iGEM 2019 — Team TU_Eindhoven

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# Team:TU Eindhoven/Notebook

* [![](https://static.igem.org/mediawiki/2019/1/18/T--TU_Eindhoven--smallLogo.png)](https://2019.igem.org/Team%3ATU_Eindhoven)
* PROJECT

  [Description](https://2019.igem.org/Team%3ATU_Eindhoven/Description)
  [Design](https://2019.igem.org/Team%3ATU_Eindhoven/Design)
  [Demonstrate](https://2019.igem.org/Team%3ATU_Eindhoven/Demonstrate)
  [Model](https://2019.igem.org/Team%3ATU_Eindhoven/Model)
  [Device](https://2019.igem.org/Team%3ATU_Eindhoven/Device)
  [Safety](https://2019.igem.org/Team%3ATU_Eindhoven/Safety)
* LAB

  [Experiments](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
  [Notebook](https://2019.igem.org/Team%3ATU_Eindhoven/Notebook)
  [Contribution](https://2019.igem.org/Team%3ATU_Eindhoven/Contribution)
  [Results](https://2019.igem.org/Team%3ATU_Eindhoven/Results)
  [Measurement](https://2019.igem.org/Team%3ATU_Eindhoven/Measurement)
* PARTS

  [Parts overview](https://2019.igem.org/Team%3ATU_Eindhoven/Parts)
  [Basic parts](https://2019.igem.org/Team%3ATU_Eindhoven/Basic_Part)
  [Composite parts](https://2019.igem.org/Team%3ATU_Eindhoven/Composite_Part)
  [Part collection](https://2019.igem.org/Team%3ATU_Eindhoven/Part_Collection)
  [Improve](https://2019.igem.org/Team%3ATU_Eindhoven/Improve)
* HUMAN PRACTICES

  [Integrated human practices](https://2019.igem.org/Team%3ATU_Eindhoven/Human_Practices)
  [Education & engagement](https://2019.igem.org/Team%3ATU_Eindhoven/Public_Engagement)
* TEAM

  [Team Members](https://2019.igem.org/Team%3ATU_Eindhoven/Team)
  [Attributions](https://2019.igem.org/Team%3ATU_Eindhoven/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ATU_Eindhoven/Collaborations)
  [Sponsors](https://2019.igem.org/Team%3ATU_Eindhoven/Sponsors)
* MEDALS

  [Medal requirements](https://2019.igem.org/Team%3ATU_Eindhoven/Medal_Requirements)
  [Awards](https://2019.igem.org/Team%3ATU_Eindhoven/Awards)
* [CONTACT](#footer)

# Notebook

* [Lab Notebook](#labnotebook)
* [Milestones](#milestones)

## Lab Notebook

Here you can find short summaries of all the work we did in the lab. You can click on the boxes for more information.
More information about our achievements outside the lab is shown on the Milestones page.

### June

Monday June 17, 2019 - Stocks

×

## Monday June 17, 2019 - Stocks

The general stocks (IPTG, Kanamycin, LB medium and LB agar for the plates) were prepared.

Tuesday June 18, 2019 - Transformation

×

## Tuesday June 18, 2019 - Transformation

The vector ordered at Genscript (pET28a(+)) with our insert dCas9-Sbit-Lbit was dissolved and
transformed into BL21 and NovaBlue. The vector transformed into BL21 will be used for proteins
expression of dCas9-Sbit. The vector transformed into NovaBlue will be used for restriction and
ligation to form dCas9-Lbit. In the end the bacteria were plated and incubated overnight.

Wednesday June 19, 2019 - Small culture

×

## Wednesday June 19, 2019 - Small culture

The plates were taken out of the incubator and were put in the fridge. Not that many colonies were formed, however
there were enough colonies for the small cultures. In the afternoon the small cultures were prepared and incubated
overnight. Furthermore, the media for the large cultures were prepared. We do not know yet which medium will be
most optimal for protein expression, so LB, 2YT and TB medium were prepared.

![Process](https://static.igem.org/mediawiki/2019/9/97/T--TU_Eindhoven--Lab20190619.png)

Thursday June 20, 2019 - Miniprep & protein expression

×

## Thursday June 20, 2019 - Miniprep & protein expression

The small cultures were taken out of the incubator. The two cultures with BL21(DE3) cells were put on ice for protein expression later in the morning.
The plasmids from the other four cultures with NovaBlue cells were isolated using the QIAprep miniprep kit. The following DNA concentrations were obtained:
215.8 ng/μL, 225.0 ng/μL, 202.0 ng/μL, 215.2 ng/μL. The samples were put in the freezer (-20 °C). Tomorrow these samples will be digested and ligated to form dCas9-Lbit.

After the two small cultures on ice were at room temperature, they were divided over the three large cultures (LB, 2YT, TB). When the OD600 had reached 0.6-0.7,
protein expression was initiated by adding IPTG. The large cultures were incubated overnight.

Friday June 21, 2019 - Digestion & protein pellets

×

## Friday June 21, 2019 - Digestion & protein pellets

The three large cultures were centrifuged in several rounds.
The volumes were too big for just one round. After each round, the supernatant was discarded. In the end the pellets were taken out of the centrifuge bottles
and put into three falcon tubes (pellets from TB, 2YT and LB medium). The pellets were snap freezed and will be purified after the weekend.

Furthermore, the DNA retrieved yesterday (miniprep) was digested to form the dCas9-Lbit. The digested DNA was put on an agarose gel
to separate the digested part from the rest of the plasmid. The ladder had leaked into the samples, however we were still able to cut out the DNA of dCas9-Lbit.
The DNA was retrieved from the agarose gel using the QIAquick Gel Extraction Kit. Due to lack of time, the sample will be ligated after the weekend.

Monday June 24, 2019 - Ligation & column preparation

×

## Monday June 24, 2019 - Ligation & column preparation

The digested DNA (from 21-06) was ligated, transformed into BL21(DE3) bacteria (for protein expression) and plated.
The plates are incubated overnight (dCas9-Lbit). Furthermore, the strep buffer W and IMAC columns were prepared for protein purification (of dCas9-Sbit),
which will be done tomorrow. The Strep columns will be used from Eva van Aalen (PhD supervisor).

Tuesday June 25, 2019 - Purification & small culture

×

## Tuesday June 25, 2019 - Purification & small culture

The pellets of dCas9-Sbit were taken out of the -80 °C freezer and put on ice to thaw. Subsequently, the pellets were dissolved and lysed (chemically).
When the lysing was finished, the proteins were purified using IMAC columns. The elution from the IMAC columns were then put on the strep columns.
Amicon filters were used to concentrate the elution from the strep columns. NanoDrop was used to measure if there were any proteins in the solution.
Unfortunately there were no proteins after the purification. The question is if the proteins were expressed. Samples were taken during the whole purification process,
these will be put on an SDS-page gel tomorrow to check if the protein was lost during the purification. If this is not the case,
the expression protocol needs to be further looked into to optimize it for this protein.

NcoI–His tag–GGS– SacI–dCas9–AgeI–(GGS)5–HindIII–SpeI–SBit–BamHI–(GGS)2–Strep tag–stop

Ten small cultures were also made for the dCas9-Lbit which was ligated and transformed in NovaBlue yesterday.

Wednesday June 26, 2019 - Miniprep & SDS-PAGE

×

## Wednesday June 26, 2019 - Miniprep & SDS-PAGE

The small cultures were taken out of the incubator and the DNA for the dCas9-Lbit was isolated using the QIAprep miniprep kit. The final DNA concentrations are shown below.
Furthermore, SDS-PAGE samples were made from the flow through samples from the purification proces; His bind, His wash, Strep lysate, Strep wash, Strep elution.
The samples were loaded on the gel to evaluate the purification steps. To be able to evaluate the results, the gel was washed and stained with Coomassie for approximately 40 minutes.
After this, the gel is washed in water overnight on the shaking table.

Thursday June 27, 2019 - SDS-PAGE, sequencing & new LB stock

×

## Thursday June 27, 2019 - SDS-PAGE, sequencing & new LB stock

![Process](https://static.igem.org/mediawiki/2019/4/49/T--TU_Eindhoven--Lab20190627.png)

Friday June 28, 2019 - 2nd Transformation Sbit, IPTG stock & New plan expression

×

## Friday June 28, 2019 - 2nd Transformation Sbit, IPTG stock & New plan expression

Since the first round of expression and purification of the dCas9-NL\_Sbit protein was unfortunately unsuccessful, we adapted the expression and purification protocol
to now test 4 different sets of expression conditions and 2 different lysis methods. To start the second trial round of protein expression of our sensor we again
transformed our plasmid into E. coli BL21 (DE3).

For transformation, a 4x dilution of the miniprepped plasmid DNA was prepared, taking 1 μl from plasmid tube 1 and adding together with 3 μl in another tube.
Final plasmid concentration ~50 ng/μl. 1 μl of this was added to the bacteria, the remainder was placed in the freezer. The bacteria were heat-shocked for 40 s.
100 μl of the final LB/bacteria solution was spread out over an agar plate and left to incubate at room temperature over the weekend.

Also, new IPTG stock solution was prepared. We added 6.7 ml MQ water to 1.6 g IPTG, totalling 8.0 ml solution
(in hind sight the total solution should have been 6.7 ml to obtain 1M conc.), resulting in a **0.84M stock**.
Hence, for 1mM final concentration, add **1.2 ml stock IPTG to 1L medium**. For 200nM final concentration, add **0.25 ml stock IPTG**.

### New plan Expression

From the SDS-PAGE results we concluded that no dCas9-Sbit was formed. Therefore a new plan was made to test different combinations of expression conditions based on
information we gathered. The plan is to test four different condition combinations.

The following conditions will be the same:

* Host: BL21(DE3)
* Medium: 750 mL LB
* OD600: 0.5
* Half of the culture will be lysed with ultrasound, the other half with Homogenizer

### July

Monday July 1, 2019 - Small culture

×

## Monday July 1, 2019 - Small culture

### Split-NL

Two small 8 mL LB liquid cultures (incl. 8 μL Kanamycin)of two of the dCas9-NL-Sbit transformants were started for growing overnight, to act as starter
cultures for protein expression tomorrow. Two large erlenmeyer flasks with 750 mL LB medium were prepared and placed in the autoclave queue for expression
tomorrow.

Tuesday July 2, 2019 - Small culture

×

## Tuesday July 2, 2019 - Small culture

### Split-NL

Since the large culture media couldn't be autoclaved before the end of today, we decided to postpone expression one day and to prepare a fresh small culture.
Hence, another two small 8 mL LB liquid cultures (incl. 8 μL Kanamycin)of two of the remaining dCas9-NL-Sbit transformants were started for growing overnight,
to act as starter culture for protein expression tomorrow.

Wednesday July 3, 2019 - Expression

×

## Friday June 28, 2019 - Expression

### Split-NL

As the initial attempt of expressing the dCas9-NL-Sbit fusion protein failed, we now try 2 sets of different expression conditions in parallel (we narrowed
down to only 2 condition sets, in contrast to the 4 we planned to do initially to save work and time, while still using the most interesting conditions).
Namely:

|  | [IPTG] | Temperature (°C) | Color label |
| --- | --- | --- | --- |
| 1. | 0.2 mM | 16 | Green |
| 2. | 1 mM | 16 | Blue |

Both cultures were at 750 mL scale, with 0.75 mL 1000x IPTG added. The cultures were grown at 37 °C, 160 rpm until reaching OD600 ~0.5-0.6, upon
which IPTG was added according to the scheme below.

|  | Color label | [IPTG] | Add this volume of 0.84M IPTG stock solution to 750 ml medium |
| --- | --- | --- | --- |
| 1. | Green | 0.2 mM | 0.18 ml |
| 2. | Blue | 1 mM | 0.90 ml |

The cultures were the incubated overnight at 16°C, 160 rpm.

Thursday July 4, 2019 - Centrifugation

×

## Thursday July 4, 2019 - Centrifugation

### Split-NL

The overnight Sbit expression cultures were distributed over two 500 mL ultracentrifuge tubes each and spun down at 8600 x g for 15 + 10 minutes at 4 °C.
The resulting pellets were dissolved with 5 mL MQ H2O each. This was then added together per culture (keeping the two different cultures apart) in
a Falcon tube and centrifuged for 10 minutes at 3000 rpm at 4 °C. The pellet was then stored at -80 °C for lysis and protein purification to be performed
tomorrow. We also received instructions on how to use the ultrasound sonication cell lysis device for use tomorrow. We also received instructions on how
to use the ultrasound sonication cell lysis device for use tomorrow. We also prepared the basis of a sonication lysis buffer (50 mM Tris, pH 8.0, 500 mM NaCl).

Friday July 5, 2019 - Sonication lysis & protein purification

×

## Friday July 5, 2019 - Sonication lysis & protein purification

### Split-NL

Yesterday's cell pellets, hopefully containing dCas9-NL-Sbit, were thawed on ice and resuspended in sonication lysis buffer, resulting in about 25 mL total
volume, to which a protease inhibitor mix tablet (Merck, without EDTA!) was added, as well as lysozyme. These suspensions were then lysed by sonication
according to the sonication protocol using a Branson 150 sonifier.

The resulting lysates were then centrifuged at 20k g for 30 min at 4 °C and the resulting supernatants were then added onto separate washed NiNTA columns for
His-tag affinity purification. Afterwards, the eluates were further purified using strep tag columns. Nanodrop measurement of the resulting eluates (using
protein A280 setting, with exctinction coefficient and MW obtained through the ExPASy online tool) unfortunately indicated that basically no dCas9-Sbit
protein was present. We then also put the flow through from the His purification over the Strep columns, to test for presence of protein that might not have
bound tightly to the NiNTA column. Unfortunately, also here the Nanodrop determined concentrations were very low.

Monday July 8, 2019 - SDS-PAGE

×

## Monday July 8, 2019 - SDS-PAGE

### Split-NL

An SDS-PAGE gel was prepared for all purification fractions of both cultures of last week. We also had a meeting with some of our supervisors to discuss the
expression problem we are facing as well as our general progress. Moreover, our new idea for an additional interesting new dCas9 based dsDNA sensor part was
presented.

Tuesday July 9, 2019 - SDS-PAGE

×

## Tuesday July 9, 2019 - SDS-PAGE

### Split-NL

Yesterday's gels were photographed and analyzed. We would expect a band at the MW, namely 164 kDa, to indicate the presence of our dCas9-Sbit fusion protein.
We do see very dim bands in both the pellet and His flow through fractions around this height, but not in the elute fractions were they would be expected.

### Peking constructs

We designed and ordered split Firefly luciferase DNA constructs from Twist Bioscience to be able to express the dCas9 split-luciferase sensor designed by iGEM Peking 2015 (not available from the Bio Brick repository) to compare it to ours.

Wednesday July 10, 2019 - Denaturing His-tag purification

×

## Wednesday July 10, 2019 - Denaturing His-tag purification

### Split-NL

We performed another round of His-tag purification with fractions from the previous purification rounds, now under denaturing conditions, namely with 8M
urea added. This in order to test the hypothesis that the His-tag might be unavailable for binding the NiNTA column in the protein's native condition. We
only saw a very dim band, so we think this is a rather unlikely explanation, and that the problem rather lies in codon related issues. Still, we'd like to
test this hypothesis. The resulting fractions were put on an SDS-PAGE gel, which was washed overnight.

