iGEM 2018 — Team NTNU_Trondheim

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# Team:NTNU Trondheim/Notebook

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* Team

  [Team Members](https://2018.igem.org/Team%3ANTNU_Trondheim/Team)
  [Collaborations](https://2018.igem.org/Team%3ANTNU_Trondheim/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3ANTNU_Trondheim/Description)
  [Design](https://2018.igem.org/Team%3ANTNU_Trondheim/Design)
  [Model](https://2018.igem.org/Team%3ANTNU_Trondheim/Model)
  [Results](https://2018.igem.org/Team%3ANTNU_Trondheim/Results)
  [Demonstrate](https://2018.igem.org/Team%3ANTNU_Trondheim/Demonstrate)
* Lab

  [Lab Journal](https://2018.igem.org/Team%3ANTNU_Trondheim/Experiments)
  [Protocols](https://2018.igem.org/Team%3ANTNU_Trondheim/Protocols)
  [Measurement](https://2018.igem.org/Team%3ANTNU_Trondheim/Measurement)
  [Safety](https://2018.igem.org/Team%3ANTNU_Trondheim/Safety)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3ANTNU_Trondheim/Parts)
  [New Part](https://2018.igem.org/Team%3ANTNU_Trondheim/Improve)
* [InterLab](https://2018.igem.org/Team%3ANTNU_Trondheim/InterLab)
* Human Practices

  [Integrated Human Practices](https://2018.igem.org/Team%3ANTNU_Trondheim/Human_Practices)
  [Education and Engangement](https://2018.igem.org/Team%3ANTNU_Trondheim/Public_Engagement)
* Acknowledgements

  [Sponsors](https://2018.igem.org/Team%3ANTNU_Trondheim/Sponsors)
  [Attributions](https://2018.igem.org/Team%3ANTNU_Trondheim/Attributions)
* [Awards](https://2018.igem.org/Team%3ANTNU_Trondheim/Awards)

![](https://static.igem.org/mediawiki/2018/9/92/T--NTNU_Trondheim--labjournalheader.png)

![](https://static.igem.org/mediawiki/2018/d/df/T--NTNU_Trondheim--labjournalmobile.png)

In our lab journal we have thoroughly documented what we have done in the laboratory, what our goals each session has been, how we have worked and results from each session. If you want a closer look on our methods/receipies, take a look at our protocol.

* Week

  25

  Week 25

  + Jun

    20

    Our first day in the laboratory!

    **Goal:** Incubate the Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 on agar plates separately with antibiotics.

    1. LA- medium (1.5%) was made.

    2. Two different agar plates were made – one containing ampicillin (AMP) and one containing chloramphenicol (CM).

    **Incubation of bacteria**

    Two Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 plasmids respectively, were ordered from Addgene. E.coli carrying the pgRNA plasmid were plated on agar plates with AMP, while E.coli carrying the pdCas9 plasmid were plated on agar plates with CM. Incubation for 24 h at 37℃.

    **Results:**

    There was growth and single colonies on all the plates.
  + Jun

    21

    Inoculation of E.coli

    **Goal:** Inoculate a colony from each agar plate in LB- medium with antibiotics.

    **Procedure**
    One colony of pgRNA- and pdCas9- bacteria were picked and inoculated in LB-media (25 mL) with 25 µL of the associated antibiotic (AMP or CM). The cell cultures were incubated at 37℃ in a shaking incubator at 204 rpm.
  + Jun

    22

    Isolation and verification of pgRNA and pdCas9

    **Goal:** Isolate and verify that the E.coli carry pgRNA and pdCas9.

    **Plasmid isolation**
    pgRNA and pdCas9 were isolated from the incubated bacteria prepared yesterday (June 21) by following the miniprep protocol.
    **Determine plasmid concentration and purity**
    The concentration of plasmids and the purity of each sample were determined by using Nanodrop.

    **Restriction digest and plasmid verification**
    The presence of pgRNA and pdCas9 in the cell cultures were verified after following the restriction digest protocol and separation of the DNA fragments by gel electrophoresis.
* Week

  26

  Week 26

  + Jun

    25

    Transformation of pgRNA and pdCas9 into competent cells

    **Goal:** Transform pgRNA and pdCas9 into competent cells (E.coli K-12 DH5α).

    **Procedure:**
    The extracted pgRNA and pdCas9 plasmid DNA were transformed into E.coli K-12 DH5α cells.
  + Jun

    26

    Transfer successful transformed cells to LB-medium and preparation of standard curves for the InterLab study

    **Goal:**

    Inoculate successful transformed competent cells to LB-medium, and prepare the standard curves for InterLab study.

