mL collection tube.
10. Open the lid and centrifuge at full speed for 5 minutes. Discard the flow through and the collection tube.
11. Place the spin column in a new 1.5mL collection tube. Add 14µL RNase-free water to the center of the spin column.
12. Close the lid and centrifuge for 1 minute at full speed to elute the RNA.
13. Store the purified RNA at -20 °C

RNA Gel Electrophoresis

1. Add 1:1 volume ratio of a [purified RNA](#RNAcleanup-scroll) sample to 2X RNA loading buffer.
2. Boil at 90 °C for 5 minutes to denature potential secondary structures and temporarily inactivate RNAse.
3. Directly put on ice for ~2 minutes.
4. Spin down and electrophorese on a 2% agarose gel (in TBE buffer) for visualization with the gel documentation system using UV light.

SDS-PAGE Electrophoresis

An SDS-PAGE electrophoresis is used to separate proteins based on their size by using an electric current. This protocol describes how to prepare SDS-PAGE gels, how to prepare samples and how to run the electrophoresis.

#### Preparing of SDS-PAGE Gel

*NOTE: An SDS-PAGE gel consist of a stacking gel and a separation gel. In this protocol we use a 6% Stack gel and a 12% separation gel (this is for protein samples between 15 kDa and 250 kDa).*

1. Prepare the following solutions for 2 gels:

   | Component | Volume 6% stacking gel | Volume 12% separation gel |
   | --- | --- | --- |
   | 40% acrylamide | 750 µL | 3 mL |
   | 0.25 M Tris-HCl pH 6.8 | 2.5 mL | - |
   | 0.55 M Tris-HCl pH 8.8 | - | 5 mL |
   | 10% SDS | 50 µL | 100 µL |
   | 10% APS | 50 µL | 100 µL |
   | TEMED | 5 µL | 10 µL |
   | MilliQ | 1.645 mL | 1.79 mL |
   | Total volume | 5 mL | 10 mL |

   *NOTE: Polymerization will start when 10% APS and TEMED are added.*
2. Assemble the casting station and pour the separation gel between the glass plates leaving enough space for the stacking gel.
3. Add 75% ethanol on top of the solidifying stacking gel.
4. When the gel is solidified, remove the ethanol and pour the stacking gel on top of the separation gel and immediately place the gel comb on top of it.
5. Allow the stacking gel to solidify, then remove the plates from the casting station.
6. The gels are ready for usage, or can be stored at 4 °C in liquid for later usage.

#### Preparing samples

7. Add 5μL protein Loading Dye to 20μL of protein sample.- Incubate the samples for 10 minutes at 90 °C.

     #### Running SDS-PAGE gel

     - Run the gel at 150 V and 25 mA.- The gel is finished when the blue line is at the end of the gel.

         #### Processing SDS-PAGE gel

         - Turn off the power pack before opening the gel box.
         - Remove the gel from the cassette and place the gel in a clean staining tray of the appropriate size filled with a layer of MilliQ.
         - Gently allow the gel to slide from the glass plate into the water.
         - Microwave the gel for 40 seconds on low heat.
         - Allow the gel to shake on a moving platform for 2 minutes.
         - Discard the water.
         - Add clean MilliQ water and repeat step 15-17 two times.
         - Cover the gel with a layer of SimplyBlue SafeStain.
         - Let the gel stain on a moving plate for approximately 20 minutes.
         - Discard the SimplyBlue SafeStain in the waste container.
         - Cover the gel with MilliQ water and allow the gel to shake on a moving platform until the gel is destained.
           *NOTE: the water can be replaced frequently to remove all the stain.*
         - Take a picture with the Gel doc system.

Sequencing with MinION

This protocol is based on the protocol supplied with the  [MinION device from Oxford Nanopore Technologies](https://nanoporetech.com/products/minion).

#### Flow cell QC and check

1. Connect MinION device to a USB 3.0 port of a computer with recommended 1 Terabyte free space.
2. Open MinKNOW GUI from desktop and execute quality control (QC) of the flow cell.
   *NOTE: The number of available active pores to work with Flow cell from ONT is > 800 pores.*

#### Library preparation rapid sequencing (lambda DNA)

3. Thaw the components of the kit for Rapid Sequencing on ice and keep on ice.
4. Add 2.5µL of DNA to 7.5µL of FRA (fragmentation mix).
5. Mix the content well by inversion and spin down.
6. Incubate tube at 30 ºC for 1 minute and then at 80 ºC for another minute.
7. After incubation, add 1µL of RAP (Rapid Adapter) to the tube and incubate for 5 minutes at room temperature.

#### Priming of Flow Cell

8. Turn the lid of the Flow Cell clockwise in order to make the priming port visible.
9. Remove bubbles from the priming port (not by directly pipetting but by changing pipette volume).
10. Pipette 30µL of Flush Tether into a tube of Flush Buffer and mix well by pipetting (priming mix).
11. Add 800µL of this priming mix to the priming port.

#### Loading the SpotON Flow Cell

12. Prepare the following reaction mixture:

    | Component | Volume (µL) |
    | --- | --- |
    | Sequencing Buffer (SQB) | 34 |
    | Loading Beads (LB) | 25.5 |
    | Nuclease-free MilliQ | 4.5 |
    | Prepared DNA library | 11 |
13. Open the SpotON port making the sample port available.
14. Add 200µL of priming mix.
15. Add 75µL of sample in the sample port in a dropwise fashion, allowing every drop to enter the port before adding another drop.
16. Close the priming port and replace the MinION lid.

#### Sequencing run and base calling

17. Start the sequencing run once the sample is loaded on the Flow cell using the Desktop Agent.
18. Check the progression of the upload and download of files, together with the network speed.

#### Close down MinKNOW and Desktop Agent

19. The run is stopped once the live base calling shows no DNA (or very little DNA) is being sequenced.
20. Quit Desktop Agent and MinKNOW and disconnect MinION.
21. To store the flow cell for a next run, follow the Wash Kit MinION protocol.

Sequence Verification

For sequence verification, the sequencing platform [EZ-Seq](https://dna.macrogen.com/eng/support/ces/ezseq_intro.jsp) established by Macrogen was used. This protocol is based on their instructions.

1. Prepare a sequencing sample in a 1.5 mL microcentrifuge tube, labelled with a Macrogen sequencing sticker (QR-code) as follows:

   | Component | Volume (µL) |
   | --- | --- |
   | DNA | X (~500 ng is required\*) |
   | Primer (10 µM) | 2.5 (final concentration 5-10 pmol/µL) |
   | Sterile MilliQ | 10-X |

   *\* NOTE: The volume depends on the concentration of the sample.*
2. Make sure to keep the QR-code and note down what sample it is affiliated with. Deliver the tubes for sequencing to the manufacturer and await results.
3. Results are evaluated *in silico* by aligning expected sequences with obtained sequences and calculating the match percentage.

Serum DNA Extraction

This protocol is an adaptation of the original [QIAGEN QIAamp DNA Blood Mini Kit protocol](https://www.qiagen.com/us/shop/sample-technologies/dna/genomic-dna/qiaamp-dna-blood-mini-kit/#orderinginformation).
Important points before starting

* All centrifugation steps are carried out at room temperature (15–25°C).
* Use carrier DNA if the sample contains <10,000 genome equivalents (see page 17).
* 200 μl of whole blood yields 3–12 μg of DNA. Preparation of buffy coat (see page 18) is recommended if a higher yield is required.

Things to do before starting

* Equilibrate samples to room temperature (15–25°C).
* Heat a water bath or heating block to 56°C for use in step 4.
* Equilibrate Buffer AE or distilled water to room temperature for elution in step 11.
* Ensure that Buffer AW1, Buffer AW2, and QIAGEN Protease have been prepared according to the instructions on page 16.
* If a precipitate has formed in Buffer AL, dissolve by incubating at 56°C.