Thursday July 11, 2019 - Denaturation

×

## Thursday July 11, 2019 - Denaturation

### Split-NL

The gel made yesterday doesn't appear to support the unavailable His-tag hypothesis, hence we will need to troubleshoot in a different direction. We discussed
the situation with supervisors and concluded the problem most likely lies in the alternative codons we used in our expression plasmid (which was codon optimized
for E. coli, which frankly appears to work counteractive for expression). We therefore decided to order a dCas9 (wild type codons) containing plasmid from
Addgene (Plasmid #62935)to extract this sequence by overhang PCR and replace the dCas9 sequence in our own construct by restriction/ligation.

### BRET

In the meantime, we started designing the DNA construct for our dCas9-NanoLuc BRET sensor part. We decided, taking the large size of the dCas9 coding sequence
into account, that it would be a good and economical idea to orderthe construct as 3 gBlocks from IDT, to be ligated to each other and into our pET-28a(+)
expression plasmid.

Friday July 12, 2019 - Design

×

## Friday July 12, 2019 - Design

We worked on the design of the BRET sensor construct, the test target DNA needed for future testing using both sensors and ordered the selected dCas9 construct from Addgene.

Monday July 15, 2019 - General

×

## Monday July 15, 2019 - General

### July 15 - 19, 2019

We continued working on the design of the DNA construct for our BRET sensor idea this week, as well as on the synthetic test target dsDNA strands and crRNA.
Moreover, protocols were prepared for cloning the dCas9 sequence from the Liu Addgene construct into our vector.

On Friday, the designed BRET constructs (3 gBlocks) were ordered at IDT, as well as primers for overhang PCR of the Liu Addgene construct. The Addgene order
was delivered and the bacterial stab was plated for growing over the weekend.

Monday July 22, 2019 - Small culture

×

## Monday July 22, 2019 - Small culture

### Split-NL

A Mach1 bacterial colony from the plate grown over the weekend containing the Liu plasmid obtained through Addgene was cultured in 5 mL LB with 5 μl Ampicillin
overnight. Moreover, some additional stocks were prepared, like ampicillin and chloramphenicol (for use with Rosetta strain cells) stocks. Also LB-Agar plates
were poured, with different antibiotics (Kanamycin, Ampicillin, Chloramphenicol and combinations).

Tuesday July 23, 2019 - Miniprep & transformation

×

## Tuesday July 23, 2019 - Miniprep & transformation

### Split-NL

The Liu Addgene plasmid was purified from the overnight culture by an alternative "miniprep" protocol (due to the Qiagen kit being
out of stock for the moment), giving the below peculiar concentrations:

|  | concentration (ng/μL) |
| --- | --- |
| Liu1a | 1808.2 |
| Liu2a | 13.5 |
| Liu3a | 1415.8 |
| Liu4a | 518.7 |

We diluted Liu4a sample 20x and transformed it in both BL21 and Rosetta strains to test the expression of this dCas9 later on. We decided not to use these
samples for PCR, first awaiting the expression results and hopefully we can soon redo the miniprep to obtain more reliably isolated DNA.

Wednesday July 24, 2019 - Small culture

×

## Wednesday July 24, 2019 - Small culture

### Split-NL

Transformations in BL21 and Rosetta appeared successful. Small cultures of the Liu Addgene plasmid in BL21, Rosetta and the original carrier strain (Mach1)
were prepared (5 mL LB + Amp for Mach1, 8 mL LB + Amp for BL21 and 8 mL LB + Amp + Cam for Rosetta).

Thursday July 25, 2019 - PCR, digestion & expression

×

## Thursday July 25, 2019 - PCR, digestion & expression

### Split-NL

The Mach1 small culture was miniprepped, obtaining below plasmid concentrations:

|  | concentration (ng/μL) |
| --- | --- |
| mpLiu1 | 131.9 |
| mpLiu2 | 192.9 |
| mpLiu3 | 136.4 |
| mpLiu4 | 124.2 |

Sample 4 was diluted 20x and used for overhang PCR to extract the dCas9 coding sequence including the relevant restriction sites for ligating it in our plasmid.
A sample of the PCR mixture was analysed on agarose gel, showing a band at the expected length. The remainder of the mixture was cleaned-up using the Qiagen PCR
purification kit. Final concentration: 85.5 ng/μL.

Our original Lbit and Sbit vectors were also digested to remove dCas9 and allow for insertion of the new dCas9 sequence.

The Liu Addgene construct was expressed (induced by 0.2 mM IPTG) in BL21 and Rosetta in LB at 18 °C, 180 rpm.

### BRET

The original Lbit vector was digested with XhoI and NcoI to replace the full original construct and allow for insertion of the 3 BRET gBlocks. Together with
the Split-NL digested vectors, this was purified by agarose gel, with subsequent gel extraction Qiagen kit.

Friday July 26, 2019 - Centrifugation, lysis, SDS-page,...

×

## Friday July 26, 2019 - Centrifugation, lysis, SDS-page,...

### Split-NL

The overnight expression cultures were pelleted, resuspended in sonication lysis buffer en lysed by sonication. Subsequently, the lysate was centrifuged and
SDS-PAGE samples were made of both the supernatant and pellet and run on a gel to check for clear presence of the dCas9 protein.

The PCRed insert was digested, as was the His-MBP gBlock to be coupled to the dCas9 in the plasmid.

### BRET

The 3 gBlocks were digested with the relevant restriction enzymes. However, we only have 1 μg of each, which would all be used in a single regular digestion
reaction! Therefore we tried with 0.1 μg and decided we would order primers and PCR amplify the gBlocks next week.

Monday July 29, 2019 - DNA struggles

×

## Monday July 29, 2019 - DNA struggles

### July 29 - August 2

### Split-NL

This week, we attempted to ligate the digested Liu Addgene dCas9 construct into our digested pET28a(+) vector together with the His-MBP tag gBlock.
Unfortunately, even after multiple attempts, this was unsuccessful. We discussed this with our lab supervisor and then decided to order a new primer to be
able to PCR the dCas9 sequence from the Addgene plasmid over again, now including a restriction site allowing us to insert the dCas9 into the vector directly,
without the need for the tag gBlock.

### BRET

Similar issues were faced for the BRET sensor construct. Multiple attempts at ligating the 3 gBlocks to each other and into the vector, either all at once,
or sequentially, were all unsuccessful. Primers for amplifying the gBlocks were designed and ordered for use next week.

### August

Monday August 5, 2019 - More DNA struggles, success for Split-NL

×

## Monday August 5, 2019 - More DNA struggles, success for Split-NL

### Split-NL

Using the new primers, overhang PCR was again performed on the Liu Addgene plasmid to extract the dCas9 sequence. This was performed successfully, as indicated by agarose gel analysis. Subsequently, this DNA was digested with the relevant restriction enzymes and ligated into the correspondingly digested Sbit and Lbit vectors. Transformation of both ligation mixtures in NovaBlue resulted in colonies, which were screened for presence of the correct insert by colony PCR. The colony PCR was positive for most of the colonies and many of these were miniprepped, resulting in multiple DNA samples of both Liu-dCas9-Lbit and -Sbit. These were transformed in BL21 for subsequent expression later on.

### BRET

After successful PCR amplification of all gBlocks, again multiple attempts at ligating the 3 gBlocks to each other and into the vector, either all at once, or sequentially, were all unsuccessful. A ligation of only the two most relevant CP-dCas9 spanning gBlocks into a correspondingly digested backbone appeared successful at first based on colony PCR, but later on turned out to be unsuccessful as well. We will look into other options next week.

Tuesday August 13, 2019 - Reorganization

×

## Tuesday August 13, 2019 - Reorganization

We reorganized the blue boxes containing all DNA, enzymes and reagents in the -20 °C freezer.

Wednesday August 14, 2019 - Digestion, PCR amplification

×

## Wednesday August 14, 2019 - Digestion, PCR amplification

### Split-NL

The expressed Liu Addgene construct was purified using IMAC. Afterwards, SDS-PAGE analysis was performed to check whether the correct protein
was retrieved. A band around 170 kDa was expected and the SDS-PAGE gel indeed shows a band just above 150 kDa.

### BRET

Parts 2 and 3 were PCR amplified to retrieve enough DNA for digestion and ligation. Subsequently, both parts were digested using the restriction
site NheI. The digested parts were separated using an agarose gel and excised.

Thursday August 15, 2019 - Meeting with supervisors

×

## Thursday August 15, 2019 - Meeting with supervisors

### General

Today we had a meeting with our supervisors. We presented our progress and discussed about some problems we encountered in the lab.

### BRET

We performed gel extraction of BRET part 2 and BRET part 3 after NheI digestion. The DNA concentrations were measured using NanoDrop.

Friday August 16, 2019 - Gibson Assembly primers design

×

## Friday August 16, 2019 - Gibson Assembly primers design

### BRET

For BRET it was decided that Gibson Assembly would be performed instead of regular digestion and ligation. The gBlocks we ordered cannot directly be used for Gibson,
since the gBlocks do not contain overlap. We designed overhang primers for the gBlocks to make them suitable for Gibson assembly and ordered them from IDT.

Monday August 19, 2019 - Transformation & ligation

×

## Monday August 19, 2019 - Transformation & ligation

### Split-NL

After successful ligation of the Liu Addgene dCas9 sequence into our original vector, the plasmid was transformed into BL21 cells. We used four plates:
2 for Sbit and 2 for Lbit. They were incubated overnight at 37 °C.

### BRET

After successful NheI digestion, BRET parts 2 and 3 were ligated to each other. This was done to be able to perform a 1:1 ligation instead of a 2:1 ligation.
However, the agarose gel made after ligation did not show any successful results.

Tuesday August 20, 2019 - Small culture & digestion

×

## Tuesday August 20, 2019 - Small culture & digestion

### Split-NL

Transformation was successful, so small cultures were made using kanamycin and LB medium. From each plate 1 colony was taken, so 2 small cultures containing Sbit
and 2 small cultures containing Lbit. In addition, a positive control was made containing just a pipette tip without tipping a colony.

### BRET

Since ligation of part 2 and 3 was not successful, digestion was performed again to prepare the parts for 2:1 ligation (part 2 and 3 separately into the
digested vector). Part 2 was restricted with SacI & NheI restriction enzymes and part 3 was restricted with NheI & XhoI restriction enzymes. In addition,
the vector was digested using the SacI & XhoI restriction enzymes. The digested samples were then ran on an agarose gel and cut out.

Wednesay August 21, 2019 - Large culture, ligation & digestion

×

## Wednesay August 21, 2019 - Large culture, ligation & digestion

### Split-NL

2 L large cultures were made from 1 Sbit and 1 Lbit small culture. The bacteria were grown at 37 °C and 160 rpm until OD600 = 0.6-0.7. At OD600 = 0.6, protein
expression was initiated by adding IPTG. The cultures were then grown overnight at 18 °C and 160 rpm.

### BRET

Gel extraction was performed for the digested part 2 and 3 and the digested vector. A 2:1 ligation was performed to ligate digested parts 2 and 3 into the digested
vector. The ligation product was transformed into NovaBlue cells.

Thursday August 22, 2019 - Purification, PCR

×

## Thursday August 22, 2019 - Purification, PCR

### Split-NL

The large cultures were centrifuged and the pellets containing bacteria were lysed using ultrasound. After this the samples were centrifuged again to spin
down the lysed bacteria. The lysate was purified with a strep column. The elution was captured in 1.5 ml fractions and protein concentrations were measured
with NanoDrop.

### BRET

Transformation was successful. 14 colonies were selected for colony PCR and this was put on an agarose gel. Colonies 1, 2, 3, 4, 7, 8, 9, 10,
11, 12 & 14 seem to be successful. 14 small cultures were made with all selected colonies. In addition, parts 1 and 2 were PCR amplified using
overhang PCR and the designed primers to prepare them for Gibson Assembly.

### Peking Split Luciferase

The inserts which were ordered at Twist Biosience were multiplied with PCR, because we will need 1 μg for digestion. These inserts will be ligated
in our own vector after dCas9. The sequence for our linker, NanoLuc small bit and NanoLuc large bit will be cut out.

Friday August 23, 2019 - SDS & Bioluminescence test

×

## Friday August 23, 2019 - SDS & Bioluminescence test

### Split-NL

To determine the purity of the samples that were purified a SDS page gel was made. Furthermore, Lbit of NanoLuc is slightly bioluminescent
itself. This was tested with the plate reader.

### BRET

The small cultures were miniprepped and the DNA concentrations were measured using NanoDrop.

Monday August 26, 2019 - Transformation

×

## Monday August 26, 2019 - Transformation

### Split-NL

We ordered three gBlocks containing target DNA at IDT for testing. We PCR amplified these gBlocks to use them for all bioluminescence tests.
After PCR amplification and PCR purification, we measured the DNA concentrations using NanoDrop.

### BRET

The successfully ligated DNA for our BRET sensor was transformed into BL21 cells and plated out. The plates were incubated overnight at 37 °C.
In addition, LB medium for large cultures was prepared for expression later this week.

Tuesday August 27, 2019 - Sequencing & purification

×

## Tuesday August 27, 2019 - Sequencing & purification

### Split-NL

The second batch of dCa9s-Lbit and dCas9-Sbit were purified with strap tag columns. The concentrations were measured using NanoDrop.
Transformation into NovaBlue was done, because there was not enough DNA for sequencing samples.

### BRET

Sequencing samples were made to confirm the ligation was successful. The samples were sent to BaseClear for sequencing. Furthermore,
colonies were picked and small cultures were made.

Wednesday August 28, 2019 - Small cultures & SDS gel

×

## Wednesday August 28, 2019 - Small cultures & SDS gel

### Split-NL

A SDS gel was made to confirm the purification of yesterday. Furthermore, NB small cultures were made which will be miniprepped tomorrow.
As you can see, a big blob can be seen for the elution fractions. A band would be expected at 179 kDa for Lbit and at 163 kDa for Sbit.

### BRET

The large culture media turned out to be infected. New culture media and new small cultures were made.

Thursday August 29, 2019 - Expression & Q-Tof sample

×

## Thursday August 29, 2019 - Expression & Q-Tof sample

### Split-NL

A Q-Tof sample was made for Split NL. However, our proteins are too big for Q-Tof, so no results were retrieved.
In addition, miniprepping of the small cultures was done to retrieve more DNA for sequencing. The DNA concentrations were measured using NanoDrop.

### BRET

The small cultures were added to the large cultures (1.5L) after adding kanamycin. Expression was initiated at an OD of approximately 0.6 with 200 μL/L IPTG.

Friday August 30, 2019 - Purification

×

## Friday August 30, 2019 - Purification

### BRET

The large cultures were spinned down in 2 bottles per culture and lysed using sonication. Per large culture 1 bottle was purified and the other lysed sample was
stored at -80 °C. His and Strep purification was performed. After His purification, no protein was present in the eluted samples.

### September

Monday September 2, 2019 - Testing

×

## Monday September 2, 2019 - Testing

### Split-NL

Started the first tests with the dCas9-Split-NanoLuc sensor. We tested with 50 μL samples in a 384-well plate:

1. Only PBS + 0.1% BSA
2. 10 nM SmallBit
3. 10 nM LargeBit
4. 10 nM SmallBit + 10 nM LargeBit
5. 10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2
6. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA
7. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2
8. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2
9. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2

NanoGlo in 1000x dilution was added to each sample before testing.