    **Procedure:**

    Inoculate colony from agar plates to LB-medium:

    A colony from each agar plate was selected and inoculated to LB- medium with antibiotics. Competent cells with pgRNA were transferred to LB- medium with AMP, while competent cells with pdCas9 were inoculated into LB- medium with CM. The cell cultures were incubated at 37℃ with shaker.

    Preparation of 1xPBS solution for the InterLab-study.

    Preparation of agar plates with CM, LA- and LB-medium.

    Preparation of standard curves for InterLab study:

    Calibration 1: OD 600 reference point – LUDOX protocol

    A conversion factor to transform absorbance data from plate reader into OD600 was obtained in spectrometer by measuring four replicates of LUDOX CL-X and ddH2O. The data were imported into Excel sheet.

    Calibration 2: Particle standard curve – Microsphere protocol

    The solution of silica beads provided in the kit from iGEM HQ, was vortexed before dilution with water (96 μL Silica beads in 904 μL dd H2O). A serial dilution of microspheres was obtained by adding 100 μL dd H2O into 4x11 wells (E2, F2, G2, H2…E12, F12, G12, H12) on a 96 well plate. Microsphere stock solution (200 μL) was pipetted into E1 and 100 μL of the solution was transferred to E2 and mixed well before transferring 100 μL to E3. The procedure repeated until 100 μL solution was pipetted into E11, and 100 μL was transferred to liquid waste. The dilution series was repeated for row F, G and H. The absorbance of the samples was measured by a plate reader with shaker at 24℃. The data were exported to Excel sheet.

    **Results:**

    Sett inn bilde her.
  + Jun

    27

    Isolation of pgRNA and pdCas9 from compentent cells (DH5α) and preparation of stock solution for calibration 3 for InterLab study

    **Goal:**

    Isolate plasmids from competent cells (DH5α), and prepare the fluorecein stock solution for InterLab study.

    **Procedure:**

    Dilution of cell cultures:

    Each cell cultures (successful transformed competent cells from day 5 – 26.06.2018) (2.5 mL) were transferred into fresh LB-medium (22.5 mL) with CM (22.5 μL). The new cultures were incubated for about 2 hours at 37℃ with shaking .

    Measurement of the cell density:

    The cell density of the new cultures was measured at OD 600, after calibration with water (blank sample).

    Plasmid isolation and determination of concentration:

    The plasmids were isolated from the competent cell after the protocol of ZR plasmid miniprep kit (see lab journal from day 3 - 22. June. 2018).

    The concentration of the plasmids was determined by using Nanodrop.

    Restriction digest:

    The plasmids from each culture (5 μL) were digested by mixing with 10xbuffer (2 μL), distilled water (12.5 μL) and restriction enzyme BspHI (0.5 μL). The solution was centrifuged down before the samples were incubated over night at 37℃.

    **Results:**
  + Jun

    28

    Plasmid verification and storing the cell cultures

    **Goal:**

    Verify that the successfully transformed DH5α cells carry pgRNA or pdCas9, and store the cell cultures for future use.

    **Procedure:**

    Plasmid verification:

    The digested plasmid samples from yesterday’s incubation (lab 6 – 27. June.2018) were separated by gel electrophoresis.

    Bacteria glycerol stocks:

    The cell cultures made from 27. June were stored in a glycerol stock (500 µL cell culture and 500 µL 50% glycerol) in the freezer at -80℃.

    **Results:**

    Plasmid verification:

    L, pR1, pR2, pC1 and pC2 are the DNA-fragments from ladder solution, the two samples of pgRNA and pdCas9, respectively. From **Figure 1** the two fragments with 1.5 kb and 1.0 kb were found from the fragments of pgRNA after digestion by BspHI. The fragment with 100 bp from the digested pgRNA is not visible on the gel, probably due to masking of the dye colour at the bottom of the gel. Two bands from the digested pdCas9 with 2.7 kb and 4 kb were also detected. By comparing the results og gel elecrophoresis obtained from today's experiment with the results from day 3 (22. June. 2018), one can clearly see that the bands with the expected length from successful transformed competent cells, are much clearer. Hence, this also indicate the samples from the successful transformed DH5α cells are purer, which is also true by comparing the results from Nanodrop (see day 6 - 27. June. 2018, and day 3 - 22. June. 2018).
  + Jun

    29

    Insertion of anti-luxS via PCR and fluorescence standard curve for InterLab study

    **Goal:**

    Insert anti-luxS in pgRNA plasmid, and generate fluorescence standard curve for the InterLab study.

    **Procedure:**

    Insertion of anti-luxS gene and amplification of pgRNA with PCR.

    InterLab – Calibration 3

    A fluorescence standard curve was generated from the data obtained after measuring the fluorescence of the serial dilution of fluorescein.