Serum DNA Extraction Procedure

1. Pipet 10 μl QIAGEN Protease (or proteinase K) into the bottom of two 1.5 ml microcentrifuge tubes.
2. Add 500 μl sample (plasma or serum) to the microcentrifuge tubes.
   *NOTE: It is possible to add QIAGEN Protease (or proteinase K) to samples that have already been dispensed into microcentrifuge tubes. In this case, it is important to ensure proper mixing after adding the enzyme.*
3. Add 500 μl Buffer AL to the sample (the same amount as the sample). Mix by pulse-vortexing for 15 s. It is a quite syrupy consistency. To ensure efficient lysis, it is essential that the sample and Buffer AL are mixed thoroughly to yield a homogeneous solution.
   *NOTE: Do not add QIAGEN Protease or proteinase K directly to Buffer AL.*
4. Incubate at 56°C for 10 min. DNA yield reaches a maximum after lysis for 10 min at 56°C. Longer incubation times have no effect on yield or quality of the purified DNA.
5. Briefly centrifuge the 1.5 ml microcentrifuge tube to remove drops from the inside of the lid.
6. Add 500 μl ethanol (96–100%) to the sample (the same amount as the sample). Pipet it into the tube via the side of tube. This will let the foam of the centrifugation go away. The ethanol will let the proteins precipitate. Mix again by pulse-vortexing.
7. Carefully apply 600 μl of the mixture from step 6 to the QIAamp Mini spin column (in a 2 ml collection tube) without wetting the rim (pipet via the side at the top of the tube). Close the cap, and centrifuge at 6000 x g (8000 rpm) for 1 min. Discard the filtrate en place the QIAamp Mini spin column in the same 2 ml collection tube. Repeat this 5 times untill the 3mL of the mixture is used (split over 2 1.5 ml microcentrifugation tubes). After the last time centrifugation place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the tube containing the filtrate.
   Close each spin column to avoid aerosol formation during centrifugation. Centrifugation is performed at 6000 x g (8000 rpm) to reduce noise. Centrifugation at full speed will not affect the yield or purity of the DNA. If the lysate has not completely passed through the column after centrifugation, centrifuge again at higher speed until the QIAamp Mini spin column is empty. Note: When preparing DNA from buffy coat or lymphocytes, centrifugation at full speed is recommended to avoid clogging.
8. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW1 without wetting the rim. Close the cap and centrifuge at 6000 x g (8000 rpm) for 1 min. Place the QIAamp Mini spin column in a clean 2 ml collection tube (provided), and discard the collection tube containing the filtrate.\*
   *NOTE: It is not necessary to increase the volume of Buffer AW1 if the original sample volume is larger than 200 μl.
   \*NOTE: Flow-through contains Buffer AL or Buffer AW1 and is therefore not compatible with bleach. See page 6 for safety information.*
9. Carefully open the QIAamp Mini spin column and add 500 μl Buffer AW2 without wetting the rim. Close the cap and centrifuge at full speed (20,000 x g; 14,000 rpm) for 3 min.
10. Place the QIAamp Mini spin column in a new 1.5 ml microcentrifuge tube (not provided) and discard the old collection tube with the filtrate. Centrifuge at full speed for 1 min.
    This step helps to eliminate the chance of possible Buffer AW2 carryover.
11. Place the QIAamp Mini spin column in a clean 1.5 ml microcentrifuge tube (not provided), and discard the collection tube containing the filtrate. Carefully open the QIAamp Mini spin column and add 60 μl Buffer AE or distilled (MilliQ) water. Incubate at room temperature (15–25°C) for 5 min, and then centrifuge at 6000 x g (8000 rpm) for 1 min.
    Take the eluate from the eppendorf tube and put it again on top of the filter. Centrifuge a second time at 14.000 rpm for 1 min.

sgRNA Transcription

This protocol is based on the [standard RNA synthesis of the New England Biolabs T7 RNA Polymerase.](https://international.neb.com/products/m0251-t7-rna-polymerase#Product%20Information)

1. The following components should be assembled at room temperature in the following order:

   | Component | Volume (µL) |
   | --- | --- |
   | 10x Reaction Buffer | 2 |
   | NTP (each 25mM) | 4 |
   | Template DNA | X (0.2 - 1 µg) |
   | RNase Inhibitor | 1 U/µL (final) |
   | DTT | 5 mM (final) |
   | T7 RNA Polymerase | 2 |
   | MilliQ | add up to 20 µL |
2. The reaction is incubated at 37oC for 4-16 hours.
3. The following sequence is used as the template DNA, in which “n” can be substituted to match the target DNA.

   TAATACGACTCACTATAGGnnnnnnnnnnnnnnnnnnnGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT

   - Clean up sgRNA product(s) according to the [RNA Clean Up](#RNAcleanup-scroll) protocol.

Solid Medium Preparation

1. Dissolve the LB-agar powder in sterile MilliQ as directed by the manufacturer’s protocol.
2. Autoclave the LB-agar solution (121 °C).
3. Cool the agar down to hand-warm temperature.
4. Supplement the medium with any relevant antibiotics and/or inducers etc.
   *NOTE: With our antibiotic stock solutions of 1000x we used 1 µL of antibiotic solution per mL of LB-agar.*
5. Under aseptic conditions, pour the medium in empty Petri dishes.
6. Close the Petri dishes and let the agar solidify at room temperature.
7. Store the Petri dishes upside down at 4 °C.
   *NOTE: When complimenting medium with X-gal, be sure to store the plates protected from light.*

Tn5 or dxCas9-Tn5 Fusion Protein Functionality Testing: Mobility Shift

This protocol describes how to evaluate the mobility shift of the Tn5:adapter DNA complex or the dxCas9-Tn5:adapter DNA complex.

#### Adapter DNA Sequence

To create 25µM adapter DNA molecule. Two primers shown on table 1 were annealed to each other as follow:1. Add 25µl of each primer (from 100µM stock)
2. Add 50µl of MilliQ water
3. Place tube in 90-95 °C heating block and leave for 3-5 minutes to denature any secondary structure
4. Remove the heating block from the heat source allowing for slow cooling to room temperature (~45 minutes)

Table 1. Sequence of the adapter DNA. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence (5’ - 3’) |
| --- | --- |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

#### Tn5:adapter DNA or dxCas9-Tn5:adapter DNA complex (transposome) formation

1. Load the purified Tn5 or dxCas9-Tn5 protein with adapter DNA shown above. Note that all samples and reagents are kept on ice during preparation.
2. Calculate all the volumes of the reagents required:
   * 1-6µM Tn5 or dxCas9-Tn5
   * 2.5µM adapter
   * Functionality buffer
   + dxCas9-Tn5 buffer: 20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol, 10mM MgCl2 pH 7.5
   + Tn5 buffer: 25mM Tris-HCl, 500mM NaCl, 1mM DTT, 0.1mM EDTA, pH 7.5
   + Water to bring the final volume to 10µl* Pipette the reagents in the following order: Water, 10x functionality buffer, Tn5 or Tn5-dxCas9 and adapter
   * Incubate at 23 °C for 1 hour.
     *NOTE: The total reaction volume can be adjusted as long as molar ratios of the reagents are kept constant.*
   * Analyse the samples on a 8% native TBE polyacrylamide gel to observe the mobility shift

Tn5 or dxCas9-Tn5 Fusion Protein Functionality Testing: Integration

This protocol describes how to evaluate the integration of adapter DNA by Tn5 or the dxCas9-Tn5 fusion protein.

#### Adapter DNA Sequence

To create 25µM adapter DNA molecule. Two primers shown on table 1 were annealed to each other as follow:1. Add 25µl of each primer (from 100µM stock)
2. Add 50µl of MilliQ water
3. Place tube in 90-95 °C heating block and leave for 3-5 minutes to denature any secondary structure
4. Remove the heating block from the heat source allowing for slow cooling to room temperature (~45 minutes)

This protocol describes how to evaluate the integration of the Tn5 or the Tn5-dxCas9.