### BRET

Last Friday, no protein was present after His purification. Since we circularly permutated the dCas9 protein, it would be possible that the His tag
is not available. Therefore, we decided to only perform Strep purification using the His flow-through from last time. We measured the concentrations
using NanoDrop.

### Peking

To be able to compare our new sensor with the sensor from Peking 2015, we decided to produce the Peking sensor containing Firefly luciferase instead
of Split NanoLuc. We ordered the sequences of the split firefly luciferases with their linker and will put these insert behind our own dCas9. First
digestion was performed with the original vector, Cluc insert, Nluc insert. PCR purification was done and the concentrations were measured using NanoDrop.
The samples were put on an agarose gel and the correct bands were cut out.

Tuesday September 3, 2019 - Testing

×

## Tuesday September 3, 2019 - Testing

### Split-NL

We did more testing in triplo with 50 μL samples in a 384-well plate:

1. Only PBS + 0.1% BSA
2. 10 nM SmallBit
3. 10 nM LargeBit
4. 10 nM SmallBit + 10 nM LargeBit
5. 10 nM SmallBit + 10 nM LargeBit + 1.2nM DNA 2
6. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM unspecific DNA
7. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 0.6 nM DNA 2
8. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 1.2 nM DNA 2
9. 10 nM SmallBit + 10 nM LargeBit + sgRNA 1 + 2.4 nM DNA 2
10. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 0.6 nM DNA 2
11. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 1.2 nM DNA 2
12. 10 nM SmallBit + 10 nM LargeBit + sgRNA 2 + 2.4 nM DNA 2
13. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 0.6 nM DNA 2
14. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 1.2 nM DNA 2
15. 10 nM SmallBit + 10 nM LargeBit + sgRNA 3 + 2.4 nM DNA 2

NanoGlo in 2000x dilution was added to each sample before testing.

In the afternoon, we tested with lower sensor concentrations in duplo:

1. Only PBS + 0.1% BSA
2. 1 nM SmallBit + 1 nM LargeBit
3. 0.5 nM SmallBit + 0.5 nM LargeBit
4. 1 nM SmallBit + 1 nM LargeBit + 1.2 nM DNA 2
5. 0.5 nM SmallBit + 0.5 nM LargeBit + 1.2 nM DNA 2
6. 1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA
7. 0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2 nM unspecific DNA
8. 1 nM SmallBit + 1 nM LargeBit + sgRNA 2 + 1.2nM DNA 2
9. 0.5 nM SmallBit + 0.5 nM LargeBit + sgRNA 2 + 1.2nM DNA 2

NanoGlo in 2000x dilution was added to each sample before testing.

When analyzing the results, the control samples 4 and 5 in test 1 and control samples 3, 4 and 5 in test 2 gave a very high signal. The signal was much higher
than the samples containing sgRNA and target DNA. Therefore, we put samples on the SDS gel. However, we used a relatively high concentration of BSA in the buffer,
which can clearly be seen on gel due to a band at the height of 50 kDa. Some smears can be observed around 300 kDa, indicating aggregation, but it cannot be seen
clearly due to the presence of BSA.

### Peking

The DNA from the piece of gel which was cut out yesterday was extracted. Nluc and Cluc were each separately ligated into the vector containing dCas9. After ligation,
the DNA was transformed into NovaBlue.

Wednesay September 4, 2019 - Colony PCR

×

## Wednesay September 4, 2019 - Colony PCR

### Peking

Ligation was successful since colonies were present. Colony PCR was performed, but the results did not show anything. The positive result had failed as well, so we
cannot use it. Random colonies were selected and small cultures were made.

Tuesday September 5, 2019 - Testing

×

## Tuesday September 5, 2019 - Testing

### Split-NL

We read that dCas9 shows aggregation when incubated at 37 °C, and that this can be prevented by incubation at room temperature for at least 10 minutes.
Therefore, we tested the sensor activity under different incubation conditions. Tests in duplo:

1. 10 nM Sbit + 10 nM Lbit | *incubated for 10 minutes at room temperature*
2. 10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | *incubated for 10 minutes at room temperature*
3. 10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | *incubated for 10 minutes at room temperature*
4. 10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | *incubated for 10 minutes at room temperature*
5. 10 nM Sbit + 10 nM Lbit | *incubated for 30 minutes at room temperature*
6. 10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | *incubated for 30 minutes at room temperature*
7. 10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | *incubated for 30 minutes at room temperature*
8. 10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | *incubated for 30 minutes at room temperature*
9. 10 nM Sbit + 10 nM Lbit | *incubated for 30 minutes at 37 °C*
10. 10 nM Sbit + 10 nM Lbit + 1.2 nM target DNA | *incubated for 30 minutes at 37 °C*
11. 10 nM Sbit + 10 nM Lbit + sgRNA + unspecific DNA | *incubated for 30 minutes at 37 °C*
12. 10 nM Sbit + 10 nM Lbit + sgRNA + 1.2 nM target DNA | *incubated for 30 minutes at 37 °C*

NanoGlo in 2000x dilution was added to each sample.

### BRET

The sequencing for the BRET sensor showed a completely wrong sequence for all samples. However, when we further analyzed the sequence it completely
aligns with the sequence for our Split-NL sensor. When our original vector was digested using the correct restriction enzymes, the bands on the gel
were difficult to distinguish from each other. Probably both our original insert (of the Split-NL sensor) and the vector were present in the sliced gel.
Therefore, the original insert was ligated into the vector. We tested both sample 10 and 11 for luminescence and BRET sample 10 showed a big peak,
indicating the presence of LargeBit. BRET sample 11 did not show any luminescence.

### Peking

Small cultures were miniprepped and transformed into BL21(DE3).

Monday September 9, 2019 - Small culture & Gibson

×

## Monday September 9, 2019 - Small culture & Gibson

### BRET

Since BRET parts 10 and 11 did not show the correct sequence for sequencing, we decided to check the sequence for other colonies. We decided to check the
sequence for colonies 3 and 8. Small cultures in NovaBlue were made for these colonies.

Furthermore Gibson Assembly was performed to ligate the three G-blocks. The mixture was transformed into XL10-Gold cells and plated. The samples of the
Gibson reaction were put on an agarose gel.

### Peking

Small cultures were made. Furthermore, large culture medium, LB medium and LB agar medium were prepared and autoclaved.

Tuesday September 10, 2019 - Sequencing samples & testing

×

## Tuesday September 10, 2019 - Sequencing samples & testing

### Split-NL

#### Sequencing

Sequencing samples were made and sent to BaseClear:

* Sample 1: Sbit 6 + dCas9-G247-FOR
* Sample 2: Sbit 6 + dCas9-G485-FOR
* Sample 3: Sbit 6 + dCas9-G717-FOR
* Sample 4: Sbit 6 + dCas9-A932-FOR
* Sample 5: Sbit 6 + dCas9-T1167-FOR
* Sample 6: Lbit 13 + dCas9-G247-FOR
* Sample 7: Lbit 13 + dCas9-G485-FOR
* Sample 8: Lbit 13 + dCas9-G717-FOR
* Sample 9: Lbit 13 + dCas9-A932-FOR
* Sample 10: Lbit 13 + dCas9-T1167-FOR

#### testing

A test was performed where the interspace distance was varied. Incubation was done for 30 minutes at room temperature. Order of addition
in the plate: (1) DNA (2) Sbit (3) Lbit. NanoGlo was added in a 2000x dilution. Samples tested:

1. 10 nM Sbit + 10 nM Lbit
2. 10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 2
3. 10 nM Sbit + 10 nM Lbit + 1.2 nM non-target DNA
4. 10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
5. 10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
6. 10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
7. 10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nMDNA 2 (interspace distance 17)
8. 10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nMDNA 2 (interspace distance 20)
9. 10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
10. 10 nM Sbit + 10 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
11. 10 nM Sbit + 10 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
12. 10 nM Sbit + 10 nM Lbit + sgRNA 3 + 1.2 nM DNA 3 (interspace distance 30)

### BRET

Miniprepping of BRET no.3 & BRET no.8 (small cultures) was done. These are 2 colonies with the correct vector length during colony PCR.
Afterwards, sequencing samples were made of these BRET samples and sent to BaseClear:

* Sample 11: BRET 10 + T7.FOR
* Sample 12: BRET 10 + dCas9-S1338-FOR
* Sample 13: BRET 10 + dCas9-G247-FOR
* Sample 14: BRET 10 + dCas9-G485-FOR
* Sample 15: BRET 10 + dCas9-G717-FOR
* Sample 16: BRET 10 + dCas9-A932-FOR
* Sample 17: BRET 3 + T7.FOR
* Sample 18: BRET 8 + T7.FOR

No colonies were present after transformation, so Gibson Assembly failed. When looking at the gel, this is not suprising.

### Peking

The small cultures were added to the large cultures. At the end of the day the cultures were induced with IPTG to activate protein expression.
Furthermore, new LB agar plates were made.

Wednesay September 11, 2019 - Overhang PCR

×

## Wednesay September 11, 2019 - Overhang PCR

### BRET

The overhang PCR of G-block 3 had failed before. This was done again (G3Bv2) because the overhangs are needed to perform Gibson Assembly of
G-block 2 and 3 into the backbone (SacI-AgeI). After PCR amplification and purification, Gibson Assembly was performed. The samples for Gibson
were also put on an agarose gel to check whether PCR was successful.

G-block 2 and 3 were also digested for regular ligation. However, this had failed due to too low concentrations.

### Peking

Due to many changes in our original vector we discovered that there were no tags in the Peking construct for purification. The His tag was
lost when the original dCas9 was replaced by the dCas9 from Addgene, due to the restriction sites. Therefore the large cultures were thrown
away and we decided not to use these samples anymore.

Tuesday September 12, 2019 - Testing & Transformation

×

## Tuesday September 12, 2019 - Testing & Transformation

### Split-NL

A test was performed where the samples were incubated for 30 minutes at room temperature. Afterwards, the samples were centrifuged at 13,400 rpm for 5 minutes
to get rid of aggregates. The supernatant was used for testing. NanoGlo was added in 2000x dilution and samples of 30 μL were tested. Samples:

1. 10 nM Sbit + 10 nM Lbit
2. 10 nM Sbit + 10 nM Lbit + 1.2 nM DNA 1
3. 10 nM Sbit + 10 nM Lbit + gRNA 3 + 1.2 nM non-target DNA
4. 10 nM Sbit + 10 nM Lbit + gRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
5. 10 nM Sbit + 10 nM Lbit + gRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
6. 10 nM Sbit + 10 nM Lbit + gRNA 3 + 1.2 nM DNA 1 (interspace distance 15)

In addition, a test was performed where the gRNA was added in a 6x excess (final concentration of 13 nM). This was based on 2 papers. The samples were incubated
for 30 minutes at room temperature. NanoGlo was added in 2000x dilution and samples of 30 μL were tested. Samples:

1. 2 nM Sbit + 2 nM Lbit
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM DNA 1
3. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM non-target DNA
4. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
5. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
6. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
7. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 2 (interspace distance 17)
8. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 2 (interspace distance 20)
9. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
10. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
11. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
12. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 1.2 nM DNA 3 (interspace distance 30)

### BRET

The Gibson Assembly mixture was transformed into XL10-Gold competent cells.

Monday September 16, 2019 - Ligation & transformation

×

## Monday September 16, 2019 - Ligation & transformation

### BRET

Transformation of the Gibson Assembly mixture had failed, since no colonies were present. We decided to do a 1 insert ligation with part 2 into the backbone.
In addition, a 2 insert ligation with both parts 2 and 3 was also performed.

Tuesday September 17, 2019 - Ligation & transformation

×

## Tuesday September 17, 2019 - Ligation & transformation

### BRET

Yesterday's 1 insert and 2 insert ligations of gBlocks BRETp2 and BRETp2 + BRETp3 respectively failed. The agar plates were devoid of colonies,
whereas the positive control plate was not. Therefore, the ligations were repeated with alternative molar ratios (4:1 and 5:5:1 respectively,
insert:(insert:)vector), starting from the same restriction digested building blocks.

Moreover, restriction digestions were performed to form a new set of digested gBlocks, which were subsequently purified by agarose gel.

### Characterization

For a Bronze medal we need to make a contribution in the form of a characterization to an existing part in the Registry. iGEM Eindhoven
2016 created a CT52-mNeonGreen part they did not express due to lack of time. They expressed CT52-Sbit and CT52-Lbit, so therefore it
would be plausible that expression of this vector would also be possible. We found the correct DNA in a pET28a vector and transformed
it in BL21 cells. Two different mutants of CT52 were found, so both were transformed.

In addition, a NanoLuc part was registered in the Part Registry. We want to also characterize NanoLuc, so we need to express this protein
as well. We were able to get the DNA sequence of NanoLuc in a pET-28a vector.

Wednesday September 18, 2019 - Small cultures, digestion, ligation & transformation

×

## Wednesday September 18, 2019 - Small cultures, digestion, ligation & transformation

### BRET

There were no colonies on the plates inoculated with the new ligation mixtures made yesterday, except for only a single one resulting from the BRETp2 + BRETp3 + vector
ligation. We suspect the ligation failed to yield the desired plasmid and will perform colony PCR tomorrow.

DNA was extracted from yesterday's gel slices containing the relevant digested parts (/SacI/-BRETp2-/NheI/; /NheI/-BRETp3-/XhoI/;
/AgeI/-pET28a(+)-dCas9-Lbit-/SacI/) using the Qiagen gel extraction kit. With these, new ligation reactions were performed for ligating
/SacI/-BRETp2-/NheI/ in /NheI/-pET28a(+)-dCas9-Lbit-/SacI/ as well as /NheI/-BRETp3-/XhoI/ in /XhoI/-pET28a(+)-dCas9-Lbit-/NheI/ prepared previously.

### Characterization

#### CT52-mNeonGreen

Transformation was successful for both mutant forms of CT52. Small cultures were made using kanamycin and LB medium.

#### NanoLuc

The vector for NanoLuc did not contain enough DNA, so we first need to replicate the plasmid DNA. Therefore, transformation in NovaBlue was performed.

Thursday September 19, 2019 - Testing & expression

×

## Thursday September 19, 2019 - Testing & expression

### Split-NL

It would be possible that the crRNA + tracrRNA is not working. Therefore, we decided to order complete sgRNA with IDT. Today we tested with
the new sgRNA. The samples were incubated for 30 minutes at 37 °C and sgRNA was added in a 6x molar excess. NanoGlo was added in a 2000x dilution.

1. 2 nM Sbit + 2 nM Lbit | *5mM DTT present in PBS*
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | *5 mM DTT present in PBS*
3. 2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA |5 mM DTT present in PBS
4. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 |*5 mM DTT present in PBS*
5. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | *5 mM DTT present in PBS*
6. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 | *5 mM DTT present in PBS*
7. 2 nM Sbit + 2 nM Lbit | *no DTT*
8. 2 nM Sbit + 2 nM Lbit + 1.2 nM target DNA | *no DTT*
9. 2 nM Sbit + 2 nM Lbit + sgRNA + non-target DNA | *no DTT*
10. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 10 | *no DTT*
11. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 17 | *no DTT*
12. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM target DNA interspace distance 25 | *no DTT*

**Native-PAGE** to determine if dCas9 binds to the target sequence.

Native PAGE sample and running buffer were prepared. Samples were made, many controls were included (see below). Samples were loaded on the gel
and afterwards the gel was stained with SYBRGold to stain the DNA. Furthermore, the gel was stained with Coomassie overnight.