    The fluorescence was measured with the following settings:

    Temperature: 24℃

    Gain (manual): 56

    |  | Wave length [nm] | Band width [nm] |
    | --- | --- | --- |
    | Excitation | 494 | 4 |
    | Emission | 525 | 20 |

    **Results:**

    InterLab – Calibration 3:

    Figure 1 shows the florescence standard curve in the concentration interval [0, 2.5 μM], with a R2 value of 0,9956. The model of the fluorescence standard curve is:
* Week

  27

  Week 27

  + Jul

    2

    Separation, isolation and transformation of linear DNA (pgRNA with anti-luxS)

    **Goal:**

    1. Separate the PCR product from circular DNA
    2. Isolate the linear DNA (pgRNA with anti-luxS from the gel
    3. Transform the linear DNA into competent cells
    4. InterLab study - transformation of plasmids to competent cells
    **Procedure:**

    Separation of linear DNA from circular DNA:

    The leaner DNA obtained from PCR (from day 8 – 29. June. 2018), was separated from the circular DNA (original plasmids) after running the sample (25 μL) mixed with loading dye (5 μl) on gel electrophoresis (gelGreen) simultaneously with a ladder (1 μL gene ruler + 1 μL loading dye + 4 μL dH2O) for 50 min at 90 V. The sample and the ladder were prepared by the gel electrophoresis protocol.

    Gel digestion and DNA isolation:

    The agarose was digested and the DNA was isolated from the sample following the gel purification protocol.

    Determine the DNA concentration and purity:

    The concentration and the purity of the isolated DNA was determined by using Nanodrop.

    Transformation of PCR product to competent cells:
    The PCR product (5 μL) was transformed to competent cells after following the transformation protocol.

    InterLab – transformation of plasmids to competent cells:

    The plasmids listed on the protocol, was transformed into competent cells (DH5α) after the following protocol:

    1. Thaw the competent cells on ice (10 min), and pre-chill the eppendorf tubes.
    2. Resuspend the DNA in the wells (provided by iGEM HQ) with dH2O (10 μL).
    3. Add competent cells (50 μL) to the eppendorf tubes.
    4. Add resuspended DNA (1 μL) to the Eppendorf tubes.
    5. Incubate on ice for 25 min
    6. Heat shock the cells for 45 s at 42℃ in water bath
    7. Incubate on ice for 5 min.
    8. Add LB-medium (950μL) to the tubes
    9. Incubate for 1h at 37℃ with shacker
    10. Pipette each sample (100 μL) to agar plate with CM
    11. Centrifuge the samples for 3 min at 6800g, discard supernatant (800 μL) and resuspend the pellet with the remaining supernatant
    12. Pipette each sample (100 μL) to agar plate with CM
    13. Incubate overnight at 37℃.
    **Results:**

    Separation of linear DNA from circular DNA:

    **Figure 1** shows the image of the gel where the linearized DNA was separated from the circular DNA. The linearized DNA is approximately 2 kb. The linearized DNA fragment was then cut out from the gel and stored in eppendorf tube.

    Note! Due to UV-light exposure, the DNA may mutate. In this case, this may affect the later steps of our project. The worse scenario, mutation of anti-luxS. Hence our CSRPRi system may not recognize luxS in the bacteria. Biofilm production will not be inhibited…

    Determine the DNA concentration and purity:

    **Table 1 shows the results of the linear pgRNA (2) from PCR (probably with anti-luxS gene).**

    **Table 1:** The concentration and the relative purity of the pgRNA (2) from PCR.

    | Concentration [ng/μL] | 260 nm/280 nm | 260 nm/230 nm |
    | --- | --- | --- |
    | 52.8 | 1.90 | 0.27 |

    From the 260/230 ratio, the sample has a relatively a small amount of nucleic acids. Probably, it is contaminated by the agarose from the gel electrophoresis. On the other hand, the 260/280 ratio is ok, indicating that the sample contains a relatively larger amount of RNA than DNA.
    InterLab – transformation of plasmids to competent cells:

    No visible colonies were detected on agar plates, except the positive and negative control cells.
  + Jul

    3

    Inoculation of successful transformed competent cells and InterLab study

    **Goal:**

    1. Inoculate colonies of successful transformed competent cells
    2. InterLab study - transformation of plasmids to competent cells
    **Procedure:**

    InterLab – transformation of plasmids to competent cells:

    No visible cell colonies were formed on the agar plates that were incubated on day 9 - 02. Junly. 2018. Only the positive and negative control cells formed colonies.