Table 1. Sequence of the adapter DNA. Note that 5’ of the upper strand of the adapter has to be phosphorylated for successful integration.

| Name | Sequence (5’ - 3’) |
| --- | --- |
| Adapter upper strand | P-CTGTCTCTTATACACATCT |
| Adapter lower strand | GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG |

#### Tn5:adapter DNA or dxCas9-Tn5:adapter DNA complex (transposome) formation

1. Load the purified Tn5 or dxCas9-Tn5 protein with adapter DNA shown above. Note that all samples and reagents are kept on ice during preparation.
2. Calculate all the volumes of the reagents required:
   * 2.5µM Tn5 or Tn5-dxCas9
   * 2.5µM adapter
   * Functionality buffer (20mM Tris-HCl, 100mM KCl, 5% v/v glycerol, 1mM beta-mercaptoethanol, 0.1mM EDTA pH 7.5)
   * Water to bring the final volume to 10µl- Pipette the reagents in the following order: Water, 10X functionality buffer, Tn5 or dxCas9-Tn5 and adapter.
   - Incubate at 23 °C for 1 hour.
     *NOTE: The total reaction volume can be adjusted as long as molar ratios of the reagents are kept constant.*

   #### gRNA:Transposome complex formation (only for fusion protein)

   - Load dxCas9-Tn5:adapter complex with gRNA provided by Arbor Biotechnologies.
     *NOTE: all samples and reagents are kept on ice during preparation.*
   - Add the following reagents to 10µl transposome
   * 2µl sgRNA (100ng/µl stock solution) to make 1:1 sgRNA:transposome molar ratio
   * 0.5µl buffer (add concentration)- Incubate at 37 °C for 10 minutes.

   #### Target DNA integration

   - Add 50ng target DNA, in our case EPO cDNA to 12.5µl of gRNA:Transposome complex.
   - Incubate at 37 °C for 1 hour.

   #### Amplification of integration products by PCR

   - Targeted integration of adapter DNA to EPO cDNA (632bp) will result in two fragments of ~250bp and ~450bp. To visualize these fragments, PCR was done to amplify the two products separately. The sequence of the primers are found on table 2. Primer set 1 will amplify the first fragment, while primer set 2 will amplify the second fragment (figure 1).

   Table 2. Primer lists used for the functionality assay

   | Primer set | Fw primer | Information | Rv primer | Information |
   | --- | --- | --- | --- | --- |
   | 1 | Fw EPO  GATCGAATTCGCGGCCGCTTC | This will bind to the start of the EPO gene (target gene) | Rv adapter  GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG | This will bind to the end of the adapter |
   | 2 | Rv adapter GCATGGTAAACAGTTCATCCATTTCGCCAATAGATGTGTATAAGAGACAG | This will bind to the end of the adapter (start of the product) | Rv EPO  CGATTCTGCAGCGGCCGCTAC | This will bind to the end of EPO gene (target gene) |  |

   ![functionality assay](https://static.igem.org/mediawiki/2018/6/61/T--TUDelft--2018_Figure10_PrimerDesign_Tn5.png)
   Figure 1. Depiction of the binding location of the primer sets 1 (left) and 2 (right) for functionality assay PCR.
   - The PCR reaction composition is shown in Table 3.

   Table 3. Pipetting scheme for PCR of integration products.

   | Component | Volume (µL) |
   | --- | --- |
   | 5X GoTaq buffer | 4 |
   | MgCl2 (25µM) | 2 |
   | 10 mM dNTPs | 0.4 |
   | Primer forward (10µM) | 0.4 |
   | Primer reverse (10µM) | 0.4 |
   | GoTaq polymerase | 0.1 |
   | DNA template | 2.5 |
   | MilliQ | up to 20µL |

   - Close all tubes thoroughly and place them in a thermocycler with the following protocol:

     | Step | Time (s) | Temperature (°C) |
     | --- | --- | --- |
     | Initial denaturation | 150 | 98 |
     | Denaturation | 60 | 94 |
     | Annealing | 60 | 60 |
     | Extension | 60 | 72 |
     | Final extension | 480 | 72 |
     | Hold | ∞ | 20 |

     - The PCR product are analysed on 5% native TBE polyacrylamide gel. The resulting gel is either stained with ethidium bromide and imaged with GelDoc system, or directly imaged with Typhoon Imaging System. In order to see the position of the protein in the gel the gel can be stained with silver quest staining.

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:TUDelft/Results

ADOPE

[![](https://static.igem.org/mediawiki/2018/0/09/T--TUDelft--2018_adope_logo_white.png)](https://2018.igem.org/Team%3ATUDelft)

* Project
  + [Description](https://2018.igem.org/Team%3ATUDelft/Description)
  + [Design](https://2018.igem.org/Team%3ATUDelft/Design)
  + [Results](https://2018.igem.org/Team%3ATUDelft/Results)
  + [Demonstration](https://2018.igem.org/Team%3ATUDelft/Demonstrate)
* Wetlab
  + [Experiments (Protocols)](https://2018.igem.org/Team%3ATUDelft/Experiments)
  + [Notebook](https://2018.igem.org/Team%3ATUDelft/Notebook)
  + [InterLab](https://2018.igem.org/Team%3ATUDelft/InterLab)
  + [Safety](https://2018.igem.org/Team%3ATUDelft/Safety)
  + [Measurement](https://2018.igem.org/Team%3ATUDelft/Measurement)
* Parts
  + [Parts Overview](https://2018.igem.org/Team%3ATUDelft/Parts)
  + [Basic Parts](https://2018.igem.org/Team%3ATUDelft/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ATUDelft/Composite_Parts)
  + [Part Collection](https://2018.igem.org/Team%3ATUDelft/Part_Collection)
  + [Sequences](https://2018.igem.org/Team%3ATUDelft/Primers)
* Modeling
  + [Model](https://2018.igem.org/Team%3ATUDelft/Model)
  + [Software Tool](https://2018.igem.org/Team%3ATUDelft/Software)
  + [Improvement](https://2018.igem.org/Team%3ATUDelft/Improve)
* Human Practices
  + [Integrated Human Practices](https://2018.igem.org/Team%3ATUDelft/Human_Practices)
  + [Education and Engagement](https://2018.igem.org/Team%3ATUDelft/Public_Engagement)
  + [Entrepreneurship](https://2018.igem.org/Team%3ATUDelft/Entrepreneurship)
  + [Applied Design](https://2018.igem.org/Team%3ATUDelft/Applied_Design)
* [EurAsian Meetup](https://2018.igem.org/Team%3ATUDelft/EurAsianMeetup)
* Team
  + [Team Members](https://2018.igem.org/Team%3ATUDelft/Team)
  + [Collaborations](https://2018.igem.org/Team%3ATUDelft/Collaborations)
  + [Contributions](https://2018.igem.org/Team%3ATUDelft/Contributions)
  + [Team Building](https://2018.igem.org/Team%3ATUDelft/Team_Building)
* Acknowledgements
  + [Attributions](https://2018.igem.org/Team%3ATUDelft/Attributions)
  + [Sponsors](https://2018.igem.org/Team%3ATUDelft/Sponsors)
* + [Judging form](https://igem.org/2018_Judging_Form?team=TUDelft)
  + [Medal Requirements](https://2018.igem.org/Team%3ATUDelft/Medals)
  + [Awards](https://2018.igem.org/Team%3ATUDelft/Awards)

![Responsive image](https://static.igem.org/mediawiki/2018/a/a8/T--TUDelft--2018_ProjResultshead.png)

* [ADOPE](#adope)
* 1. Sample Preparation
  + [Sample Preparation](#sampleprep)
  + [1.1 Gene doping Model](#sp2)
  + [1.2 Extraction Verification](#sp3)
  + [1.3 Gene Doping DNA Extraction](#sp4)
* 2. Prescreening
  + [Prescreening](#prescreening)
  + [2.1 D-AuNPs: Prescreening with Visible Readout](#pr2)
  + [2.2 D-AuNPs: SsDNAp effect on d-AuNPs’ stability](#pr3)
  + [2.3 D-AuNPs: Visual difference between target and non-target DNA](#pr4)
* 3. Fusion Protein -
  dxCas9-Tn5
  + [Fusion Protein - dxCas9-Tn5](#fusionprotein)
  + [3.1 Fusion Protein Plasmid Construction](#fp2)
  + [3.2 Protein Expression and Purification](#fp3)
  + [3.3 *In Vivo* Functionality of
    the Fusion Protein](#fp4)
  + [3.4 *In Vitro* Functionality of
    the Fusion Protein](#fp5)
* 4. Targeted Sequencing
  with dxCas9-Tn5
  + [Targeted Sequencing with dxCas9-Tn5](#targetedseq)
  + [4.1 sgRNA Array Determination
    for Gene Doping Sequences](#ts2)
  + [4.2 Barcode DNA for Nanopore Sequencing](#ts3)
  + [4.3 Nanopore Targeted Sequencing](#ts4)
  + [4.4 Data Analysis Software](#ts5)* [References](#references)

# ADOPE

The aim of ADOPE is to develop a complete method for gene doping detection. The final method consists of four main steps: sample preparation, prescreening, targeted library preparation and DNA sequencing and analysis.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/3/37/T--TUDelft--2018_Figure3_DescriptionPage.png)

**Figure 1.** A flow diagram of our detection method ADOPE. With the four steps sample preparation, prescreening, library preparation and sequencing.