### BRET

No colonies were present, indicating that ligation had failed once again. The ligation of part 2 was repeated and the ligation mixtures of yesterday
were transformed again in NovaBlue cells. In addition, colony PCR of the single colony on the plate of 17/09 was performed. The positive control
showed the correct band on the gel, but the colony did not.

### Characterization

#### CT52-mNeonGreen

Two 1L large cultures were made (1 for each small culture) using kanamycin and LB medium. Small cultures were added to the large cultures and
incubated at 37 °C and 160 rpm until OD600 = 0.6-0.7. Expression was induced at OD600 = 0.6 by adding IPTG.

#### NanoLuc

Transformation was not successful. The transformation of BRET ligation was not successful as well, so it would be possible that something
went wrong during the transformation experiment. Therefore, transformation was performed again, in BL21.

Friday September 20, 2019 - Purification

×

## Friday September 20, 2019 - Purification

### Split-NL

The native PAGE gel was destained with water and images were taken using ImageQuant. However, the gel took very long to run and we encountered
some problems. Therefore, the gel did not run long enough to be able to distinguish between bands.

### BRET

No colonies were present after transformation of the 1:1 ligation. There were some small colonies on the 2:1 ligation plate, which will be analyzed
using colony PCR next week.

### Characterization

#### NanoLuc

The transformation was successful.

#### mNeonGreen

The large cultures were centrifuged, the pellets were dissolved and lysed with BugBuster. The lysate was purified with IMAC chromatography. The
protein concentration were measured with NanoDrop. No protein was measured and the pellet after expression was not green, so the expression had
probably failed.

Monday September 23, 2019 - Colony PCR & testing

×

## Monday September 23, 2019 - Colony PCR & testing

### Split-NL

Sequencing was not successful last week, so new sequencing samples were made.

In addition, additional tests were performed. A new buffer was used containing 20 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2and 5% glycerol.
30 μL samples were made and 2000x diluted NanoGlo was added. We used 2 different protein batches; one batch that was purified on 22-08
and one batch that was purified on 27-08.

1. 2 nM Sbit + 2 nM Lbit | *Proteins from 22-08*
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM DNA 1 | *Proteins from 22-08*
3. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | *Proteins from 22-08*
4. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | *Proteins from 22-08*
5. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 1 | *Proteins from 22-08*
6. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 2| *Proteins from 22-08*
7. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 3 | *Proteins from 22-08*
8. 2 nM Sbit + 2 nM Lbit | *Proteins from 27-08*
9. 2 nM Sbit + 2 nM Lbit + DNA 1| *Proteins from 27-08*
10. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | *Proteins from 27-08*
11. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA| *Proteins from 27-08*
12. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 1 | *Proteins from 27-08*
13. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 2 | *Proteins from 27-08*
14. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + DNA 3 | *Proteins from 27-08*

### BRET

Colony PCR was performed using the colonies from the transformation of 20/09. However, colony PCR failed
since the positive control showed a band at the correct height, but all samples did not show any band.

### Characterization

#### mNeonGreen

Protein expression was not successful, so sequencing samples were made to check whether the DNA sequence is correct. Sequencing samples
were made using the T7 FOR primer. The sequencing samples showed that a part of the mNeonGreen sequence was not correct. Therefore, we
decided to use a different part for characterization.

Tuesday September 24, 2019 - Small cultures

×

## Tuesday September 24, 2019 - Small cultures

### Characterization

Small cultures were made for NanoLuc.

Wednesday September 25, 2019 - Testing

×

## Wednesday September 25, 2019 - Testing

### Split-NL

We found out that we miscalculated the DNA concentration, so we have been using 6 pM DNA instead of 6 nM DNA. Therefore, we have only been measuring
background signal. New tests were performed using the correct DNA amounts. 30 μL samples were created with a 2 nM protein concentration, a 6x molar
excess of sgRNA and a 6 nM DNA concentration. The protein with sgRNA was incubated at 37 °C for 10 minutes and incubation of the complex with DNA was
done for 30 minutes at room temperature. For half of the samples the sgRNA was incubated at 50 °C for 5 minutes before use, which could avoid secondary
structure formation of the sgRNA before incubation with dCas9.

1. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | *sgRNA incubation at 37 °C*
2. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | *sgRNA incubation at 37 °C*
3. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | *sgRNA incubation at 37 °C*
4. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | *sgRNA incubation at 37 °C*
5. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | *sgRNA incubation at 37 °C*
6. 2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
7. 2 nM Sbit + 2 nM Lbit
8. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25) | *sgRNA incubation at 50 °C*
9. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17) | *sgRNA incubation at 50 °C*
10. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10) | *sgRNA incubation at 50 °C*
11. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA | *sgRNA incubation at 50 °C*
12. 2 nM Sbit + 2 nM Lbit + sgRNA 1 | *sgRNA incubation at 50 °C*
13. 2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
14. 2 nM Sbit + 2 nM Lbit
15. 2 nM Sbit + 2 nM Lbit + sgRNA 2A + sgRNA 1B + target DNA | *sgRNA incubation at 50 °C*

### BRET

After extracting the digested parts and backbones from yesterday's agarose gel using the Qiagen gel extraction kit , we tried two more
single insert ligations for creating our BRET sensor construct:

* /NheI/-BRETp3 -/XhoI/ in /XhoI/-pET28a(+) -/NheI/
* /SacI/-BRETp2 -/NheI/ in /NheI/-pET28a(+) -/SacI/

Both were performed using T4 DNA ligase, in 20 and 25 μL reactions for BRETp2 and BRETp3 respectively and 50 ng backbone was used.

Thursday September 26, 2019 - Testing

×

## Thursday September 26, 2019 - Testing

### Split-NL

More tests were performed to find the optimal interspace distance. 30 μL samples were made in triplo. The sgRNA was incubated for 10 minutes
at 50 °C before use. Subsequently, sgRNA and Sbit or Lbit was incubated for 10 minutes at 37 °C. The sgRNA:protein complex was incubated with
DNA for 30 minutes at room temperature. NanoGlo was added in 2000x dilution just before testing.

1. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 3 (interspace distance 30)
2. 2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 3 (interspace distance 27)
3. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 3 (interspace distance 25)
4. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 2 (interspace distance 22)
5. 2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 2 (interspace distance 20)
6. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 2 (interspace distance 17)
7. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM DNA 1 (interspace distance 15)
8. 2 nM Sbit + 2 nM Lbit + sgRNA 2 + 1.2 nM DNA 1 (interspace distance 12)
9. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM DNA 1 (interspace distance 10)
10. 2 nM Sbit + 2 nM Lbit + sgRNA 1 + 1.2 nM non-target DNA
11. 2 nM Sbit + 2 nM Lbit + sgRNA 1
12. 2 nM Sbit + 2 nM Lbit + 1.2 nM non-target DNA
13. 2 nM Sbit + 2 nM Lbit

### BRET

Unfortunately, the ligation reactions tried yesterday appear to have failed, again no colonies had grown on the plates. We ordered new
gBlocks coding for the BRET sensor from IDT last night that are ready for Gibson assembly in our pET28a(+) vector, linearized by PCR.

### Characterization

We had a meeting with one of our supervisors and we decided to characterize mCherry.

Monday September 30, 2019 - Testing

×

## Monday September 30, 2019 - Testing

### Split-NL

Bioluminescent tests were performed with centrifugation before testing to hopefully prevent aggregation. Samples of 30 μL
were made using 2 nM protein, 1.2 nM DNA and a 6x molar excess of gRNA. NanoGlo was added in 2000x dilution to each sample.
gRNA was incubated at 50 °C for 5 minutes prior to use. Subsequently, gRNA and protein were incubated at 37 °C for 20 minutes.
DNA with the protein-gRNA complex was incubated at room temperature for 45 minutes.

1. 2 nM Sbit + 2 nM Lbit | *5 min centrifugation after incubation*
2. 2 nM Sbit + 2 nM Lbit + 1.2 non-target DNA | *5 min centrifugation after incubation*
3. 2 nM Sbit + 2 nM Lbit + sgRNA 3| *5 min centrifugation after incubation*
4. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM non-target DNA | *5 min centrifugation after incubation*
5. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + DNA 3 | *5 min centrifugation after incubation*
6. 2 nM Sbit + 2 nM Lbit
7. 2 nM Sbit + 2 nM Lbit + 1.2 non-target DNA
8. 2 nM Sbit + 2 nM Lbit + sgRNA 3
9. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + 1.2 nM non-target DNA
10. 2 nM Sbit + 2 nM Lbit + sgRNA 3 + DNA 3

### Characterization preparation

#### Cysteine free NanoLuc

Transformation in BL21 was done.

### October

Tuesday October 1, 2019 - Small cultures

×

## Tuesday October 1, 2019 - Small cultures

### Characterization preparation

#### Cysteine free NanoLuc

Colonies were present, so small cultures were made.

#### mCherry

Small cultures were made from a glycerol stock with mCherry in BL21.
Furthermore, large culture media were prepared for expression of cysteine free NanoLuc and mCherry.

Wednesday October 2, 2019 - Expression & testing

×

## Wednesday October 2, 2019 - Expression & testing

### Split-NL

We performed test with a variable protein concentrations. We used the concentrations 2 nM, 1 nM and 0.5 nM. We used 30 μL samples using the Tris-HCl
buffer containing BSA and DTT. DNA concentrations of 1.2 nM and a gDNA 6x molar excess were used. gRNA was incubated at 50 °C for 10 minutes.
Incubation of Sbit and Lbit with gRNA was performed for 20 minutes at 37 °C, and subsequently incubation with DNA was done for 45 minutes at room
temperature. NanoGlo was added in 2000x excess.

1. 2 nM Sbit + 2 nM Lbit
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA
3. 2 nM Sbit + 2 nM Lbit + gRNA
4. 2 nM Sbit + 2 nM Lbit + gRNA + 1.2 nM nontarget DNA
5. 2 nM Sbit + 2 nM Lbit + gRNA + 1.2 nM DNA 3 (interspace distance 30)
6. 1 nM Sbit + 1 nM Lbit
7. 1 nM Sbit + 1 nM Lbit + 1.2 nM nontarget DNA
8. 1 nM Sbit + 1 nM Lbit + gRNA
9. 1 nM Sbit + 1 nM Lbit + gRNA + 1.2 nM nontarget DNA
10. 1 nM Sbit + 1 nM Lbit + gRNA + 1.2 nm DNA 3 (interspace distance 30)
11. 0.5 nM Sbit + 0.5 nM Lbit
12. 0.5 nM Sbit + 0.5 nM Lbit + 1.2 nM nontarget DNA
13. 0.5 nM Sbit + 0.5 nM Lbit + gRNA
14. 0.5 nM Sbit + 0.5 nM Lbit + gRNA + 1.2 nM nontarget DNA
15. 0.5 nM Sbit + 0.5 nM Lbit + gRNA + 1.2 nM DNA 3 (interspace distance 30)

### Characterization

#### mCherry & Cysteine-free NanoLuc

The expression of cysteine free NanoLuc and mCherry was activated by adding 1 mM IPTG at OD 0.5. The large cultures were incubated overnight
18 °C at 160 rpm.

Thursday October 3, 2019 - Testing & purification

×

## Thursday October 3, 2019 - Testing & purification

### Split-NL

We performed tests using variable concentrations of mNeonGreen-NanoLuc to determine which concentration we would be using in our experiments later on.
30 μL samples were used in the Tris-HCl, MgCl2buffer containing DTT and BSA. NanoGlo was added in 2000X dilution.

1. 2 nM Sbit + 2 nM Lbit + sgRNA + 1.2 nM DNA 3 (interspace distance 30)
2. 0.1 pM mNeonGreen-NanoLuc
3. 0.2 pM mNeonGreen-NanoLuc
4. 1 pM mNeonGreen-NanoLuc
5. 2 pM mNeonGreen-NanoLuc
6. 5 pM mNeonGreen-NanoLuc
7. 10 pM mNeonGreen-NanoLuc
8. 20 pM mNeonGreen-NanoLuc

### mCherry

The large culture of mCherry was purified with Nickle affinity chromatography.

Fluorescence was measured.

* 96 Well plate
* A7. 1nM mCherry
* A8. 1pM mCherry
* A9. 10 pM mCherry
* A10. 100 pM mCherry
* A11. 10 nM mCherry

### Cysteine free NanoLuc

The large culture was purified with Nickle affinity chromatography. After elution
a concentration of 1.36 mg/ml was measured with Nanodrop. Tomorrow the elution will be further purified with the strep tag.

### Phage experiments

One of us went to the MCT lab of the QAMH Brussels to discuss with Dr. Pirnay about the experiments we would like to perform
with phages. A 100 μL sample of T7 phages with a high titer of 1011PFU/mL was also taken back to Eindhoven to denature by heat
and allow for testing our split-NL sensor on whole T7 genome.

Friday October 4, 2019 - PCR for Gibson & Testing

×

## Friday October 4, 2019 - PCR for Gibson & Testing

### Split-NL

T7 phages were heat denatured at 70 °C for 2 hours. The phage DNA was then tested. A 2 nM protein concentration, ±0.1 nM phage DNA concentration and
6x excess of gRNA was used. The gRNA was incubated at 50 °C before use. The gRNA with protein was incubated at 37 °C for 10 minutes and subsequently
the gRNA-protein complex was incubated with DNA for 30 minutes at room temperature. NanoGlo was then added in a 1000x dilution.

1. 2 nM Sbit + 2 nM Lbit
2. 2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA
3. 2 nM Sbit + 2 nM Lbit + gRNA
4. 2 nM Sbit + 2 nM Lbit + 1.2 nM nontarget DNA + gRNA
5. 2 nM Sbit + 2 nM Lbit + gRNA 1 + 0.1 nM T7 phage DNA (interspace distance 17)
6. 2 nM Sbit + 2 nM Lbit + gRNA 2 + 0.1 nM T7 phage DNA (interspace distance 20)
7. 2 nM Sbit + 2 nM Lbit + gRNA 3 + 0.1 nM T7 phage DNA (interspace distance 22)

### BRET

The backbone required for the new Gibson assembly strategy we will try next week were linearized from the original pET28a(+)-split-NL vector by PCR
in two variants:

* One complete backbone was made (A, ca. 5.3 kb)
* The same backbone was prepared as two parts with a 20 bp mutual overlap for Gibson assembly (B and C, ca. 2.7 and 2.6 bp respectively).

Afterwards, the resulting PCR mixtures were PCR purified and samples were run on an agarose gel to check for correct PCR products.

### mCherry & Cysteine free NanoLuc

The samples from the purification were put on an SDS-PAGE gel.

Monday October 7, 2019 - Gibson Assembly & QAMH

×

## Monday October 7, 2019 - Gibson Assembly & QAMH

### BRET

Gibson assembly was performed, one sample with one backbone and one sample with the backbone in 2 parts. A gibson assembly control was also included.
The samples were transformed in XL10-Gold and plated.

### mCherry

Dilutions were made of 100 nm, 1 μM and 10 μM. The excitation and emission spectrum were measured. The effect of photobleaching after 10 min, 20 min and
30 min UV light exposure was also measured.