    Therefore, the remaining plasmids (6 μL) were transformed to the competent cells and plated on agar based on the same protocol from day 2. Jul. 2018. During the incubation at 37℃ with shaker, the incubation time was extended to 2 h. The cell cultures were centrifuged, and 700 μL supernatant was discarded, and the pellets were resuspended with the remaining supernatant. The cell cultures were then plated on agar plated with CM, and incubated overnight at 37℃.

    Inoculation of successful transformed competent cells:

    Colonies of successful transformed competent cells with pgRNA (probably containing anti-luxS) were inoculated from the agar plates (from day 9 – 02. July. 2018) to LB-medium with AMP. The cell cultures were incubated with shaker overnight at 37℃.

    **Results:**
  + Jul

    4

    Verification of successful inserted anti-luxS in pgRNA

    **Goal:**

    1. InterLab - transformation of plasmids into competent cells
    2. Verification of succsesful insertion of anti-luxS in pgRNA(2)
    **Procedure:**

    InterLab – Transformation of plasmids to competent cells

    There has been a mistake of numbering the plates with plasmids provided by iGEM HQ. Therefore, we did not get any cell colonies from the previous two transformations…

    The plasmids (1.5 μL) from the right wells were transformed to competent cells by following the transformation protocol from day – 02. July. 2018, after resuspension of the plasmids in the wells.

    Cell density:

    The cell cultures with pgRNA(2) (probably with anti-luxS), inoculated from day 10 – 04. July. 2018, were diluted 10 times by following the same dilution procedure from day 6 – 27. June. 2018. The cells were incubated with shaker for 1h before measuring at OD 600.

    Plasmid isolation:

    The plasmids from the two cell cultures were isolated after the same mini-prep protocol from day 3 – 22. June. 2018. The concentration and the purity of the isolated plasmids were measured by Nanodrop.

    Restriction digest and gel elecrophoresis:

    Insertion of anti-luxS gene was verified after the protocol of restriction digest and gel electrophoresis (see the protocols from day 3 – 22. June. 2018). The restriction enzyme PstI was chosen, and the expected length of the plasmid fragments are 490 bp and 2094 bp.

    Note! PstI has a cut site at the anti-luxS gene in the plasmid.

    Bacteria glycerol stock:

    Two samples of the diluted cell cultures (made at this morning), were stored in 50% glycerol solution at -80℃ (the same green box with the other successful transformed cells with pgRNA (without anti-luxS) or cells with pdCas9 plasmids).

    **Results:**

    InterLab - Transformation:

    Colonies were formed on agar plates with CM.

    Cell density:

    Table 1: Absorbance of the cell cultures with pgRNA(2) after 1 h incubation at 37℃ with shaker.

    | Culture | OD 600 |
    | --- | --- |
    | pR2 (1) | 0.954 |
    | pR2 (2) | 0.998 |

    Plasmid isolation:
    Table 2: Concentration and the relative purity of isolated pgRNA(2) from the two cell cultures.

    |  | Concentration (ng/μL) | 260 nm/280 nm | 260 nm/230 nm |
    | --- | --- | --- | --- |
    | pR2(1) | 37.2 | 2.06 | 4.29 |
    | pR2(2) | 123.3 | 1.77 | 1.01 |
  + Jul

    5

    Double transformation and InterLab study - Inoculation

    **Goal:**

    1. Transform both pdCas9 and pgRNA with anti-luxS into competent cells
    2. InterLab study - inoculate colonies in LB-medium
    **Procedure:**

    Double transformation of pdCas9 and pgRNA with anti-luxS:

    Both pdCas9(2) and pgRNA(2) with anti-luxS (isolated from day 11 – 05. July. 2018) were simultaneously transformed into competent cells (DH5α). The amount of added pdCas9(2) (5 μL) and pgRNA(2) with anti-luxS was 5 µL per/plasmid, and it was added to the same tube containing competent cells. The cells were plated on agar with both AMP and CM. A couple of steps were added at the end, and the purpose was to increase the cell concentration plated on agar, and probably increase the possibility of colony formation. The cell culture was therefore centrifuged for 3 minutes at 6800g, and the supernatant discarded. The pellet was then resuspended and cells were plated.

    InterLab – Inoculation of colonies

    A colony from each agar plate was inoculated in LB-medium (5 mL) with CM (5 μL). The cell cultures were incubated overnight at 37℃ with shaker.

    **Results:**
  + Jul

    6

    Inoculation of successful double transformed cells, interLab study - Cell measurement

    **Goal:**

    1. Inoculate successful double transformed cells (probably carrying pdCas9 and pgRNA with anti-luxS)
    2. InterLab study - measure absorbance and the fluorescence of each cell cultures
    **Procedure:**

    Inoculation of successful double transformed cells

    1. A colony from each agar plates with AMP and CM (made from day 13 – 05. July 2018) was picked and inoculated in LB medium (20 mL) with AMP (25 μL) and CM (25 μL).
    2. The cell cultures were incubated at 37℃ with shaker.
    InterLab – Cell measurement

    We were not able to measure the cell cultures today, since the TECAN device was under reparation.