# 1. Sample Preparation

In the sample preparation module we developed a workflow to extract the target DNA from blood. As a proof of concept, we verified the extraction of the cell free DNA (cfDNA) from bovine serum. Finally, we successfully extracted gene doping DNA that we spiked to the serum, guided by the concentration value generated by the gene doping model.

1.1 Gene Doping Model: Understanding Gene Doping

**Design:**
We modeled the process of infection and degradation of gene doping DNA in blood to determine its concentration overtime. We use this model to provide approximate sensitivity requirement for DNA extraction.

**Results and interpretation:**
Based on our [model](https://2018.igem.org/Team%3ATUDelft/Model), we determined microdosing was the best doping method for doping athletes, as it avoids high EPO fluctuation, and hence avoids detection through the biological passport. **Figure 2** displays the predicted amount of doping DNA fragments in the blood over time as a result of intramuscular (IM) injection every 20 days. Based on this [model](https://2018.igem.org/Team%3ATUDelft/Model), we predicted that there will be 40,000 to 50,000 fragments of DNA per mL blood. We based the sensitivity requirement of DNA extraction on this value.

 ![Model Figure 2](https://static.igem.org/mediawiki/2018/e/e7/T--TUDelft--Doping_DNA.png)
 **Figure 2.** Concentration of EPO gene doping DNA in the blood during intramuscular microdosing with an initial dose of 96 billion vectors followed by smaller doses of 27 billion vectors every 20 days.

1.2 cfDNA Extraction

**Design:** We extracted cfDNA from bovine serum and confirmed successful extraction with Qubit dsDNA HS assay and nested PCR.

**Results and interpretation:**
We performed cfDNA isolation from the serum using QIAamp DNA Blood Mini Kit. To verify extraction, we quantified cfDNA on the serum using Qubit. We obtained 0.394+/- 0.0149 ng/uL of cfDNA after extraction. To verify the extraction further, we performed nested PCR on albumin gene. Albumin gene is often used for verification of the presence of genomic mammalian DNA and was recommended by Sanquin. The nested albumin PCR consists of a first round of PCR using [primers 091 and 092](https://2018.igem.org/Team%3ATUDelft/Primers), amplifying an internal fragment of the bovine albumin gene of 229 bp. We deliberately chose to amplify a short fragment since cfDNA in blood is fragmented. The product of the first round of PCR was used as the template for the second round. We used internal [primers 066 and 067](https://2018.igem.org/Team%3ATUDelft/Primers) to amplify a 150 bp internal fragment of the first PCR product.The product of the first and second round of PCR are shown on **figure 3**. The bands on **figure 3** lane 2-7 confirmed the success of the extraction.

 ![Model Figure 2](https://static.igem.org/mediawiki/2018/0/09/T--TUDelft--SP_Albumin_pure_serum.png)
 **Figure 3:** Albumin verification by nested PCR on extracted cfDNA. 2% Agarose gel. **A.** Round 1, expected band size 229 bp. **B.** Round 2, expected band size 153 bp. Both gels: Lane 1: Ladder (bp). Lane 2: Extraction Sample 1, non spiked serum. Lane 3: Extraction Sample 2, non spiked serum. Lane 4: Extraction Sample 3, linear intronless EPO spiked. Lane 5: Extraction Sample 4, linear intronless EPO spiked. Lane 6: Extraction Sample 5, BBa\_K2643004 spiked. Lane 7: Extraction Sample 6, BBa\_K2643004 spiked. Lane 8: Negative control using miliQ as template. Lane 9: Positive control extraction sample of 03/10

1.3 Gene Doping DNA Extraction

**Design:** We spiked samples with artificial gene doping DNA and confirmed its extraction with PCR.

**Results and interpretation:**
Guided by our gene doping model, we spiked samples with 2x10^8 fragments/ml of either linear or plasmid EPO cDNA. We performed extraction, followed by PCR to verify it. Two EPO internal primer combinations were used. For the [primer set 072 & 073](https://2018.igem.org/Team%3ATUDelft/Primers) the expected band size was 297 bp and for the primer set 074 & 075 the expected band size was 140 bp. **figure 4** confirmed the successful extraction of gene doping EPO DNA. The difference in intensity between the linear DNA spiked (sample 3 & 4) and the plasmid spiked (sample 5 & 6) indicates that the QIAamp DNA Blood Mini Kit has a lower extraction efficiency for plasmids, since the same amount of fragments were spiked.

 ![Model Figure 2](https://static.igem.org/mediawiki/2018/a/a5/T--TUDelft--SP_EPO_yeah.png)
 **Figure 4:** Artificial gene doping EPO DNA verification extraction of spiked EPO. 2% Agarose gel. Lane 1&11: Extraction Sample 1, non spiked serum. Lane 2&12: Extraction Sample 2, non spiked serum. Lane 3&13: Extraction Sample 3, linear intronless EPO spiked. Lane 4&14: Extraction Sample 4, linear intronless EPO spiked. Lane 5&15: Extraction Sample 5, BBa\_K2643004 spiked. Lane 6&16: Extraction Sample 6, BBa\_K2643004 spiked. Lane 7&17: Negative control, MilliQ template. Lane 8&18: Positive control, BBa\_K2643004 template. Lane 9&19: Positive control, BBa\_K2643004 spiked. Lane 10&20 Ladder

##### Conclusion

We showed successful extraction of artificial gene doping DNA at concentration predicted by our model using the QIAamp DNA Blood Mini Kit. Further extraction optimization is advisable to maximize extraction efficiency.

# 2. Prescreening

In this module, we developed a high throughput screening method to detect gene doping. The method is based on the concept of color changes induced by d-AuNPs aggregation. In this module, we first analysed the stability of d-AuNPs upon introduction of NaCl with and without single stranded DNA probe. Finally, we were able to visually differentiate between samples where target DNA is presence or absence.

2.1 NaCl Effect on d-AuNPs’ Stability

**Design:**
We generated batches of dextrin-capped gold nanoparticles and tested their aggregation upon NaCl introduction to determine the optimal NaCl concentration.

**Results and interpretation:**
Upon increasing NaCl concentration, d-AuNPs destabilize and aggregate, changing the color of the solution from red to purple. We scanned the visual light spectra from 450nm to 650nm for a range of 0mM to 333mM NaCl. The important absorbance wavelengths are 520nm (red color) and 620 nm (purple color). We calculated the ratio of 620nm and 520nm to quantify the expect of color changes (red to purple) (**figure 5**). We observed an increase in the 620/520 with an increase in NaCl concentration, indicating a color change from red to purple.