### Phage experiments at QAMH Brussels

The first day of our experimental work at the laboratory for molecular and cellular technology (MCT lab) at Queen Astrid Military Hospital (QAMH) in Brussels!
Today after some introduction to the lab, meeting its researchers and getting to know where to find the things we needed, we prepared the M9 based minimal medium
we will use for culturing the *E. coli B* DSM 613 (ATCC 11303) bacteria. Some unexpected difficulties arose, as at first we added all components together
directly in random order, resulting in precipitation. We adapted the protocol after some more trial and error and doing some more research to finally obtain
the medium we needed. We also poured some petri dish LB-Agar plates for the experiments to be performed later this week and started two 100 ml liquid overnight
cultures (37 °C, 120 RPM) of the *E coli B* DSM 613 (from a collaboration between Dr. Pirnay's lab and the iGEM Munich 2018 team), taken from the plate Maya
Merabishvili prepared for us.

Tuesday October 8, 2019 - Gibson Assembly, testing & QAMH

×

## Tuesday October 8, 2019 - Gibson Assembly, testing & QAMH

### Split-NL

We ordered new DNA gBlocks, so we would be able to test interspace distances 30-60 bp. Tests were performed using 2 nM protein, 1.2 nM DNA and a 6x
molar excess of gRNA. gRNA was incubated at 50 °C before use and the gRNA and protein were incubated at 37 °C for 20 minutes. Subsequently, the gRNA-protein
complex was incubated with DNA for 45 minutes at room temperature. NanoGlo was added in 2000x dilution.

### BRET

No colonies were present on all the plates. The control plates also had no colonies, but we discovered that the wrong plates were used because the control was
not kanamycin resistant. The gBlocks were PCRed and Gibson assembly was tried again and the samples were transformed in XL10-Gold ultracompetent cells. The
samples from Gibson assembly yesterday were put on an agarose gel.

### Phage experiments at QAMH Brussels

We planned to start the bacterial growth rate determination experiment today, yet unfortunately the overnight cultures still appeared very transparent, whereas
one would expect them to be rather turbid because of a high cell density. The only turbidity we could see was some sediment, which we believe was some
precipitated calcium-or magnesium sulfate. OD600 measurements confirmed the lack of an appreciable amount of bacteria. We discussed the apparent
problem with others at the lab and looked into the recipe we used once more and made slight adjustments (added a trace amount of FeSO4, kept MgCl
out and made some with tryptone added as control) and started some small new liquid cultures using these media. We also prepared and autoclaved 400 mL
semisolid LB Agar (0.8%) for later use.

Wednesday October 9, 2019 - Gibson Assembly, testing & QAMH

×

## Wednesday October 9, 2019 - Gibson Assembly, testing & QAMH

### Split-NL

To improve the signal-to-noise ratio, we decided to test with higher protein concentrations. We used interspace distance 30 for testing, since this gave the
highest signal in previous tests. We under the same conditions as the previous tests, but we used the protein concentrations 2, 5 and 10 nM.

### BRET

No colonies were present on all the plates. The agarose gel of the gBlocks which were PCRed showed unexpected results for part 2. The weight was lower as expected.
The PCR reaction was tried again. However, the same results came out of the gel.

### Phage experiments at QAMH Brussels

Luckily we did not throw away our seemingly failed cultures yesterday, but kept them in the shaking incubator instead, as this morning the media of one of the
flasks was all turbid! OD600 measurement confirmed the presence of a high bacterial cell density, with an OD600 of 1.525. The other culture
had obtained an OD600 of 0.081 and appeared slightly turbid to the naked eye, hence it was put back in the incubator. At the end of the day this
culture had reached an OD600 of 0.604. To check the linearity of the OD600 vs. cell concentration we prepared a serial two-fold dilution
series and measured the OD600 giving the below, nicely linear, results:

| Dilution | OD600 culture 1 | OD600 culture 2 |
| --- | --- | --- |
| 1x | 1.475 | 0.601 |
| 2x | 0.825 | 0.304 |
| 4x | 0.433 | 0.159 |
| 8x | 0.224 | 0.075 |
| 16x | 0.114 | 0.040 |
| 32x | 0.057 | 0.019 |
| 20x | 0.090 |  |

![Process](https://static.igem.org/mediawiki/2019/e/e3/T--TU_Eindhoven--Lab20191009Linearity.png)
Figure 3: linearity of the OD600 vs. cell concentration of culture 1 with R2 = 0.9934 and culture 2 with R2 = 0.9997.

We then started the bacterial growth rate determination experiment according to the protocol. Eight 70 mL cultures were used, being 20x dilutions of the culture
described above, resulting in a starting OD600 of 0.13 (measured in flask 8 just before start of the actual experiment). The 8 cultures differed in
starting concentration of glucose:

1. 0.0 g/L Glucose
2. 0.01 g/L Glucose
3. 0.25 g/L Glucose
4. 0.5 g/L Glucose
5. 1.0 g/L Glucose
6. 2.0 g/L Glucose
7. 4.0 g/L Glucose
8. 8.0 g/L Glucose

A 1 mL sample was taken every 20 minutes to measure the OD600 of each individual culture. At the same time, another 1 mL sample was stored in an
Eppendorf tube and quickly frozen using liquid nitrogen to be analyzed for remaining glucose concentration later on. A set of calibration samples was also
prepared covering the starting glucose concentrations of the experimental cultures and included *E. coli* at an OD600 of 0.163.

![Process](https://static.igem.org/mediawiki/2019/2/25/T--TU_Eindhoven--Lab20191009BacterialGrowthVSGlucose.png)

Thursday October 10, 2019 - Transformation, testing & QAMH

×

## Thursday October 10, 2019 - Transformation, testing & QAMH

### Split-NL

We tested multiple protein concentrations to see what range can be used for testing. We expected that the background signal would increase for high protein
concentrations. Yesterday we saw that 10 nM protein concentration gave a good signal-to-noise ratio. Today we tested 2, 10, 20, 50 and 100 nM.

### BRET

Gibson assembly was tried again from the original gBlock stocks and the samples were transformed in XL10-Gold ultracompetent cells. Two controls were includes;
one for the Gibson assembly and one for the transformation.

### Phage experiments at QAMH Brussels

Yesterday's glucose concentration calibration samples were analyzed using a Roche Hitachi cobas 6000 / c 501 (set at blood serum and GLUC-3 analysis setting)
made available to us at QAMH. From the results, it became clear that calibration wasn't needed as reported concentration values closely matched the expected
values. Due to a lack of time, the experimental samples will be analyzed tomorrow.

We also performed a first trial of the experiment for determination of the bacteriophage adsorption rate. A dilution of a mid-log phase culture
(OD600 0.549) was made (resulting in OD600 0.188) and used for the actual experiment. We performed the experiment with two different
phage titers in parallel, both with a final dilution of 10-7 and 10-6 of the 3∙1011 stock. The resulting solutions were
plated and incubated overnight.

Yesterday's plates for enumerating the amount of colonies to calculate the bacteria titer showed inconsistent results, hence we will make multiple sets of
new plates in the upcoming days to be able to convert OD600to CFU/ml reliably.

Friday October 11, 2019 - Testing

×

## Friday October 11, 2019 - Testing

### Split-NL

We tested the limit of detection of our sensor using 2 nM and 10 nM protein. A range of DNA concentrations from 1 pM to 10 nM was tested. The incubation
conditions were the same as in previous tests and NanoGlo was added in 2000x dilution.

### BRET

Colonies were present one of the Gibson assembly samples, the control for Gibson and the control for transformation! The colonies from Gibson were very small
at first, so they were put back into the stove and taken out at the end of the day.

### Phage experiments at QAMH Brussels

This morning we found our very first phage plaques! The plaques of yesterday's 24 plates were counted, but unfortunately no clear decline in unadsorbed phage
could be seen over the 10 minutes sampling time. Therefore, we decided to plate the samples we stored in the fridge again today, to see if the unexpected
result might be due to plating errors. Only the samples from the 10-6 dilution were plated again, and using 200μL instead of 100μL this time to
increase the number of plaques, aiding counting. We also plated a 10-fold serial dilution series of the T7 phage stock in order to re-titer the stock. Later
the same day we could already count the plaques, but unfortunately we found the same overall result for the adsorption rate experiment. For the stock we found:

| Dilution | Plaques | PFU/ml |
| --- | --- | --- |
| 10-7 | 518 | 5E+10 |
| 10-7 | 68 | 7E+10 |
| 10-7 | 9 | 9E+10 |
| Average |  | 7E+10 |

The glucose concentrations of the samples of 9 October were also measured using the Hitachi Roche cobas 6000. The results showed some dubious deviations from
the expected curve, but we will still try to use it to fit our model.

We also performed the infection assay experiment, first growing a diluted overnight culture, starting from OD600 0.104, reaching 0.126 when starting
the actual experiment. To a 66 mL culture, 7\*106PFU/ml phage was added, resulting in a 7\*104PFU/ml solution. 3.8 ml growth medium and 15
droplets of chloroform were added in multiple falcon tubes to collect samples of 0.2 ml cell/phage suspension in. Moreover, 1 ml samples were collected in
cuvettes for OD600 measurements as well as 1 ml samples for glucose concentration measurements. The glucose samples will be analyzed next week,
while part of the chloroformed samples were already plated (plaque assay) in various dilutions. The remainder will be plated next week.

Monday October 14, 2019 - Testing, small cultures & miniprepping

×

## Monday October 14, 2019 - Testing, small cultures & miniprepping

### Split-NL

Tests using 1 nM and 0.5 nM protein concentrations were performed to test whether the limit of detection could be lowered. DNA concentrations from 0.5 pM to
10 pM were used and the same incubation conditions were used as in previous tests.

### BRET

Colony PCR was performed and small cultures were made early in the morning. The small cultures were miniprepped at the end of the day. However, some samples
had no DNA at all.

Tuesday October 15, 2019 - Testing & PCR

×

## Tuesday October 15, 2019 - Testing & PCR

### Split-NL

We wanted to make a calibration curve using NanoLuc-mNeonGreen. We tested a range of DNA concentrations using just our sensor without NanoLuc-mNeonGreen and
we tested the same range of DNA concentrations with the addition of NanoLuc-mNeonGreen.

### BRET

To confirm the negative colony PCR, a regular PCR reaction was performed to check the ligation of the gBlocks. T7 reverse and forward primers were also used
the look at what is inside the vector.

### Phage experiments at QAMH Brussels

The plaques of last Friday's plates were counted and the remaining samples that were not yet plated, were plated at several relevant dilutions. Later that day
we could already count the plaques on the plates with a countable number of plaques. Unfortunately, not of all sampling times the titer could be determined,
but with the available datapoints we found a clear overall pattern that matched our expectations.

| Time (min) | Phage [PFU/ml] |
| --- | --- |
| 0 | 7.00E+3 |
| 20 | 3.67E+03 |
| 40 | 6.67E+03 |
| 60 | 9.41E+04 |
| 80 | 9.99E+05 |
| 120 | 1.80E+07 |
| 160 | 4.60E+07 |
| 180 | 4.00E+07 |
| 200 | 7.00E+07 |
| 220 | 3.49E+08 |

![Process](https://static.igem.org/mediawiki/2019/5/5e/T--TU_Eindhoven--Lab20191015PhageGrowth.jpg)
Figure 5: Phage growth plotted over time.

Griet Steurs helped us out by starting a bacterial culture from the plated DSM 613 for us on Sunday already. This culture was subcultured and grown for use in
a repetition of the adsorption rate determination experiment. Unfortunately the bacteria appeared to grow only very slowly, so we didn't perform the experiment
and instead started some additional liquid cultures for use tomorrow.

We also heat denatured (70°C, 2 hours) a 10-fold serial dilution (from 100 to 10-9) of the phage stock for experiments in Eindhoven, to try
to detect the freed phage DNA.

The glucose measurement samples collected last Friday during the infection assay were also measured using the Roche Hitachi cobas 6000.

Wednesday October 16, 2019 - Testing

×

## Wednesday October 16, 2019 - Testing

### Split-NL

The proof-of-concept was tested using denatured phages that had infected bacteria for a variable amount of time. However, we did not exactly know the DNA
concentration of these samples. Bioluminescent tests were performed using 2 nM sensor protein and a 3x molar excess of gRNA. The results show that we are
able to detect the phage DNA after infection and denaturation. However, no difference in signal can be observed for the different samples. We do not yet know
the cause, but it would be possible that the DNA concentration does not vary much between the samples.

### Phage experiments at QAMH Brussels

A successfully grown overnight culture was used to repeat the phage adsorption rate determination experiment, with a start OD600 of 0.308 and phage
titer 7∙104. After discussing the results of the previous trial of this experiment with Dr. Pirnay and Dr. Merabishvili, we discovered we hadn't
strictly controlled the temperature, which could have influenced the adsorption. Therefore, this time we used a water bath in which the flask could remain fixed.

We also took pictures of all our plates that we accumulated thus far, one examples clearly showing plaques is shown below.

![Process](https://static.igem.org/mediawiki/2019/0/09/T--TU_Eindhoven--Lab20191016PlaquePlate.jpg)
Figure 6: One of the accumulated plates.

Thursday October 17, 2019 - Testing

×

## Thursday October 17, 2019 - Testing

### Split-NL

We performed bioluminescence tests to check whether the limit of detection can be lowered when multiple sensor protein pairs bind to multiple recognition
sites on one target. We used the test gBlocks and all three gRNA pairs to test the bioluminescent signal for a range of DNA concentrations when three sensor
proteins bind to one target. Results show that the limit of detection can be lowered to 5 pM in this situation.

### Phage experiments at QAMH Brussels

Unfortunately, all plates of the adsorption rate determination experiment contained an uncountable number of phages, in fact only a web like structure
remained with some resistant bacterial colonies. Clearly an error was made, as more phages than expected were present.

Some plates of samples of the infection assay of last week that were plated yesterday did show countable numbers of plaques, consistent over multiple dilutions.
Results obtained from these plates were added to the ones obtained previously (see 15-10-2019).

The adsorption rate determination experiment was performed one last time, over a period of 20 minutes this time. Starting OD600was 0.096, with an
OD600 0.142 reached 30 minutes after finishing a sampling period of 20 minutes. Note: The origin culture grew remarkably well this time, obtaining
an OD600 of 1.237 from 0.962 in 1,25 hours. A 10-fold serial dilution series was made of this culture and plated to convert the OD600
values to CFU/ml.

The next day, Griet Steurs counted all plates for us (for which many thanks!). Unfortunately, the results of the adsorption rate determination experiment were
inconsistent again and didn’t show the expected decrease in unadsorbed phage. The plates for determining the cell titer were nicely consistent over multiple
dilutions and duplo's however, so we could reliably convert all OD600 values.

Friday October 18, 2019 - Testing

×

## Friday October 18, 2019 - Testing

### Split-NL

Three different bioluminescence tests were performed: variable interspace distance, a range of phage DNA concentrations and a calibration test using
NanoLuc-mNeonGreen.

#### Variable interspace distance

We performed bioluminescence tests using interspace distances from 20 - 150 bp. A 2 nM protein concentration, 3x molar excess of gRNA and 250 pM DNA
concentration was used. The same incubation conditions as in previous experiments were used. The results showed a high bioluminescent signal for the
controls and the bioluminescent signal for many samples was lower than the controls. Therefore, we decided to perform this test again on Monday.

#### Range of phage DNA concentrations

From Brussels we retrieved phage samples containing a range of CFU/mL. These samples were denatured by heating them for 2 hours at 70 °C and measured
using our sensor. The results show a decrease in bioluminescent signal when the CFU/mL is lower. However, some outliers were present.