    Each cell cultures were stored with 50% glycerol (see day 7 – 28. June. 2018) at -80℃.

    **Results:**
  + Jul

    7

    Verification of pdCas9 and pgRNA with anti-luxS after double tranformation

    **Goal:**

    Verify that both pdCas9 and pgRNA with anti-luxS are tranformed into DH5α cells

    **Procedure:**

    Dilution of cell culture:

    Each cell cultures, inoculated and incubated from day 13 – 07. July. 2018, were diluted (1mL) with LB medium with antibiotics until the OD 600 reached 1.9.

    **Plasmid verification:**

    Plasmid isolation:

    The plasmids were isolated from each cell cultures after following the mini-prep plasmid protocol from day 3 – 22. June. 2018. The concentration and the purity of the samples were determined by Nanodrop.

    Restriction digest and gel electrophoresis:

    The plasmids in each sample were digested with restriction enzymes BamHI-HF and PstI with the following ingredients and amounts:

    - 7.1 μL Plasmids
    - 0.5 μL PstI
    - 0.5 μL BamHI-HF
    - 2 μL NEBuffer 1.1
    - 10.4 μL ddH2OThe samples were incubated for 1.5 h at 37℃.

    Note! NEBbuffer 1.1 was chosen instead of Cut smart since the activity of PstI in Cut smart was 50%.

    The activity of the enzymes in NEBbuffer 1.1:

    BamHI-HF: 100%

    PstI:75%

    After the plasmids have been degraded by restriction enzymes, the fragments were separated by gel electrophoresis.

    **Results:**

    **Plasmid verification:**

    Plasmid isolation:

    | Sample | Concentration [ng/μL] | 260 nm/280 nm | 260 nm/230 nm |
    | --- | --- | --- | --- |
    | 1 | 219.9 | 2.01 | 1.82 |
    | 2 | 35.3 | 2.03 | 3.86 |

    Restriction digest and gel electrophoresis:

    The bands on the gel, corresponded with the expected fragments (6705bp, 2094bp, 465bp, 25bp) from digestion of the plasmids by PstI and BamHI-HF. Therefore, we concluded that both of pdCas9 and pgRNA with anti-luxS have successfully been transformed into DH5α cells.
* Week

  28

  Week 28

  + Jul

    9

    No lab
  + Jul

    10

    No lab
  + Jul

    11

    No lab
  + Jul

    12

    InterLab Study: Revive bacteria glycerol stock

    **Goal** : InterLab Study: Revive bacteria glycerol stock

    **Procedure:**
    The frozen bacteria glycerol stock made from 6th of July were thawed on ice. Each culture were inoculated in LB medium with CM (5 mL), and incubated over night at 37°C.
  + Jul

    13

    InterLab Study: CFU/mL/OD calculations

    **Goal:**

    Count colony forming units on plates spread the day before.

    **Procedure:**

    Counted CFU on all 36 plates incubated 13. July.

    **Results:**

    The colonies on agar plates, from yesterday’s incubation, were counted and registered:

    | CFU | Dilution 3 | Dilution 4 | Dilution 5 |
    | --- | --- | --- | --- |
    | 1.1 | 195 | 6 | 1 |
    | 1.2 | 185 | 2 | 0 |
    | 1.3 203 | 15 | 3 | |
    | 2.1 | 222 | 19 | 2 |
    | 2.2 | 38 | 22 | 1 |
    | 2.4? | 52 | 15 | 1 |
    | 3.1 | 113 | 56 | 3 |
    | 3.2 | 112 | 12 | 2 |
    | 3.3 | 127 | 25 | 2 |
    | 4.1 | 79 | 4 | 0 |
    | 4.2 | 75 | 10 | 1 |
    | 4.3 | 107 | 3 | 1 |

    where 1 and 2 are positive controls, 3 and 4 are negative controls. (= 2.1 means the second dilution of the second positive control culture)
  + Jul

    14

    InterLab – CFU/mL/OD calculation

    **Goal:**

    Counting colonies on each agar plate, and calculate CFU/mL in starting samples with an OD600 = 0.1.

    **Procedure:**

    The colonies on the agar plates, prepared from yesterday’s incubation, were counted and registered.