![sample prep workflow](https://static.igem.org/mediawiki/2018/c/c7/T--TUDelft--d-AuNPs_saltaggregation.png)

**Figure 5.** Influence of NaCl on d-AuNPs aggregation. At a concentration of 133 mM NaCl, the d-AuNPs start to aggregate; the peak in the spectra shift from 520nm to 620nm and the ratio 620/520 wavelengths start to increase indicating color change from red to purple. **A)** Spectrum of d-AuNPs solutions with NaCl concentrations 0, 167 and 333mM. **B)** Wavelength ratios of 620/520 of d-AuNPs solutions with NaCl concentrations ranging from 0 to 333mM. **C)** Visual outcome of the d-AuNPs solution with NaCl concentrations ranging from 0 to 333mM.

2.2 ssDNAp Effect on d-AuNPs’ Stability

**Design:**
We tested the effect of single stranded DNA probe (ssDNAp) on d-AuNPs aggregation.

**Results and interpretation:**
ssDNAp are known to stabilize d-AuNPs from aggregating ([Baetsen-Young, et al, 2018](#references)). We incubated the d-AuNP's with and without a ssDNAp and compared the d-AuNP aggregation at a range of NaCl concentrations. The stabilization effect can be observed on figure 6. The addition of ssDNAp causes a lower 620/520 ratio shift with increasing NaCl concentration.

![sample prep workflow](https://static.igem.org/mediawiki/2018/0/0d/T--TUDelft--d-AuNPs_ssDNAp.png)

**Figure 6.** Influence of ssDNAp on d-AuNPs aggregation. At a concentration of 133mM NaCl, the d-AuNPs start to destabilize while with presence of ssDNAp the destabilization occurs at higher NaCl concentrations. **A)** Wavelength ratios of 620/520 of d-AuNPs solutions with NaCl concentrations ranging from 0 to 333mM with and without ssDNAp. **B)** Visual outcome of the d-AuNPs solution with NaCl concentrations ranging from 0 to 333mM without (top) and with (bottom) ssDNAp.

To further evaluate the influence of ssDNAp concentration on the d-AuNPs stabilization, we measured the stability at different NaCl concentrations with varying ssDNAp concentrations. Increasing ssDNAp concentration stabilizes the d-AuNPs, reducing the aggregation and changing the color of the solution from red to purple.

![sample prep workflow](https://static.igem.org/mediawiki/2018/c/ce/T--TUDelft--d-AuNPs_ssDNAp_variation.png)

**Figure 7.** Influence of ssDNAp concentration on stabilization of d-AuNPs. At higher primer concentrations, the d-AuNPs are more stabilized. **A)** Spectrum of d-AuNPs solutions with NaCl concentration of 333mM incubated with a ssDNAp concentration ranging from 0 nM to 2.5nM. **B)** Visual outcome of the d-AuNPs solution with NaCl concentration of 333mM with increasing ssDNAp concentrations.

2.3 Visual Difference between Target and non-Target DNA

**Design:**
We incubated DNA with targeting ssDNAp or non-targeting ssDNAp to show the visual difference between the two.

**Results and interpretation:**
The ssDNAp was incubated with the corresponding target gene doping DNA to let the ssDNAp anneal and form the secondary structure. Subsequently, the d-AuNPs were added and their aggregation was analysed. The ssDNAp targeting EPO was compared with a ssDNAp that is not targeting EPO. The on-target ssDNAp stabilized the d-AuNPs as the ratio 620/520 wavelength was lower compared to off-target ssDNAp (**figure 8**).

![sample prep workflow](https://static.igem.org/mediawiki/2018/0/0c/T--TUDelft--d-AuNPs_ssDNAp%2Btarget.png)

**Figure 8.** **A)** Wavelength ratios of 620/520 of d-AuNPs solutions with NaCl concentration of 333 mM with on and off target ssDNAp. **B)** Visual outcome of the d-AuNPs solution with ranging NaCl concentrations of 0 to 333mM with on- and off-target ssDNAp targeting EPO.

After showing the functionality of the d-AuNPs to detect target DNA, we aimed to determine the sensitivity by using variations of target EPO DNA concentrations while keeping the total amount of DNA constant. We tested concentrations ranging from 1 pM to 1 nM comparing on- and off-target primers and determined our lower limit of detection is 1nM. To achieve comparable results to [Baetsen-Young et al. 2018](#references) (29 fM) further optimization are required.

Additionally, we evaluated the influence of background DNA present in the sample. We used 38 ng/µL background DNA, equal to the expected cfDNA extraced during sample preparation. We were able to show that the presence of background DNA does not affect the sensitivity of the prescreening method. Further sensitivity evaluation are required before implementing the prescreening method.

##### Conclusion

The functionality of the prescreen was proven. We were able to visually detect the presence of target EPO DNA. However, to implement this prescreening, protocol optimization is required to reach a lower detection limit of at least 9 fM, based on data from our model. Thus, if the results of [Baetsen-Young et al. 2018](#references) can be reproduced, the detection limit can be lowered to 2.94 fM.

# 3. Fusion Protein - dxCas9-Tn5

In order to achieve targeted sequencing, we constructed a fusion protein by linking dxCas9 with Tn5 to be used in library preparation for sequencing platform. We constructed the strain that expressed our fusion protein, successfully purified them and tested their functionality in vivo and in vitro. We were able to show targeted integration by our fusion protein in vitro and sequence verified the integration products.

3.1 Strain Construction

**Design:**
We constructed a fusion protein by linking dxCas9 cDNA to Tn5 cDNA with a linker coding for glycine helical peptide.

**Results and interpretation:**
The sequences encoding the dxCas9 (3.7) and Tn5 were obtained via Addgene with plasmid numbers #60240 from [Picelli et al. (2014)](#references) and #108383 from [Liu et al (2018)](#references) respectively. First, we introduced a linker coding for glycine helical peptide to Tn5 cDNA and cloned it to pACYCDuet. We screened colonies with the correct insert by colony PCR, followed by visualization of amplified insert on a 0.8% TAE agarose gel (**figure 9**). Plasmid was isolated from colony 8 and sequence verified.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/7/71/T--TUDelft--Lin-Tn5.png)

**Figure 9.** Colony PCR of Linker-Tn5 in pACYCDuet-1 (1508 bp). The ladder represents the size of DNA in bps.

Next, we introduce dxCas9 into pACYCDuet-1\_Lin-Tn5. We screened colonies with the correct insert by colony PCR, followed by visualization of amplified insert on a 0.8% TAE agarose gel (**figure 10, 11, 12**). After plasmid isolation from positive colony 2, we sequence verified plasmid pACYCDuet-1\_dxCas9-Lin-Tn5 (**figure 10**).

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/0/02/T--TUDelft--19_07_PCRfusion.png)

**Figure 10.** Colony PCR of dxCas9-Tn5 in pACYCDuet-1\_dxCas9-Lin-Tn5, expected size 5924 bp. The ladder represents the size of DNA in bps.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/1/1b/T--TUDelft--19_07_PCR_fusionCas9.png)

**Figure 11.** Colony PCR of dxCas9 in pACYCDuet-1\_dxCas9-Lin-Tn5, expected size 4172bp. The ladder represents the size of DNA in bps.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/a/a4/T--TUDelft--19_07_PCR_fusionTn5.png)

**Figure 12.** Colony PCR of Lin-Tn5 in pACYCDuet-1\_dxCas9-Lin-Tn5, expected size 1452bp. The ladder represents the size of DNA in bps.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/9/94/T--TUDelft--FusionProtein_seq_ver.png)

**Figure 13.** The alignment of the sequencing results of the plasmid dxCas9-Lin-Tn5. This image shows the alignment of the sequencing results of plasmids from colony 2 with the original insert design in SnapGene.

3.2 Fusion Protein Expression and Purification

**Design:**
We developed a new production process for the fusion protein BBa\_K2643002. We followed existing dxCas9 expression protocols ([Huai et al. 2017](#references)), but developed a new downstream process for the fusion. We evaluated three different types of chromatography:

* Nickel affinity chromatography: The fusion protein has a HIS-tag (used to purify both Tn5 and dxCas9 individually).
* Heparin chromatography: The fusion protein is a DNA-binding protein (used to purify both Tn5 and dxCas9 individually).
* MonoQ chromatography: It’s an anion exchange resin, utilizing the charge of the fusion protein as a purification property.