#### Calibration test using NanoLuc-mNeonGreen

We already performed a calibration test, which had quite good results. However, we did not test enough concentrations to be able to make a good calibration
curve containing many data points. Therefore, we did a calibration test using 2 nM sensor concentration, a 3x molar excess of gRNA, 2 pM NanoLuc-mNeonGreen
and a range of DNA concentrations. However, the resulting data points could not be fitted into a good calibration curve.

Monday October 21, 2019 - Testing

×

## Monday October 21, 2019 - Testing

### Split-NL

Since the test using variable interspace distances was not successful last week, we performed the test again today. We used 1.2 nM DNA concentrations instead
of 250 pM. The same incubation conditions and concentrations were used. We retrieved good results with an optimal interspace distance of 70 bp.

## Milestones

Throughout the competition, a lot of milestones were achieved inside and outside the lab. Below you can find the list of the most important milestones we achieved together with our team.

February 6, 2019 - Kick-off meeting

March 27, 2019 - Team complete

March 28, 2019 - Officially registered for iGEM

April 15, 2019 - First meeting with an expert, Chris Arts

April 23, 2019 - Reached the top 40 of the TU/e contest

April 24, 2019 - Arrival of the iGEM distribution kit

May 2, 2019 - First team bonding activity

May 13, 2019 - Offical iGEM TU Eindhoven 2019 design

May 20, 2019 - Reached the top 20 of the TU/e contest

May 22, 2019 - Netherlands Biotechnology Congress (NBC), Ede

May 28, 2019 - First meeting at Queen Astrid Military Hospital, Brussels, with Jean-Paul Pirnay

June 6, 2019 - 2nd place at TU/e contest final event

June 11, 2019 - New office

June 13, 2019 - iGEM Europe meetup, The Hague

June 13, 2019 - First patient talk, Queen Astrid Military Hospital, Brussels

June 14, 2019 - Phage therapy symposium, Queen Astrid Military Hospital, Brussels

June 17, 2019 - First day in the lab, because of arrival vector

June 17, 2019 - Arrival team clothing and team photoshoot

July 11, 2019 - First sponsor: DSM

July 17, 2019 - Article about iGEM TU Eindhoven on FMT gezondheidszorg

August 17, 2019 - Dutch Meet-Up Leiden and Groningen

August 22, 2019 - Formation of our dCas9-Split-NanoLuc

August 26, 2019 - Crowdfunding online

August 26, 2019 - Official GO for the team

August 29, 2019 - Booking of tickets to Boston

September 2, 2019 - Team project name: dCastect

September 7, 2019 - Reaching 50% of our crowdfunding campaign

September 19, 2019 - Second team bonding evening

September 21, 2019 - Article in the Eindhovens Dagblad (ED)

September 21, 2019 - Reaching 100% of our crowdfunding campaign

September 25, 2019 - Positive test results of our dCas9-Split-NanoLuc

October 2, 2019 - Arrival Postcards Duesseldorf collaboration

October 7, 2019 - Performing our phage experiments at the QAMH in Brussels

October 22, 2019 - Successful Wiki Freeze!

October 25, 2019 - BeNeLux Mini Jamboree, Eindhoven


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TU Eindhoven/Experiments

* [![](https://static.igem.org/mediawiki/2019/1/18/T--TU_Eindhoven--smallLogo.png)](https://2019.igem.org/Team%3ATU_Eindhoven)
* PROJECT

  [Description](https://2019.igem.org/Team%3ATU_Eindhoven/Description)
  [Design](https://2019.igem.org/Team%3ATU_Eindhoven/Design)
  [Demonstrate](https://2019.igem.org/Team%3ATU_Eindhoven/Demonstrate)
  [Model](https://2019.igem.org/Team%3ATU_Eindhoven/Model)
  [Device](https://2019.igem.org/Team%3ATU_Eindhoven/Device)
  [Safety](https://2019.igem.org/Team%3ATU_Eindhoven/Safety)
* LAB

  [Experiments](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
  [Notebook](https://2019.igem.org/Team%3ATU_Eindhoven/Notebook)
  [Contribution](https://2019.igem.org/Team%3ATU_Eindhoven/Contribution)
  [Results](https://2019.igem.org/Team%3ATU_Eindhoven/Results)
  [Measurement](https://2019.igem.org/Team%3ATU_Eindhoven/Measurement)
* PARTS

  [Parts overview](https://2019.igem.org/Team%3ATU_Eindhoven/Parts)
  [Basic parts](https://2019.igem.org/Team%3ATU_Eindhoven/Basic_Part)
  [Composite parts](https://2019.igem.org/Team%3ATU_Eindhoven/Composite_Part)
  [Part collection](https://2019.igem.org/Team%3ATU_Eindhoven/Part_Collection)
  [Improve](https://2019.igem.org/Team%3ATU_Eindhoven/Improve)
* HUMAN PRACTICES

  [Integrated human practices](https://2019.igem.org/Team%3ATU_Eindhoven/Human_Practices)
  [Education & engagement](https://2019.igem.org/Team%3ATU_Eindhoven/Public_Engagement)
* TEAM

  [Team Members](https://2019.igem.org/Team%3ATU_Eindhoven/Team)
  [Attributions](https://2019.igem.org/Team%3ATU_Eindhoven/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ATU_Eindhoven/Collaborations)
  [Sponsors](https://2019.igem.org/Team%3ATU_Eindhoven/Sponsors)
* MEDALS

  [Medal requirements](https://2019.igem.org/Team%3ATU_Eindhoven/Medal_Requirements)
  [Awards](https://2019.igem.org/Team%3ATU_Eindhoven/Awards)
* [CONTACT](#footer)

# Experiments

* [General Protocols](#general)
* [Cloning, Mutagenesis & Gibson Assembly](#cloning)
* [Transformation, Protein Expression & Purification](#transformation)
* [Testing](#testing)
* [Phage experiments](#phage)

## Experiments

Here you can find and download all the protocols that were used for our labwork.

### General Protocols

[Preparation of general necessities 🔗](https://static.igem.org/mediawiki/2019/6/66/T--TU_Eindhoven--ProtocolGeneral.pdf)
Preparation of antibiotics, growth media, stock solutions and more.
[NanoDrop 🔗](https://static.igem.org/mediawiki/2019/0/05/T--TU_Eindhoven--ProtocolNanoDrop.pdf)
NanoDrop is used to determine the concentration of DNA – and protein samples.
[Agarose Gel Electrophoresis 🔗](https://static.igem.org/mediawiki/2019/c/c6/T--TU_Eindhoven--ProtocolAgarose.pdf)
Agarose Gel Electrophoresis is used to determine the length of DNA samples.
[SDS-PAGE analysis 🔗](https://static.igem.org/mediawiki/2019/a/a3/T--TU_Eindhoven--ProtocolSDSPAGE.pdf)
SDS-PAGE is used to determine the size of a protein.
[Gel extraction 🔗](https://static.igem.org/mediawiki/2019/4/43/T--TU_Eindhoven--ProtocolGelExtraction.pdf)
Gel extraction is the silica-membrane-based purification of DNA fragments from gels.

### Cloning, Mutagenesis & Gibson Assembly

[PCR Amplification 🔗](https://static.igem.org/mediawiki/2019/3/3f/T--TU_Eindhoven--ProtocolPCRAmplification.pdf)
PCR Amplification is used to amplify a DNA sequence.
[PCR Purification 🔗](https://static.igem.org/mediawiki/2019/4/45/T--TU_Eindhoven--ProtocolPCRPurification.pdf)
PCR Purification is the purification of a product from a PCR reaction.
[Digestion 🔗](https://static.igem.org/mediawiki/2019/7/78/T--TU_Eindhoven--ProtocolDigestion.pdf)
Digestion is the cleaving of a vector and insert using restriction enzymes to prepare them for ligation.
[Ligation 🔗](https://static.igem.org/mediawiki/2019/7/70/T--TU_Eindhoven--ProtocolLigation.pdf)
Ligation is the process of ligating an insert into a vector.
[Vector Linearization 🔗](https://static.igem.org/mediawiki/2019/0/06/T--TU_Eindhoven--ProtocolVectorLinearization.pdf)
Vector Linearization is the process of linearizing a vector, after which it can be used for Gibson Assembly.
[Gibson Assembly 🔗](https://static.igem.org/mediawiki/2019/8/89/T--TU_Eindhoven--ProtocolGibson.pdf)
Gibson Assembly is the ligation of gBlocks into a linearized vector.

### Transformation, Protein Expression & Purification

[Transformation into competent cells & plating on agar plates 🔗](https://static.igem.org/mediawiki/2019/3/3d/T--TU_Eindhoven--ProtocolTransformation.pdf)
Transformation is the introduction of a desired plasmid into bacteria to amplify it.
[Colony picking & colony PCR 🔗](https://static.igem.org/mediawiki/2019/0/09/T--TU_Eindhoven--ProtocolColony.pdf)
Colony picking & colony PCR are used to determine the present of insert DNA in plasmid constructs.
[Sequencing at BaseClear 🔗](https://static.igem.org/mediawiki/2019/b/b3/T--TU_Eindhoven--ProtocolSequencing.pdf)
Sequencing is used to determine the sequence of the amplified and transformed DNA.
[Small Culturing 🔗](https://static.igem.org/mediawiki/2019/e/ee/T--TU_Eindhoven--ProtocolSmallCulturing.pdf)
Small culturing is used to amplify a specific bacterial colony after transformation.
[Protein Expression 🔗](https://static.igem.org/mediawiki/2019/6/6c/T--TU_Eindhoven--ProtocolProteinExpression.pdf)
Protein Expression is the induction of protein production in bacteria from small culture.
[Protein Purification 🔗](https://static.igem.org/mediawiki/2019/4/4b/T--TU_Eindhoven--ProtocolProteinPurification.pdf)
Protein Purification is the purification of the desired protein using purification tags.

### Testing

[Bioluminescence Measurements 🔗](https://static.igem.org/mediawiki/2019/5/58/T--TU_Eindhoven--ProtocolBioluminescence.pdf)
Bioluminescence Measurements are used to determine the bioluminescent activity of a desired compound.

### Phage experiments

[Preparation of media 🔗](https://static.igem.org/mediawiki/2019/6/61/T--TU_Eindhoven--PreparationOfMedia.pdf)
Preparation of M9-based minimal medium for bacterial growth used in phage experiments.
[Bacteria multiplication rate determination 🔗](https://static.igem.org/mediawiki/2019/3/37/T--TU_Eindhoven--BacterialMultiplicationDeterminationRate.pdf)
Bacteria multiplication rate determination is important for the determination of certain parameters used in the model.
[Determination of adsorption rate 🔗](https://static.igem.org/mediawiki/2019/d/dc/T--TU_Eindhoven--DeterminationOfAdsorptionRate.pdf)
Determination of the adsorption rate, which will be used in the model.
[Infection Assay 🔗](https://static.igem.org/mediawiki/2019/6/6f/T--TU_Eindhoven--InfectionAssay.pdf)
The Infection Assay is used to determine several parameters for the model.
[Phage Titer Determination 🔗](https://static.igem.org/mediawiki/2019/0/02/T--TU_Eindhoven--PhageTiterDetermination.pdf)
Determination of the phage titer of a certain solution.

###### +31 6-18106424

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# Team:TU Eindhoven/Results

* [![](https://static.igem.org/mediawiki/2019/1/18/T--TU_Eindhoven--smallLogo.png)](https://2019.igem.org/Team%3ATU_Eindhoven)
* PROJECT

  [Description](https://2019.igem.org/Team%3ATU_Eindhoven/Description)
  [Design](https://2019.igem.org/Team%3ATU_Eindhoven/Design)
  [Demonstrate](https://2019.igem.org/Team%3ATU_Eindhoven/Demonstrate)
  [Model](https://2019.igem.org/Team%3ATU_Eindhoven/Model)
  [Device](https://2019.igem.org/Team%3ATU_Eindhoven/Device)
  [Safety](https://2019.igem.org/Team%3ATU_Eindhoven/Safety)
* LAB

  [Experiments](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
  [Notebook](https://2019.igem.org/Team%3ATU_Eindhoven/Notebook)
  [Contribution](https://2019.igem.org/Team%3ATU_Eindhoven/Contribution)
  [Results](https://2019.igem.org/Team%3ATU_Eindhoven/Results)
  [Measurement](https://2019.igem.org/Team%3ATU_Eindhoven/Measurement)
* PARTS

  [Parts overview](https://2019.igem.org/Team%3ATU_Eindhoven/Parts)
  [Basic parts](https://2019.igem.org/Team%3ATU_Eindhoven/Basic_Part)
  [Composite parts](https://2019.igem.org/Team%3ATU_Eindhoven/Composite_Part)
  [Part collection](https://2019.igem.org/Team%3ATU_Eindhoven/Part_Collection)
  [Improve](https://2019.igem.org/Team%3ATU_Eindhoven/Improve)
* HUMAN PRACTICES

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* TEAM

  [Team Members](https://2019.igem.org/Team%3ATU_Eindhoven/Team)
  [Attributions](https://2019.igem.org/Team%3ATU_Eindhoven/Attributions)
  [Collaborations](https://2019.igem.org/Team%3ATU_Eindhoven/Collaborations)
  [Sponsors](https://2019.igem.org/Team%3ATU_Eindhoven/Sponsors)
* MEDALS

  [Medal requirements](https://2019.igem.org/Team%3ATU_Eindhoven/Medal_Requirements)
  [Awards](https://2019.igem.org/Team%3ATU_Eindhoven/Awards)
* [CONTACT](#footer)

# Results

* [Results of our project](#results)
* [Future plans](#future)
* [Project achievements](#achievements)

## Results of our project

### Results Paired dCas9-Split-NanoLuc system

To evaluate our paired dCas9-Split-NanoLuc system, test DNA in the form of gBlocks was designed ([Figure 1](#resultfig1)).
The sequence of this test DNA was based on the T7 phage genome since the T7 phage is used for the proof-of-concept of dCastect.
By using the T7 sequence, the same gRNA pairs can be used for all experiments.
Using the gBlocks, we first wanted to optimize the sensor by varying the protein concentrations and interspace distance.
Subsequently, the limit of detection could be determined, followed by tests where the sensor proteins bind to multiple binding sites on the same target
to see if an even smaller limit of detection could be reached.

![](https://static.igem.org/mediawiki/2019/1/19/T--TU_Eindhoven--PAMblocksXYZ.png)

#### Variable interspace distances

The interspace distance was varied between the two dCas9 domains to find the optimal distance for association of SmallBit and LargeBit. Because of the helical shape of
DNA, we expected to see a pattern with higher signals when the two proteins bind on the same side of the helix and lower signals when the two proteins bind on the opposite
side of the helix. Eventually for high interspace distances, we expected a lack of signal because the distance would be too big for association of SmallBit and LargeBit.
In the first test we tested interspace distances from 10 bp to 30 bp (see [Additional bioluminescence measurements dCas9-Split NanoLuc system](#bioluminescence)).
In this test it was seen that interspace distance 30 gave the highest signal.
Therefore, we decided to design gBlocks to test interspace distances 10 – 60 bp to find the optimal interspace distance ([Figure 2](#resultfig2)).
The tests were performed using a 1.2 nM DNA concentration, 2 nM protein concentration
and 6x molar excess of gRNA. The results show that the systems containing an interspace distance lower than 20 bp give a low signal. From 20-60 bp the signal is high,
which means that interspace distance is not a limitation for choosing desired gRNA sequences. Interspace distances 30, 50 and 60 give the highest signals.