    **Results:**

    **Table 1:** The average values of counted colonies from two cultures of positive and negative controls with different final dilution factor (dilution 1 = 8x104, dilution 2 = 8x105, dilution 3 = 8x106).

    |  | Positive control | Negative control |
    | --- | --- | --- |
    | Culture 1, dilution 1 | 194.3 | 117.3 |
    | Culture 1, dilution 2 | 7.7 | 31.0 |
    | Culture 1, dilution 3 | 1.3 | 2.3 |
    | Culture 2, dilution 1 | 104.0 | 87.0 |
    | Culture 2, dilution 2 | 18.7 | 5.7 |
    | Culture 2, dilution 3 | 1.3 | 0.7 |
* Week

  29

  Week 29

  + Jul

    16

    Transformation of the RFP-biobrick (test)

    **Goal:**

    Transform the RFP-biobrick (BBa\_J04450) into competent DH5α-cells.

     **Procedure :**

    Before doing the transformation the biobrick was resuspended with 10 µL dH2O in the plate (position 23O, plate 7). The cells with and without the biobrick (negative control) were both incubated and plated after the transformation. The negative control was plated on LA-petri dishes while the transformed cells was plated on LA-petri dishes with CM.

     **Results:**

    There was only one colony on each plate
  + Jul

    17

    Colony picking biobrick (test) and transformation

    **Goal :**

    1. Get colonies of the transformed DH5α (biobrick)
    2. Get colonies on the negative control plate

    **Procedure:**

    1. Due to the lack of colonies on the negative control plates, new plates was streaked.

    2. New LA-plates without antibiotics were made.

    3. Colonypicking the two colonies from the plates with the transformed bacteria in to liquid medium.

    4. The transformation of bacteria with the biobrick plasmid was redone on four plates because of the lack of growth on the plates (see 16.07.18).

    **Results:**

    There were more colonies on the four new plates, and they were light red as well (About 20 colonies).
    The new negative control plates had colonies as well.
  + Jul

    18

    RFP biobrick fluorescens measurement (test)

    **Goal:**

    1. To get a descriptive curve for the rise in fluorescens (transformed DH5α)

    2. Find out how often the measurements need to be done.

    **Procedure:**

    The second batch of plates from transformation and the negative control:

    1. Colonypicking and exchange to liquid medium

    Measurements of the first batch (Tecan):

    1. Measured the OD of the blank (LB and CM), and measured the OD of the two cultures.

    2. For the next measurements we diluted the cultures by 1:100 in separate tubes.

    3. Incubation of the tubes

    4. The first measurement was done in the Tecan, and repeated for every 30 minutes and then 1 hour (because of minor changes in values)

    5. The measurements was set overnight as well using the following script: FluorescenceLong. The data was set to 588 nm absorbance, 584 nm excitation, 607 nm emission, 37°C, 40 gain, 150 kinetic cycles, 300s shaking (orbital 3 mm amplitude).

    **Results:**

    The second batch from transformation has turned red. From the measurements of the test batch we have an idea of the increase in fluorescence intensity and the change in absorbance over time. This can be used to determine the timing of the second batch with the negative control as well.
  + Jul

    19

    Biobrick: Preparations, Fluoresence microscopy

    **Goal:**

    Prepare for measurements tomorrow.

    Learn about fluorescence microscopy

    **Procedure:**

    Preparations:

    Made LA-petri dishes with CM.
    Diluted cultures of the biobrick Bba\_J04450 and negative control and incubated them at 37°C overnight.

    Demonstration and training in fluorescence microscopy:

    Astrid Bjørkøy showed us how to use the fluorescence microscope, and we tried it out by taking photos of cultures of the Bba\_J04450 biobrick. She also told us how to count cells by adjusting a photo's threshold and calculate based on image size.

    **Results:**

    The biobrick Bba\_J004450 was very bright. We used excitation 561 nm and emission ca 625-680 nm as the biobrick was too bright at emission 607 nm. Consider using another laser further away from excitation point (584 nm) for the real photos of the biobrick, and the improved biobrick.
  + Jul

    20

    Biobrick: Red Fluorescent Protein measurement (FAILED)

    **Goal:**

    Measure the expression of the Bba\_J04450 biobrick in transformed dH5α cells. This is done by measuring fluorescence.