**Results and interpretation:**
Of the three chromatography methods, both heparin and monoQ were successful. As a result we developed a production process which includes heparin chromatography as a capture step, followed by monoQ as a final polishing step.

We used heparin chromatography to capture the fusion from the crude lysate. The fusion protein has a high affinity to the heparin resin, resulting in good initial separation of the fusion protein from contaminating and degrading species. The results suggested that the fusion protein was cleaved at the linker, resulting in a dxCas9 contamination (**figure 14**, lane 2 to 10) . The dxCas9 contaminant has a lower affinity to the heparin resin and eluted at lower concentrations of salt (**figure 14**, lane 2-10), whereas the fusion has a higher affinity to the heparin resin and eluted at high concentration of salt (**figure 14**, lane 11-13). Only the last three elution fractions were pooled (**figure 14**, lane 11-13), and further purified.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/9/9a/T--TUDelft--2018_Figure2_24082018.png )
**Figure 14.** 8% SDS PAGE of fusion of Heparin Chromatography (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 elution 1, lane 3 elution 2, lane 4 elution 3, lane 5 elution 4, lane 6 elution 5, lane 7 elution 6, lane 8 elution 7, lane 9 elution 8, lane 10 elution 9, lane 11 elution 10, lane 12 elution 11, lane 13 elution 12.

We used MonoQ chromatography to polish the fusion and remove any remaining contaminants. The fusion eluted in a broad peak (**figure 15, lane 6-14**) from the resin, however the fractions are relatively pure and the dxCas9 contamination was mostly eliminated.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/6/6b/T--TUDelft--2018_Figure1_25082018.png)
**Figure 15.** 8% SDS PAGE of MonoQ chromatography of fusion (expected size 214.7kDa). Lane 1 molecular ladder (kDa), lane 2 starting material, lane 3 flow through, lane 4 wash, lane 5 elution 1, lane 6 elution 2, lane 7 elution 3, lane 8 elution 4, lane 9 elution 5, lane 10 elution 6, lane 11 elution 7, lane 12 elution 8, lane 13 elution 9, lane 14 elution 10, lane 15 elution 11.

We also evaluated nickel affinity chromatography, however we experienced a lot of difficulty binding the HIS tag to the nickel beads. Majority of the fusion was detected in the flow through and detailed results can be found in our notebook. Additionally, the complete upstream and downstream process for Tn5 transposase (BBa\_K2643002), dxCas9 (BBa\_K2643001) and dxCas9-Tn5 fusion (BBa\_K2643002) is documented in detail on the biobrick registry.

3.3 Fusion Protein *In Vivo* Functionality Test

**Design:**
To visualize *in vivo* whether a donor DNA sequence can be integrated close to a target sequence, we built a strain that harbors the dxCas9-Tn5 fusion protein (BBa\_K2643000), as well as a lacZ gRNA expression cassette (BBa\_K2643010). The cell is electroporated with a kanamycin resistance cassette flanked by MEs (BBa\_K2643011), which can be recognized by the Tn5 transposase domain for cut and paste integration near the gRNA target. This disrupts the *lacZ* gene and renders beta-galactosidase catalytically inactive, which can be visualized by blue/white screening with X-gal. In parallel, we have electroporated an *E. coli* strain that only harbors the Tn5 transposase for comparison with untargeted integration.

**Results and interpretation:**
Prior to doing any actual genomic integration, we performed a broad scala of control experiments. **Table 1** gives an overview of the results obtained for these controls. The obtained data confirmed that, based on their blue colony phenotype, the parental BL21DE3 strain indeed contained intact and functional *lacZ*. Furthermore, the parental strain was not found to be resistant to chloramphenicol nor to kanamycin. Harboring the fusion construct and the gRNA that targets *lacZ*, while not provided with donor DNA, did not influence the functionality of the *lacZ*, resulting in remaining blue colony phenotype. This discards the potential expressional inhibition effects to to binding of the fusion complex. Furthermore, we took along some controls including Red Fluorescent Protein (RFP), which is present by default on iGEM backbone vectors (pSB1K3). Together with blue phenotype, this resulted into a purple colony phenotype.

**Table 1:** Overview of observed phenotypes of the controls taken along in the *in vivo* assay.

| Strain | DNA | LB-medium supplements | Observed phenotype |
| DH5α | - | X-gal + IPTG | white |
| BL21DE3 | - | X-gal + IPTG | blue |
| BL21DE3 | - | Cam | No growth |
| BL21DE3 | - | Kan | No growth |
| BL21DE3 | pACYCdxCas9Tn5gRNA | Cam + X-gal + IPTG | blue |
| BL21DE3 | pACYCdxCas9Tn5gRNA + pSB1K3 | Cam + Kan + X-gal + IPTG | purple |
| BL21DE3 | pSB1K3 | Kan + X-gal + IPTG | purple |

When transforming the donor DNA plasmid (BBa\_K2643011) into *E. coli* DH5α and selecting on medium complemented with chloramphenicol and kanamycin, we observed formation of red colonies. This indicates that the kanamycin cassette, as well as the RFP cassette were intact and functional. The sequence verification confirmed both sequences were intact and oriented as desired. *DH5α* was not able to grow on LB+Kan+Cam, and demonstrates the negative control.

The MEs of the donor DNA (BBa\_K2643011) were also sequence verified and confirmed to be located as designed, but we were not able to show that they were recognized and that the donor DNA was integrated, neither in a targeted nor a non-targeted manner. Surviving colonies demonstrated development of purple phenotype, sometimes requiring several days (**figure 16**), while going through an interesting metamorphosis of color changes. Based on the evaluated controls, it is very likely that a purple phenotype was due to the presence of the original RFP-containing template plasmid used for creating the linear donor DNA sequence. The RFP cassette was kept on the plasmid to observe this deviant purple color to identify growing false positives. These colonies survived in the presence of kanamycin and chloramphenicol, because this template plasmid is propagated and harbors intact and functional chloramphenicol and kanamycin resistance expression cassettes.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/d/db/T--TUDelft--COLONYMETAMORPHOSIS.jpg)

**Figure 16.** Effect of incubation time on chromogenic in vivo assays. These LB+Cam+Kan+X-gal+IPTG plates demonstrate growth of BL21DE3+pACYCdxCas9Tn5gRNA strains that are electroporated with donor DNA. (A) 1 day after electroporation with linear ME flanked kanamycin resistance cassette, blue colony formation was observed, whereas (B) a week later red phenotype has developed strongly. (C) 1 day after transformation with ME flanked kanamycin resistance cassette in a plasmid, blue colony formation was observed, whereas (D) a week later purple phenotype has developed strongly.

Evaluation of colonies by several genomic PCRs have demonstrated that integration did not take place near the desired target site. The negative control used for these genomic PCRs was purified isolated gDNA of the BL21DE3 parent strain. Even transformation of donor DNA into a competent strain harboring only Tn5 did not result in colonies, suggesting a problem related to the transposase effector function of the fusion construct.

The experimental results demonstrate that the used kanamycin cassette is functional, judging from the control results. The RFP remains unchanged and functional too, allowing for identification of false positives. Furthermore, we have shown that strain BL21DE3 contains a functional lacZ copy, based on chromogenic conversion of X-gal.

Although we have shown that MEs are present and intact on the donor DNA sequence, we were not able to show that the MEs were recognized and that the donor DNA was integrated. We would have expected either targeted or non-targeted integration, but neither occured. Even when we try out the setup with just Tn5 and donor sequence, this yielded no integration.

3.4 Fusion Protein *In Vitro* Functionality Test

**Design:**
Two assays were performed to prove the in vitro functionality of the fusion protein:

1. Electrophoretic mobility shift assay (EMSA) verified the ability of fusion protein to load DNA adapter and sgRNA, and form complex with the target DNA (EPO).
2. Integration assay verified the target specific integration of fusion protein.