![](https://static.igem.org/mediawiki/2019/0/0f/T--TU_Eindhoven--20191008VariableInterspaceDistance.png)

However, it can be observed that the different gRNA pairs have an influence on the overall signal ([Figure 2](#resultfig2)). gRNA pair 1 gives a
significantly lower signal for all measurements. To test whether this lower signal is caused by the gRNA and not by the difference in interspace distance, the system
containing interspace distance 30 was tested using all three different gRNA pairs ([Figure 3](#resultfig3)). As can be seen, gRNA pair 1
gives a significantly lower signal than the other two gRNA pairs. gRNA pair 3 gives the highest signal, so it was decided to use this gRNA pair in future measurements.

![](https://static.igem.org/mediawiki/2019/0/07/T--TU_Eindhoven--20191008Interspace30DifferentgRNAs.jpg)
Figure 3: The difference in bioluminescent signal for the three different gRNA pairs.

Subsequently, it was decided to design gBlocks to test even higher interspace distances, since interspace distances of 50 and 60 bp still give a high bioluminescent signal.
According to our calculations, interspace distances up to 70 bp should give a measurable bioluminescent signal.
In the next test, we tested interspace distances from 20 – 150 bp ([Figure 4](#resultfig4)).
It can be observed that an interspace distance of 110 bp gives the highest signal. However, we expected that ±70 bp would be the highest interspace distance that could give a signal,
taking into account the approximate distance between PAM-site and C-terminus of dCas9 as well as the length of the linker between this C-terminus and Lbit/Sbit of Split-NanoLuc.
A possible explanation for the high signal at 110 bp is the persistence length of dsDNA. The persistence length is a material property of a polymer quantifying its stiffness.
If the length of a piece of polymer, like DNA, is much longer than its persistence length, it behaves highly flexible due to thermal energy.
If significantly shorter however, the polymer is stiff. The persistence length of DNA is between 35 – 50 nm
[[1](#resultref), [2](#resultref), [3](#resultref)], which corresponds to the length of 103-147 base pairs.
The 110 bp interspace distance thus matches this persistence length, meaning this long interspace is semi-flexible.
Therefore, we expect the target sites could come closer to each other, hence it would be possible for the two dCas9 proteins to find each other
even though they do not bind close to each other on the DNA strand.

![](https://static.igem.org/mediawiki/2019/3/38/T--TU_Eindhoven--20191021InterspaceDistance.png)

Figure 4: Bioluminescence intensity with interspace distances from 20 bp to 150 bp.

#### Variable protein concentrations

Another test was executed to determine which range of protein concentrations would be most reliable for the detection of DNA.
We tested protein concentrations 2, 10, 20, 50 and 100 nM with a DNA concentration of 1.2
nM. The results show that a protein concentration of 10 nM has the best signal-to-noise ratio ([Figure 5](#resultfig5)). At higher protein concentrations, an increase in background
signal can be observed without a significant increase in signal. For 100 nM, the background signal is as high as the actual signal, indicating that higher protein
concentrations do not increase the sensitivity of the sensor.

![](https://static.igem.org/mediawiki/2019/4/45/T--TU_Eindhoven--20191010VariableProteinConcentration.jpg)
Figure 5: Bioluminescence intensity when the protein concentration was varied from 2 nM to 100 nM. Background signal containing only protein and gRNA and a control using non-target DNA were also tested.

#### Variable DNA concentrations

We wanted to test the limit of detection of our sensor. Therefore, we tested a range of DNA concentrations from 1 pM to 10 nM using a protein concentration of 2 nM
([Figure 6A](#resultfig6)) and 10 nM ([Figure 6B](#resultfig6)). It was expected that the 2 nM sensor would
be more sensitive than the 10 nM sensor and would therefore be able to detect lower DNA concentrations. It was shown that the 10 nM sensor is better at detecting higher DNA concentrations.
The limit of detection then is 50 pM. The 2 nM sensor is indeed more sensitive and can detect DNA concentrations as low as 10 pM. Also, the Hook effect can be observed for both sensors, which is the
decrease in signal at greater concentrations of the target compound. Due to the high availability of binding sites, the likelihood of either dCas9-SmallBit or dCas9-LargeBit
binding at a binding site is higher. Therefore, SmallBit and LargeBit do not associate and a decrease in bioluminescent signal can be observed.

![](https://static.igem.org/mediawiki/2019/e/e4/T--TU_Eindhoven--VariableDNAconcentrations.png)
Figure 6: A) Bioluminescence intensity when the target DNA concentration is varied at a 2 nM protein concentration. B) Bioluminescence intensity when the target DNA concentration is varied at a 10 nM protein concentration.

#### Multiple sensor proteins

The limit of detection was determined using one dCas9-SmallBit and one dCas9-LargeBit binding to the recognition sites on a target sequence.
We wanted to see if the limit of detection could be lowered when multiple sensor proteins would bind to multiple recognition sites on one target DNA strand.
We tested the limit of detection using three pairs of sensor proteins on one target ([Figure 7](#fig7)).
The results show that the limit of detection is slightly lowered to 5 pM when three pairs of sensor proteins bind to multiple recognition sites on one target.

![](https://static.igem.org/mediawiki/2019/a/a0/T--TU_Eindhoven--20191017ThreeSensorProteinsOnOneTarget.png)
Figure 7: Bioluminescent intensity when three sensor protein pairs bind to three paired recognition sites.

### dCastect proof of concept

It was not allowed to work with phages at the Eindhoven University of Technology. Therefore, we went to Brussels and denatured the phages.
In Eindhoven we measured the denatured phages with our sensor. This is further explained on the [Demonstrate](https://2019.igem.org/Team%3ATU_Eindhoven/Demonstrate) page.

## Future outlook

### Statistical relevance

First of all, most experiments need to be repeated several times to obtain more statistical relevant results.
Within our limited timeframe we have not been able to repeat all our experiments for multiple times to realize statistical relevance and more results are needed to achieve this,
however certain trends within our results have been observed and discussed.

### Paired dCas9-Split-NanoLuc system

The optimal conditions for the paired dCas9-Split-NanoLuc system have been determined. The next step would be to test with actual patient samples.
Patient samples contain a more complex medium with other proteins and DNA present.
After validation with patient samples, the detection proteins can be incorporated into a device.

### Single dCas9-BRET system

After many trials with classical ligation and Gibson assembly, we were unable to form the DNA construct of our dCas9-BRET fusion protein.
We find this very unfortunate, because for now we do not know if our design of the dCas9-BRET fusion protein could be functional.
If the design would be functional, it would be a ground-breaking ratiometric detection protein.
Hence, research will be continued at our university after the iGEM competition.

### dCastect

Our proof of concept was based on the T7 phage. The next step would be to infect bacteria with phages and measure the phage DNA after the replication and heat shock.
Subsequently we would validate the system with other phage types so other bacterial strains can be detected.
As mentioned in the [collaboration with Stockholm](https://2019.igem.org/Team%3ATU_Eindhoven/Collaborations),
we could use our system to detect which one of their phages is needed to kill a certain bacterium.

## Project achievements

### Successes

* [Additional bioluminescence measurements dCas9-Split NanoLuc system](#bioluminescence)
* [Expression dCas9 addgene](#expression)
* [dCas9 replacement and expression of dCas-Split-NanoLuc system](#dcas9)
* [Cysteine-Free-NanoLuc expression and characterization](#cysteinefree)
* [mCherry expression and characterization](https://2019.igem.org/Team%3ATU_Eindhoven/Contribution)

### Failures: The road to success

* [Expression dCas9-Split-NanoLuc system](#firstexpression)
* [Bioluminescence measurements dCas9-Split-NanoLuc system](#bioluminescencefailure)
* [Assembly of IDT gBlocks for dCas9-BRET into our vector](#gblocks)

### References

1. Gross, P., et al. (2011). Quantifying how DNA stretches, melts and changes twist under tension. Nature, 7, 731-736.
2. Cifra, P., Benková, Z. & Bleha, T. (2010). Persistence length of DNA molecules confined in nanochannels. Phys. Chem. Chem. Phys., 12, 8934-8942.
3. Brinkers, S., Dietrich, H.R.C., de Groote, F.H., Young, I.A. & Rieger, B. (2009). The persistence length of double stranded DNA determined using dark field tethered particle motion. J. Chem. Phys, 130(21), 215105.

[◄ Back](#achievements)

## Expression dCas9 addgene

The sequence of the S. pyogenes derived catalytically inactive Cas9 was a gift from David Liu ([Addgene plasmid #62935](https://www.addgene.org/62935/)).
After experiencing difficulties with expressing our own dCas9 fusion proteins, expression of this Addgene dCas9 was performed to optimize conditions for later expression of our dCas9 fusion proteins.

The protein was successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC).
All the purification steps (lysate flow through, bind flow-through, wash flow-through, elution)
and a concentrated sample of the elution were put on an SDS-PAGE gel to evaluate the purification process.
dCas9 has a molecular weight of 158 kDa. A clear band is visible in the elution around 158 kDa.
It is remarkable that there is also a band at this height in the wash flow-through.
This indicates that the 30 mM imidazole of the wash buffer already causes the protein to be released from the column.
Furthermore, a very big band is visible in the sample of the concentrated elution. To conclude, dCas9 was successfully expressed.

![SDS-PAGE gel](https://static.igem.org/mediawiki/2019/1/16/T--TU_Eindhoven--HisPurificationdCasAddgene.jpg)
Figure 1: SDS-PAGE gel of Nickle affinity chromatography samples.

[◄ Back](#achievements)

## dCas9 replacement and expression of dCas-Split-NanoLuc system

### dCas9 replacement

The dCas9 sequence of the original vector could not be expressed.
Therefore, the dCas9 sequence was replaced by the dCas9 sequence of the Addgene plasmid which was a gift from David Liu ([Addgene plasmid #62935](https://www.addgene.org/62935/)).
Overhang PCR was used to include the restriction sites needed to replace the dCas9 in the vector.
With colony PCR we confirmed that the Addgene dCas9 was ligated into the vector.
All successful colonies were transformed into NovaBlue and after small culture miniprepped.
The samples with the highest DNA concentration for dCas9-SmallBitNanoLuc and dCas9-LargeBitNanoLuc were transformed and expressed.
The complete sequences were confirmed with sequencing.

![Colony PCR gel](https://static.igem.org/mediawiki/2019/d/d1/T--TU_Eindhoven--ColonyPCRSmallBit.png)
Figure 1: Colony PCR gel of dCas9-SmallBitNanoLuc.

![Colony PCR gel](https://static.igem.org/mediawiki/2019/5/5f/T--TU_Eindhoven--ColonyPCRLargeBit.png)
Figure 2: Colony PCR gel of dCas9-LargeBitNanoLuc.

### Expression

The fusion proteins were successfully expressed in BL21(DE3) and purified with immobilized metal affinity chromatography (IMAC).
The elution was collected in 5 fractions of 1.5 mL. The three elution samples with the highest concentrations were put on an SDS-PAGE gel to evaluate the purity and molecular weight of the samples.
dCas9-SmallBitNanoLuc has a molecular weight of 162 kDa and dCas9-LargeBitNanoLuc has a molecular weight of 178 kDa.
As shown in figure 3, the blobs of dCas9-LargeBitNanoLuc are a bit higher compared to the blobs of dCas9-SmallBitNanoLuc.
This was expected when looking at the molecular weights of the two fusion proteins. There are other bands visible, so the purity is not optimal.
However, the contrast between the huge blobs and the other bands is very big.

![SDS-gel Split-NanoLuc](https://static.igem.org/mediawiki/2019/d/d3/T--TU_Eindhoven--SDSgelSplitNanoLuc.png)
Figure 3: SDS-PAGE gel of IMAC elution samples of dCas9-SmallBitNanoLuc (Elute 2: 20.79 μM, Elute 3: 7.298 μM, Elute 4: 12.08 μM) and dCas9-LargeBitNanoLuc (Elute 2: 17.90 μM, Elute 3: 6.37 μM, Elute 4: 2.88 μM).

[◄ Back](#achievements)

## Additional bioluminescence measurements dCas9-Split NanoLuc system

### Incubation conditions

First, we tested which incubation conditions were most suitable to test the sensor. The incubation of dCas9 with gRNA can best be performed for 20 minutes at 37 °C.
The subsequent incubation of the dCas9-gRNA complex with DNA should be done for at least 10 minutes at room temperature.
Furthermore, gRNA can be heated for 5-10 minutes before use to prevent the formation of secondary structures.
However, the incubation temperature can be optimized.
Therefore, we decided to test incubation temperatures of 37 °C and 50 °C ([Figure 1](#addfig1)).
As can be seen, the bioluminescent signal for all three sensors binding to target DNA is higher after gRNA incubation at 50 °C.
Also, the bioluminescent signal of both control groups is lower for 50 °C.
Therefore, in all bioluminescent experiments, an gRNA incubation condition of 50 °C has been used.

![](https://static.igem.org/mediawiki/2019/f/f8/T--TU_Eindhoven--20190924DifferentIncubationConditionsgRNA.png)

Figure 1: Bioluminescent intensity of samples when gRNA is incubated for 5-10 minutes at either 37 °C or 50 °C.

### PAM orientation

[It was explained](https://2019.igem.org/Team%3ATU_Eindhoven/Design) that the PAM-In orientation was chosen for the design of our paired dCas9-Split-NanoLuc system.
Although this design was based on literature, we still wanted to test if we made the right choice.
Thus, we tested the bioluminescent signal using the PAM-Out orientation for interspace distance 25 ([Figure 2](#addfig2)).
The results show that the mean bioluminescent signal for the PAM-Out orientation is much lower than the mean bioluminescent signal for PAM-In.
From these results, we derived that PAM-In was a good choice.

![](https://static.igem.org/mediawiki/2019/4/41/T--TU_Eindhoven--20190924PAMout.png)

Figure 2: Bioluminescent intensity when PAM-Out orientation is used.

### Variable interspace distance

We designed the first gBlocks and corresponding gRNA pairs in a way to be able to test interspace distances from 10 to 30 bp ([Figure 3](#addfig3)).
This was based on literature and PyMOL measurements of our sensor. We performed bioluminescent tests to find the optimal interspace distance
using a 2 nM protein concentration, a 1.2 nM DNA concentration and a 6x molar excess of gRNA. The results show an optimal interspace distance of 30 bp.
As expected, interspace distance 20 also gives a relatively high signal, which is due to the orientation of the helix.
The proteins then bind at the same side of the helix, resulting in a higher signal.
After this test, it could still be possible that an interspace distance higher than 30 bp would be optimal, since we were not able to test this.
Thus, in later tests, higher interspace distances were tested to check whether the optimal interspace distance is indeed 30 bp
(see [Results Paired dCas9-Split-NanoLuc system](#results)).

![](https://static.igem.org/mediawiki/2019/7/79/T--TU_Eindhoven--20190926VariableInterspaceDistance.png)

Figure 3: Bioluminescent intensity when interspace distances from 10 bp to 30 bp were tested.