    **Procedure:**

    1. Incubated dH5α, J04450 transformed cells were dilluted 1:100 with LB

    2. 200 µL, 5 replicates of LB, LB with Chloramphenicol and 5 dH5α dillutions made from different batches were applied to a 96 well plate

    3. Tecan plate reader was used to measure absorbance at 588 nm and fluorescence at excitation 584 nm and emission 607 nm with the following parameters:

    - Temperature: 37,0°C, varying from 36,5°C to 37,5°C
    - Kinetic cycles: 750
    - Gain: 40
    - Z position: 18055 µM
    - Shaking: 300 s
    - Orbital shaking amplitude: 3 mm

    **Results:**

    The wells dried out, presumably due to us choosing not to use a lid on the plate, and the measurements were therefore aborted by the machine.
* Week

  30

  Week 30

  + Jul

    23

    Make super competent cells of the TG1

    **Goal:**

    Make supercompetent cells of the E.coli - TG1 strain

    **Procedure:**

    DAY 1:

    - Made Psi media, transformation buffer 1 and transformation buffer 2.

    - Made an E.coli TG1 culture in a small flask with 10 mL Psi- medium. Inoculated culture at the end of the day and left the flask for incubating while shaking and at 37°C overnight.

    **Results:**
  + Jul

    24

    Make supercompetent cells of the TG1, day 2

    DAY 2:

    This was done from an overnight culture of TG1-cells, see protocol.
    **Results:**

    There was no growth on the plates with CM and AMP after the transformation (25. July. 2018), so the conclusion was that the cells were not supercompetent. The protocol was then updated and optimized for another trial.
  + Jul

    25

    Miniprep and transformation of TG1-cells

    **Goal:**

    1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene.

    2. Make LA-plates with both CM and AMP antibiotics

    **Procedure:**

    pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmids were isolated and extracted by the miniprep protocol. Transformation of the TG1-cells with the extracted plasmids was done, and the bacteria were incubated.

    **Results:** No bacterial growth has been observed.
  + Jul

    26

    Preparations for biofilm measurements

    **Goal:**

    1. Make media for the biofilm measurements (LB and M63B1)

    2. Prepare TG1, TG2 buffers and Psi medium for supercompetent cells.

    **Procedure:**

    1. Made three versions of the two media with pH-values at 4.5, 7.2 and 9.5. The media was then autoclaved and sugar will be added before use of the media.

    2. Made TG1, TG2 buffers and Psi medium.
  + Jul

    27

    Make supercompetent cells of TG1, second attempt

    **Goal:**

    1. Make supercompetent cells of TG1 by following an improved protocol.
* Week

  31

  Week 31

  + Jul

    30

    Transformation of TG1-cells and flow cytometry test

    **Goal:**

    1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene.
    2. Learn and conduct a flow cytometry test of the E.Coli DH5a that contains the Bba\_J04450-biobrick and a negative control. The goal was also to optimize a protocol for the biobrick measurements.

    **Procedure:**

    pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmids were isolated and extracted by the miniprep protocol, and the cells transformed and incubated. For the transformation, 5 µL of the miniprepped plasmids were used instead of 2 µL.

    2.

    **Results:** Noticeable bacterial growth was observed on all plates, with no apparent contamination.
  + Jul

    31

    TG1 colonypicking

    **Goal:**

    1. Make cultures out of plates made on the 30th of July, prepare for test digest.

    2. Make DH5α cultures for later use in biobrick measurements.

    **Procedure:**

    Colonypicking into five beakers with 25 mL LB, 25uL Amp and 25 µL CM. Incubation at 37°C, 225 RPM overnight. New LB-media was made.
  + Aug

    1

    Miniprep, Nanodrop, Test digest

    + Aug

      2

      Verification of transformation of TG1 and preparation of media for biofilm measurements

      **Goal:**

      1. Confirm successful transformation of TG1 with pgRNA and PdCas9 with anti-luxS.

      2. Prepare media for biofilm measurements

      3. Inoculate TG1 in newly made media

      **Procedure:**

      Verification of the plasmids was done by miniprep, nanodrop, test digest and gel electrophoresis. Different amounts of glucose was added to the respective media.
    + Aug

      3

      Measure OD + change medium

      **Goal:**
* Week

  32

  Week 32

  + Aug

    6

    E.Coli TG1 biofilm measurement

    The two E.Coli TG1 96-well plates made yesterday (06.08.2018) underwent the Crystal Violet Assay protocol (see Protocols), and the biofilm produced was measured by measuring absorbance at 590 nm with Tecan Infinite 200 Pro plate reader.
  + Aug

    7

    Incubate DH5α and TG1 in chosen media for biofilm measurements.

    **Goal:**

    - Incubate DH5α and TG1 (with pgRNA and pdCas) in selected media (LB and M63B1, pH 7.2, 0.4%/0.8% Glucose)
    - Make Tetracycline stock solution (5.0 mg/ml)

     **Procedure:**

    Incubated the frozen DH5α-stocks in LB (with CM and AMP) for a few hours. Inoculated into the chosen media in eppendorf tubes. Incubated TG1 into the chosen media in eppendorf tubes overnight.
    Made 5mg/mL Tetracyclin-solution (inducer). Note: light sensitive.