**Results and interpretation:**
We successfully performed EMSA to individual (non-fused) dxCas9 and Tn5 protein (see the [notebook](https://2018.igem.org/Team%3ATUDelft/Notebook)). Next, we performed EMSA to verify that our fusion protein was able to load Cy5-labbelled adapter DNA, sgRNA, and Cy5-labelled target DNA. **Figure 17** displays the result of the EMSA on 5% TBE native PAGE as shown by the Typhoon Imaging System.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/8/80/T--TUDelft--result_in_vitro_1.png)

**Figure 17.** 5% TBE native PAGE by Typhoon Imaging System. A: Lane 1: unlabelled DNA ladder (100-1000bp), lane 2: 2.5µM fusion protein, lane 3: 2.5µM adapter, lane 4: 2.5µM adapter, lane 5: 2.5µM adapter + 0.15µM fusion protein, lane 6: 2.5µM adapter + 0.5µM fusion protein, lane 7: 2.5µM adapter + 1.5µM fusion protein, lane 8: 2.5µM adapter + 2.5µM fusion protein, lane 9: 2.5µM adapter + 2.5µM fusion protein + heat, lane 10: 2.5µM adapter. B: Lane 1: unlabelled DNA ladder (100-1000bp), lane 2: 0nM Fusion w/ gRNA (1:1), lane 3: 27nM Fusion w/ gRNA (1:1), lane 4: 134nM Fusion w/ gRNA (1:1), lane 5: 402nM Fusion w/ gRNA (1:1), lane 6: 804nM Fusion w/ gRNA (1:1), lane 7: 0nM Fusion w/out gRNA , lane 8: 27nM Fusion w/out gRNA, lane 9: 134nM Fusion w/out gRNA, lane 10: 402nM Fusion w/out gRNA, lane 11: 804nM Fusion w/out gRNA, lane 12: 134nm Fusion w/ gRNA and EDTA, lane 13: 402nM Fusion w/ gRNA and EDTA, lane 14: 804nM Fusion w/ gRNA and EDTA, lane 15: 1.34uM Fusion w/ gRNA and EDTA.

We performed EMSA for Tn5 and dxCas9 portion of the fusion protein separately. **Figure 17A** displays the EMSA of adapter. Loading of adapter on the fusion protein retards DNA movement on PAGE, which was observed as higher bands on **figure 17A**, lane 7 and 8. It indicates that mobility shift occurs when >1.5µM of fusion protein is present. We performed three different negative controls in parallel:

* No adapter DNA was added (**figure 17A**, lane 2).
* No fusion protein was added (**figure 17A**, lane 3,4, and 10).
* Fusion protein was denatured by heat (**figure 17A**, lane 9).

None of these negative control reactions resulted in the band shift, confirming the specificity of our result.

With the same principle, higher bands observed on **figure 17B**, lane 5,6,14, and 15, confirmed the complex formation between sgRNA-loaded fusion protein with the target DNA. As a negative control, the same reaction was carried out without sgRNA. In this case, the dxCas9 should not be able to find the target DNA and complex should not be formed. The absence of higher bands on **figure 17B**, lane 7-11 confirmed this.

We performed integration assay based on the protocol. To amplify the integration products, we performed PCR with primers that anneal to the end of adapter DNA, and the beginning or end of target DNA. **Figure 18** displays the PCR products on 5% TBE native gel.

We designed the sgRNA to target the second exon-exon junction of EPO cDNA (position ~200bp from total length 623bp). When integration of adapter DNA (length ~50bp) occurs, 2 fragments of ~250bp and ~470bp should be generated. Indeed, **figure 18** lane 2 and 3 shows intense bands on position ~250bp and ~470bp respectively. This indicated the success of targeted integration by our fusion protein. Moreover, the absence of these bands and the stronger presence of other unspecific bands when sgRNA was omitted (**figure 18**, lane 4 and 5) or when Tn5 was used (**figure 18** lane 10 and 11) confirmed that targeted integration is specific to sgRNA targeted fusion protein. Moreover, the absence of integration products on the other negative control (without fusion protein, adapter, or target DNA) on **figure 18**, lane 6-9, 12, and 13 confirmed this further.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/d/d4/T--TUDelft--result_in_vitro_2.png)

**Figure 18.** 5% TBE native PAGE stained by EtBr and imaged by GelDoc system. A: Lane 1: DNA ladder (100-1000bp), lane 2: amplified product with fw EPO lane 3: amplified product with rv EPO, lane 4: amplified product with fw EPO without sgRNA, lane 5: amplified product with rv EPO without sgRNA, lane 6: amplified product with fw EPO without fusion protein, lane 7: amplified product with rv EPO without fusion protein, lane 8: amplified product with fw EPO without adapter DNA, lane 9: amplified product with rv EPO without adapter DNA, lane 10: amplified product with fw EPO with Tn5, lane 11: amplified product with rv EPO with Tn5, lane 12: amplified product with fw EPO without target DNA, lane 13: amplified product 2 without target DNA.

To confirm that these two bands represented the expected integration products, we isolated and sequence verified them. **Figure 19** shows the sequence alignment between the two fragments and the target EPO gene. From the sequence alignment we can confirm that integration occured upstream the sgRNA binding site. The overlapping alignment between the two products suggested that integration does not always occur at the exact base pair position (~10bp variation), which might be the result of the flexible linker between dxCas9 and Tn5.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/c/c5/T--TUDelft--result_in_vitro_3.png)
**Figure 19.** Sequence alignment of integration product (two red arrows) with the target EPO. Pink box represent the sgRNA binding site.

To examine the functionality of our fusion protein further, we studied integration activity with different sgRNA and target DNA [notebook](https://2018.igem.org/Team%3ATUDelft/Notebook)). Moreover, we repeated the reaction with longer incubation duration. With increasing incubation time, we observed fading bands representing targeted integration with increase in bands representing random integration ([notebook](https://2018.igem.org/Team%3ATUDelft/Notebook))

##### Conclusion

We successfully constructed, expressed and purified the fusion protein. We developed a method to test its functionality both *in vivo* and *in vitro*. We successfully demonstrated that our fusion protein was able to perform targeted integration *in vitro*. We sequence verified the integration products and confirmed that integration occurred in the region close to the sgRNA binding site.

# 4. Targeted Sequencing with dxCas9-Tn5

Once the *in vitro* functionality of the fusion protein was established, we used it as a tool to prepare samples for targeted sequencing using ONT MinION sequencing. We proved the targeted integration of DNA adapters compatible with Rapid Sequencing kit ([Oxford Nanopore Technologies](https://nanoporetech.com/)) and linked this activity to the pipeline of library preparation for sequencing. Data analysis proved the potential of the fusion protein to be used for targeted sequencing. To compliment our work, we also developed software tools to generate [sgRNA arrays](https://2018.igem.org/Team%3ATUDelft/Software#gRNAtool) and [adapters with barcodes](https://2018.igem.org/Team%3ATUDelft/Improve) needed for MinION sequencing and a software tool for [sequencing data analysis](https://2018.igem.org/Team%3ATUDelft/Software#method) to identify gene-doping DNA.

4.1 sgRNA Array Determination for Gene Doping Sequences

**Design:**
We developed a tool that screens through a gene coding sequence, identifies possible Protospacer Adjacent Motif (PAM) sequences for dxCas9 and analyzes the number of possible sgRNA molecules required for specific sites. One final specific site is chosen based on the lowest possible variation due to synonymous mutations, which was 12 for EPO cds, followed by generation of this array.

**Results and interpretation:**
We generated a heat map that indicates the regions of human EPO gene with the lowest possible variation due to synonymous mutations (**figure 20**). The exon-exon junction (target sites that are gene-doping DNA-specific) is indicated with arrows in **figure 20**.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/6/63/T--TUDelft--Heat_Map_gRNAs.png)

**Figure 20.** Heat map representing the total variation of EPO cds possible based on synonymous mutations. X axis represents EPO cds gene divided into subzones of 9 bp each. Subzones with darkest blue represent up to 18 possibilities of DNA (3 codons with 6 possible combinations each), whereas lightest subzones represent least number of possible DNA combinations. Junctions between exons are represented (in total 4 junctions) with their position in bp in EPO cds, number of identified PAM sequences and minimal number of sgRNAs required to cover all possibilities.