### Limit of detection using lower protein concentrations

We tested the limit of detection for 2 nM and 10 nM sensor protein concentrations (see [Results Paired dCas9-Split-NanoLuc system](#results)),
which was as low as 10 pM for a 2 nM sensor protein concentration.
To check if the limit of detection could be lowered, we tested even lower protein concentrations:
1 nM ([Figure 4A](#addfig4)) and 0.5 nM ([Figure 4B](#addfig4)).
For the 1 nM sensor protein, 5 pM is the lowest DNA concentration that can be distinguished from the background signal.
However, the difference in signal with the background signal is only minimal.
For 0.5 nM sensor protein, the limit of detection is 10 pM, which is the same as for 2 nM sensor protein.
Therefore, the limit of detection cannot be lowered significantly using lower sensor protein concentrations.

![](https://static.igem.org/mediawiki/2019/9/95/T--TU_Eindhoven--20191014VariableDNAconcentration.png)

Figure 4: The effect of testing a variety of DNA concentrations using A) 1 nM sensor and B) 0.5 nM sensor.

[◄ Back](#achievements)

## Cysteine-Free-NanoLuc expression and characterization

Cysteine-free-NanoLuc was cloned into a pET28a(+) vector, subsequently expressed in BL21 (DE3) E. coli and purified using Ni-NTA affinity chromatography and Strep-Tactin
purification. Afterwards, expression was analyzed on SDS-PAGE ([Figure 1](#cysfig1)). This shows that after the purification there is a clear
blob between 20 kDa and 25 kDa which corresponds with the molecular weight of the 6xHis-tagged and Strep-tagged Cysteine-free-NanoLuc, which is 22 kDa.

![](https://static.igem.org/mediawiki/2019/f/f6/T--TU_Eindhoven--SDSCysFreeNL.png)
Figure 1: SDS-PAGE of Cysteine-free-NanoLuc (319 µM) after purification.

### Functionality test

After successful expression and purification, measurements could be performed to determine whether cysteine-free-NanoLuc was still functional and could be used as a
replacement for NanoLuc in fusion proteins where subsequent maleimide coupling is required. Bioluminescence intensity was measured for different concentrations of both
NanoLuc proteins ([Figure 2](#cysfig2)). This shows that cysteine-free-NanoLuc has a similar shape of the emission spectrum and has its
maximum at the same wavelength as NanoLuc. It furthermore shows that cysteine-free-NanoLuc is still functional, although showing a decrease of about fifty percent in
intensity for all measured concentrations. However, due to NanoLuc’s initial brightness, cysteine-free-NanoLuc is still a lot brighter than most other luciferases while
also maintaining its small size and high stability.

![](https://static.igem.org/mediawiki/2019/6/61/T--TU_Eindhoven--NanoLucAndCysFreeNanoLuc.png)
Figure 2: Emission spectrum (A) and maximum intensity at 460 nm (B) of Cysteine-free-NanoLuc and NanoLuc.

[◄ Back](#achievements)

## Expression dCas9-Split-NanoLuc system

The vector ordered at Genscript (pET28a(+)) with our insert was dissolved and transformed into BL21.
Three different large cultures were used for protein expression; LB, 2YT and TB. When the OD600 had reached 0.6-0.7, protein expression was initiated by adding 1 mM IPTG.
After protein expression, purification was done using the His-tag at the beginning and the Strep-tag at the end of the construct. No protein concentrations were measured with NanoDrop.
All the samples from the purification were put on an SDS-PAGE gel. From the results we concluded that the expression had failed. There was no protein present at all, not even in the pellet.
Therefore, a new plan was made to test different combinations of expression conditions based on information we gathered.
We had contact with the iGEM team Dresden to troubleshoot our problem and we also looked in literature.
We suspected that dCas9 might be a bit toxic for the bacteria. Therefore, we wanted to temper protein expression by changing some expression condition.
Four different condition combinations were tested, see [Table 1](#table1). The following conditions stayed the same for all four samples; BL21(DE3) as host,
LB as grow medium, expression initiation at OD600 = 0.5. The cultures were lysed with ultrasound, a less aggressive lysis method compared to BugBuster which was used at first.

Table 1: Condition combinations for expression.

|  | IPTG (nM) | Temperature (°C) |
| --- | --- | --- |
| 1. | 0.2 | 16 |
| 2. | 1.0 | 16 |
| 3. | 0.2 | 20 |
| 4. | 1.0 | 20 |

The expression of our protein was still unsuccessful. After purification, the samples were run on an SDS-PAGE gel.
No bands were visible at the expected height. We found out that the sequence of our dCas9 was different compared to the team from Dresden and other sources from literature.
While ordering our sequence, codon optimization was applied. Apparently, this was the cause of our expression problem.
We ordered a vector from Addgene with the correct sequence and replaced our sequence for dCas9.
Now we were able to express our protein! You can find these results [here](#expression).

[◄ Back](#achievements)

## Bioluminescence measurements dCas9-Split-NanoLuc system

### First tests

The protein was successfully expressed, so the first bioluminescence tests were performed to check the function of our sensor.
The tests were performed using 10 nM protein and 1.2 nM DNA. The molar ratio of protein : gRNA was 3:4.
Several controls and dCas9 complexes containing interspace distances of 17, 20 and 22 were tested and the substrate furimazine was added in a 1000X dilution to each sample.
A PBS buffer containing 0.1% BSA was used for all samples.
The bioluminescence was tested using the Tecan Spark 10M Plate Reader in a 384 Wells Plate using 50 μL samples ([Figure 1](#figure1)).

![](https://static.igem.org/mediawiki/2019/6/61/T--TU_Eindhoven--FirstTest.jpg)
Figure 1: First bioluminescence test.

As can be seen in [Figure 1](#figure1), the background samples containing either only dCas9-SmallBit or dCas9-LargeBit give a very low signal.
However, the control sample containing both dCas9-SmallBit and dCas9-LargeBit without gRNA and DNA and the control sample containing both proteins and target DNA without gRNA give a high signal.

### Variable DNA concentration

In the first tests, the control samples give a high signal. We decided to vary the DNA concentrations while testing the dCas9 complexes with the same interspace distance.
The test was performed in triplicate with 50 μL samples. The protein concentration was 10 nM, the molar ratio of protein : gRNA was 3:4
and DNA concentrations of 0.6 nM, 1.2 nM and 2.4 nM were used. Protein and gRNA were incubated for 20 minutes at room temperature
and this complex together with DNA was incubated at 37 °C for 30 minutes. The substrate was added in a 1000X dilution to each sample.
The same buffer and plate reader were used as in the first test. The results repeatedly show high bioluminescent signal for the control samples ([Figure 3](#figure2)).
When the DNA concentration is varied, the difference in signal is not substantial ([Figure 2](#figure2)).
In addition, the controls show a much higher signal than the samples containing target DNA, so these tests are not reliable yet.

![](https://static.igem.org/mediawiki/2019/3/39/T--TU_Eindhoven--DNAconcentration%2BControls.png)

Figure 2: Relative bioluminescence of the samples tested with a DNA concentration of 0.6 nM, 1.2 nM or 2.4 nM.

Figure 3: Controls during the second bioluminescnece test.

### Lower protein concentrations

To hopefully reduce the background signal, tests were performed using lower protein concentrations.
Concentrations of 1 nM and 0.5 nM were tested, together with DNA concentrations of 1.2 nM and a 3:4 molar ratio of protein : gRNA.
Incubation of protein with gRNA was done for 20 minutes at room temperature and the subsequent incubation with DNA was done at 37 °C for 30 minutes.
The substrate was added in 2000X dilution. The same buffer and plate reader were used as previously mentioned.

![](https://static.igem.org/mediawiki/2019/1/1a/T--TU_Eindhoven--LowerProteinConcentrations.jpg)
Figure 4: Relative bioluminescence during testing with lower protein concentrations.

The results show that the background containing only the proteins without gRNA and DNA give the highest signal ([Figure 4](#figure4)).
The samples that contain target DNA give the lowest signal, which is not what we expected.

### Variable incubation conditions

We read more about dCas9 in literature and several researchers had problems with aggregation when dCas9 is incubated at 37 °C [[1](#bioref1)].
Therefore, we decided to vary the incubation conditions to reduce aggregation. In addition, DTT was added to the buffer.
Tests with 50 μL sample were performed in duplicate. 10 nM protein, 1.2 nM DNA and a 3:4 molar ratio of protein : gRNA was used.
The tests were performed using a 2000X dilution of furimazine in the same plate reader as previously mentioned.
Incubation of dCas9-gRNA and DNA was done for 10 minutes and 30 minutes at room temperature and for 30 minutes at 37 °C.

![](https://static.igem.org/mediawiki/2019/2/25/T--TU_Eindhoven--VariableIncubationConditions.jpg)
Figure 5: Relative bioluminescence of the samples when incubation conditions vary.

The background signal of the control groups did not decrease under different incubation conditions ([Figure 5](#figure5)).
It can be seen that the bioluminescent signal of the control groups is lowest in the samples that were incubated at 37 °C,
so either no aggregation occurs or aggregation is not prevented under these testing conditions.

### Variable interspace distance

In the previous tests, only the samples with interspace distances of 17, 20 and 22 were tested.
Our DNA and gRNA combinations make it possible to test samples with interspace distances of 10, 12, 15, 17, 20, 22, 25, 27 and 30.
Therefore, we decided to test all possible interspace distances in order to find the optimal distance that would give a signal higher than the background signal.
50 μL samples were tested in duplicate, using the same buffer containing DTT. The same concentrations of protein, DNA, gRNA and furimazine were used as in the previous test.
Incubation of dCas9-gRNA and DNA was performed for 30 minutes at room temperature.

![](https://static.igem.org/mediawiki/2019/3/3c/T--TU_Eindhoven--Controls%2BVariableInterspaceDistance.png)

Figure 6: Relative bioluminescence of controls.

Figure 7: Relative bioluminescence in samples containing variable interspace distances.

The samples with interspace distances 10, 12 and 15 (blue), 17, 20 and 22 (red) and 25, 27 and 30 (green) are measured using the same DNA strand ([Figure 7](#figure6)).
The samples with interspace 10, 17 and 25; 12, 20 and 27; and 15, 22 and 30 are measured using the same gRNA.
A pattern can be observed, since the samples measured with the same gRNA give approximately the same signal. Furthermore, the control samples give a high signal ([Figure 6](#figure6)).

### Centrifugation

The relative bioluminescent signal for the control samples is still high, and aggregation is the most likely explanation.
Therefore, in this test the samples were centrifuged for 5 minutes at 13,400 rpm and the supernatant was used for testing.
The same concentrations for protein, DNA, gRNA and furimazine as in the previous tests were used. 50 μL samples were used in the same buffer containing DTT.
Incubation of dCas9-gRNA and DNA was performed for 30 minutes at room temperature.

![](https://static.igem.org/mediawiki/2019/f/f2/T--TU_Eindhoven--CentrifugationBar.jpg)
Figure 8: Relative bioluminescence of samples after centrifugation for 5 minutes at 13,400 rpm.

Centrifugation of the samples did not decrease the high background signal of the controls ([Figure 8](#figure8)), since a high signal can be observed.

### Higher molar excess of gRNA

In several papers about dCas9, a higher molar excess of gRNA is used. We decided to test with a molar excess of 6x, to make sure dCas9 can successfully form a complex with gRNA.
30 μL samples were made, using the same buffer containing DTT. The same concentrations of DNA and furimazine as in previous tests were used.
A 2 nM protein concentration and 6x molar excess gRNA was used. Incubation was done for 30 minutes at room temperature.

![](https://static.igem.org/mediawiki/2019/1/1c/T--TU_Eindhoven--gRNAExcess.jpg)
Figure 9: Relative bioluminescence of the samples when tested with a 6x molar excess of gRNA.

The difference in bioluminescent signal between samples containing different interspace distances is minimal ([Figure 9](#figure9)).
The control samples give a very high signal, which is about 4x as high as the samples containing target DNA.
The control sample containing non-target DNA gives a slightly higher signal than the samples containing target DNA. Therefore, the gRNA excess did not improve the functioning of the sensor.

### Testing with sgRNA instead of gRNA

For testing, we ordered tracrRNA and crRNA, which need to be incubated to form guideRNA. To make sure the guideRNA is functional, we ordered complete sgRNA for testing.
30 μL samples were made, using the same PBS buffer containing 0.1% BSA and 5 mM DTT. Some samples were tested without DTT.
The same protein, DNA, sgRNA and furimazine concentrations were used as in the previous test. Incubation was done for 30 minutes at 37 °C.

![](https://static.igem.org/mediawiki/2019/5/56/T--TU_Eindhoven--sgRNATesting.jpg)
Figure 10: Relative bioluminescence of the samples when tested with sgRNA instead of tracrRNA + crRNA.

The results show that the sgRNA did not make a significant difference ([Figure 10](#figure10)).
The signal for the controls is still higher than the signal for the samples containing target DNA. However, this difference in signal is much smaller.

### Testing with Mg in buffer

It is suspected that we are not measuring a real signal yet in the samples containing target DNA. It is known that magnesium plays an important role in the interaction between proteins and DNA.
In the case of Cas9, it can accelerate the binding time of dCas9 to DNA up to 800x when magnesium is present [[2](#bioref2)].
We made a new buffer containing 20 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2, 5% glycerol and 5 mM DTT.
30 μL samples were made, where the same protein, DNA, RNA and furimazine concentrations were used. Incubation was performed for 30 minutes at room temperature.

![](https://static.igem.org/mediawiki/2019/1/12/T--TU_Eindhoven--MgClInBuffer.jpg)
Figure 11: Relative bioluminescence measured in samples when Mg is used in the buffer.

The relative bioluminescent signal is still high for all control samples, especially for the control sample containing non-target DNA ([Figure 11](#figure11)).

Finally, we found the mistake causing us to measure only background signal. In the beginning a mistake had been made with the calculations of the DNA concentrations.
This resulted in the addition of 1.2 pM instead of 1.2 nM DNA. We diluted the DNA samples to 1.2 nM and tested again, which resulted in a good signal when target DNA was present.

### References

1. Sternberg, S.H., LaFrance, B., Kaplan, M. & Doudna, J.A. (2015). Conformational control of DNA target cleavage by CRISPR-Cas9. Nature, 527, 110-114.
2. Raper, A.T., Stephenson, A.A. & Suo, Z. (2018). Functional Insights Revealed by the Kinetic Mechanism of CRISPR/Cas9. J. Am. Chem. Soc., 140(8), 2971-2984.

[◄ Back](#achievements)

## Assembly of IDT gBlocks for dCas9-BRET into our vector

We used the IDT offer to synthesize the dCas9-BRET construct. The construct was so big that it had to be ordered in two gBlocks.
Restriction sites were incorporated to ligate the two gBlocks and to ligate the complete construct into our expression vector.
At first, we tried to ligate the two insets at once in the vector after digestion. This was unsuccessful.
After this we tried it again. Furthermore, we also tried to ligate one insert at the time. This was also unsuccessful. We decided to try Gibson assembly.
Our gBlocks were not designed for Gibson assembly, so we performed overhang PCR to incorporate the overhangs for Gibson assembly.
However, after Gibson assembly still no colonies were present and a control for the transformation was included. We have tried it again but still no results.
From a member of the iGEM team of Stockholm we heard that it is better to use PCR linearization instead of digestion. This was also tried, but still no results.
We have designed new gBlocks with the new overhangs and order them at IDT. Due to a lack of time, we were unable to make it work.

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