The model helped us to decide the best junction to be targeted by our fusion protein (junction with the least sgRNA posibilities). For EPO, it generated an array of 12 sgRNA at Junction three, with PAM sequence at position 232 bp of EPO CDS. For detailed information on the gRNA array model, please visit the [model page](https://2018.igem.org/Team%3ATUDelft/Model).

4.2 Barcode DNA for Nanopore Sequencing

**Design:**
We developed a tool useful for the generation of DNA barcodes within the sequencing adapters to identify samples’ subjects. The resulted barcodes can be used for ONT MinION sequencing and allow the possibility of multiplexing.

**Results and interpretation:**
To use this tool, please visit our [improvement page](https://2018.igem.org/Team%3ATUDelft/Improve).

4.3 Nanopore Targeted Sequencing

##### Sequencing by ONT MinION Rapid Adapter kit: random adapter integration

**Design:** To prove the potential of targeted sequencing, we used our fusion protein during library preparation of specific DNA samples and sequenced them with ONT MinION. We run sequencing tests to determine whether different types of DNA molecules can be identified during data analysis. Once this analysis was done, we tested the DNA product from an integration reaction with our fusion protein and sequencing adapters.

**Results and interpretation:**
We prepared a solution with a mix of different DNA molecules that ranged from plasmid to linear DNA of different sizes. This solution was used during library preparation, and after transposase reaction was stopped, an extra DNA molecule was added to assess whether unadapted DNA is sequenced. The total number of aligned reads for each reference molecule, presented in **figure 21** indicated that small linear DNA molecules (600 bp) are not identified during data analysis. This does not mean that these DNA molecules were not read, but their alignment was not possible with our working pipeline, probably due to the size of the reference. These results also indicated that, when DNA molecules are added without the first transposase reaction (the case of PCR 3 that is unadapted), the reads generated are much lower (> 3%) than equimolar concentrations of molecules that were fragmented by the transposase reaction (PCR 1).

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/f/f3/T--TUDelft--Sequencing_1.png)

**Figure 21.** Number of reads aligned to reference molecules after MinION sequencing run on flow cell type FLO-MIN106 with Rapid Sequencing transposase based kit. Each bar represents a different DNA molecule added in the sample solution from either plasmid or linear DNA of different lengths. DNA molecules used were EPO biobrick (Plasmid 1), EPO + Ampicilin biobrick (Plasmid 2), pACYCduet-1 (Plasmid 3), EPO + Kanamycin amplification (PCR 1), EPO cds amplification (PCR 2) and dxCas9 amplification (PCR 3).

##### Applying the fusion protein: targeted adapter integration

**Design:**
We designed and tested DNA adapters, compatible with ONT sequencing, to be integrated by the Tn5 portion of our fusion protein. We targeted the integration of these adapters on EPO coding sequence in plasmid DNA.

**Results and interpretation:**
The initial assay for adapter (~30bp) integration on plasmid DNA with EPO coding sequence (BBa\_K2643004) was not successful (
[notebook](https://2018.igem.org/Team%3ATUDelft/Notebook)). Based on this result, we hypothesised that it might be necessary to work with linearized DNA, so we digested EPO plasmid with EcoRI enzyme and repeated the adapter integration. Adapter integration with sgRNA ([sg001](https://2018.igem.org/Team%3ATUDelft/Primers)) targeting EPO at around 232th bp should result in fragments of ~250 bp and ~430bp. We PCR amplified the products of integration using EPO and adapters’ primers. The results in **figure 22**, (Lane 1,2,5, and 6) indicated a correct integration of both adapters in a specific position in EPO cds molecule. The absence of these bands on the negative controls without sgRNA (lane 3,4,7,8) confirmed this specificity further.

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/2/28/T--TUDelft--SequencingAdapters.png)

**Figure 22.** Sequencing adapter integration by fusion protein on EPO target. 5% TBE native PAGE stained by EtBr and imaged by GelDoc system. DNA substrate: (lanes 1-4) EPO coding sequence or (lanes 4-8) EPO plasmid linearized with EcoRI. Lane L: DNA ladder (100-1000bp), lane 1, 5: amplified DNA with fw EPO lane 2, 6: amplified DNA with rv EPO, lane 3, 7: amplified DNA with fw EPO without sgRNA, lane 4, 8: amplified DNA with rv EPO without sgRNA.

##### ONT MinION sequencing with adapters integrated by fusion protein

We took our fusion protein one step further and attempted to use the integration product (**figure 23**) for ONT MinION sequencing. For this assay, we took an initial solution with equimolar concentrations of Tn5 DNA and EPO plasmid DNA and performed our integration assay, after which we continued with Rapid Sequencing Kit pipeline (without transposase fragmentation reaction). As a control, an equimolar concentration of dxCas9 DNA was prepared by ONT library preparation protocol (including transposase fragmentation).

After the sequencing run, we followed the workflow defined in [Sequencing alignment protocol](https://2018.igem.org/Team%3ATUDelft/Wetlab/Protocols#sequencingminion-scroll). The alignment results showed that most of the reads (>99 %) aligned to dxCas9 DNA used as positive control. Analysis of aligned reads to sample treated with our fusion protein showed zero sequences aligned to Tn5 DNA reference (non-target of our fusion) and 89 reads aligned to the target EPO plasmid DNA. These reads indicated that the integration of sequencing adapters preferred the linearized EPO plasmid DNA rather than Tn5 DNA for sequencing. Closer look at the aligned sequences rendered one sequence read presented in **figure 23**, where we have a clear example of targeted adapter integration next to the gRNA flanking site (**figure 23**).

![Picture of Timeline of the relevance of gene doping](https://static.igem.org/mediawiki/2018/5/5f/T--TUDelft--Alignment_miNION_read.png)

**Figure 23.** A) Alignment of DNA sequence read with target EPO cds in plasmid DNA. (B) Zoom into the sequence alignment to determine sgRNA target. Fusion protein depicted with arrows representing binding of dxCas9 position (red) and the addition of sequencing adapters (blue).

These preliminary results show the function of adapter integration directed by our fusion protein to the sgRNA complementary site. The efficiency of integration was not high and some of the aligned reads showed extended regions summing up to long reads of 2 - 11 kbp. More experimentation should be done to obtain conclusive data on these rare events, but most importantly to define better conditions for the integration assay. The low efficiency of this integration could be explained by different factors explained below:

* During the purification of the fusion protein, there could be contamination by DNA attached to some domain of the protein (as they are DNA binding proteins). This contamination could come from the cells used to produce the protein.
* Purification of the fusion protein was done with heparin column, and other DNA interacting domain-like proteins could be present in the protein mixture.
* Conditions of the integration assay could be suboptimal to favour reaction kinetics of the fusion protein.
* Incompatibility of buffers required for transposase and dxCas9 binding reactions, or incompatibility of these buffers with ONT sequencing buffers.
* Probable suboptimal compatibility of our designed sequencing adapters with ONT sequencing platform. The specific adapters’ sequence is not known due to corporate secrets.

We have, however, proven that our fusion protein is able to load sequencing adapters and integrate them at a specific site in their target DNA, allowing this DNA molecule to be sequenced with ONT sequencing platform. Moreover, we were able to identify 89 sequencing events aligned to the DNA target, and even analyse the possible direction for integration of our fusion protein based on one single DNA read out of over 200 000 reads.

4.4 Data Analysis Software

**Design:**
We developed a software tool for data analysis of sequencing data based on a machine learning algorithm to increase the database for reference alignment in an iterative process.

**Results and interpretation:**
To use this tool, please visit our [software tool page](https://2018.igem.org/Team%3ATUDelft/Software#method).

##### Conclusion

We demonstrated that our fusion protein and sequencing adapters could be used to replace the first reaction in library preparation for ONT MinION sequencing. More importantly, we proved specific targeted enrichment by our fusion protein for library preparation and ONT MinION sequencing. We obtained preliminary results on the position of adapter integration from sgRNA binding site.

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