    Protocol:

    - Tetracyclin-HCl(powder): 54 mg

    - Ethanol: 7 mL

    - dH2O: 3 mL

    After being incubated for some time tetracyclin solution was added to the eppendorf tubes do induce the transcription of Cas9, and the culture tubes were further incubated.
  + Aug

    8

    Incubation of cell cultures for biofilm assay and preparation of M63B1 medium

    **Goal :**

    1. Incubate cell cultures on 96-well plates for biofilm assay

    2. Make M63B1 medium

    **Procedure :**

    Preparation for biofilm assay:

    1. Overnight cultures (with TET) was diluted until OD600 = 0.1

    2. Each diluted culture (100μL) was added on 96-well plate and incubated for 4 h at 37℃

    3. After 4 h incubation, the non-adhered cells were removed by washing the wells with physiological saline and the supernatant was discarded.

    4. New medium (100 μL) was added to each well and the plate was incubated for 24 h at 37℃.

    Note! The media that were added to the wells right before 24 h incubation did not contain TET

    M63B1 medium:

    M63B1 medium was made after following the protocol from 26. July. 2018

    **Results:**
  + Aug

    9

    Preparation of media with TET, and incubation of cell cultures on 96-well plate for biofilm assay

    **Goal :**

    1. Preparation of media with tetracycline (2 μM)

    2. Incubate cell cultures with and without tetracycline (TET) on a 96-well plate for the 24 h biofilm assay.

    3. Measure the absorbance of CV- assay with and without TET, made from yesterdays’ incubation.

    4. Prepare TG1 and dH5α cell cultures in growth medium (LB or M631B) with or without TET (2 μM).

    **Procedure :**

    Preparation of media with TET (2 μM):

    1. Tetracycline (TET) (50 μL, 0.5 mg/mL) was added to a 25mL LB medium with 0.4% glucose, AMP and CM. The same amount of tetracycline was added to LB medium with 0.8% glucose, AMP and CM

    2. For the final M63B1 medium (0.5 L) with MgSO4\*7H2O, AMP and CM and 0.4% or 0.8% glucose, TET (1mL, 0.5 mg/mL) was added.

    Preparation of 96-wells plate for 24h biofilm assay:

    1. Overnight cultures were diluted with or without TET (2 μM) containing media (LB or M63B1) until a target OD600 = 0.1.

    2. Each diluted culture (100 μL) were incubated on a 96-wells plate for 20 h at 37℃.

    Absorbance measurement of CV-assay:

    1. The supernatant was discarded from a 24h incubation of cell cultures on a 96-wells plate (prepared from 08. August. 2018) and rinsed with PS (100μL).

    2. The biofilm was fixed by adding methanol (100 μL, 99%) and waited for 15min. The supernatant was discarded, and the plate was airdried.

    3. CV-solution (100 μL, 0.1%) was added to each well and coloured the biofilm for 20 min. The excess CV was washed away with dH2O
    4. Acetic acid (150 μL, 33%) was added to the each well.

    5. The absorbance was measured at Abs 590 nm

    **Results:**
  + Aug

    10

    Preparation of overnight cultures for biofilm assay, and storage of TG1 cells with pgRNA and pdCas9

    **Goal :**

    1. Prepare overnight cultures in LB (0.4% or 0.8% glucose, with AMP and CM) or M63B1 (0.4% or 0.8% glucose, with AMP and CM) with or without TET.

    2. Wash the 96-well plate (after 20 h adhesion - prepared from 09.08.2018) with physiological saline, add new media and incubate the plate.

    3. Make bacteria glycerol stocks of TG1 cells with pgRNA and pdCas9.

    **Procedure :**

    Preparation of overnight culture:

    TG1 and dH5α were incubated in eppendorf tubes with different media (LB or M63B1 with glucose (0.4% or 0.8%), AMP and CM) with or without TET (2 μM) for overnight at 37℃ with shaking.

    Preparation for biofilm assay:

    1. After 20 h adhesion (plate prepared from 09. August. 2018), the non-adhered cells were removed by washing the wells with physiological saline (PS) and the supernatant was discarded.

    2. New medium (100 μL) was added to each well and the plate was incubated for 24 h at 37℃.

    Bacteria glycerol stock preparation:

    TG1 cells with pgRNA and pdCas9 (500 μL) was stored with glycerol (500 μL, 50%) at -80℃.

    Results :
  + Aug

    11

    Preparation of overnight cultures for biofilm assay

    **Goal :**

    Prepare 96-well for cell adhesion and CV-assay

    **Procedure :**

    The OD of some of the overnight cultures was not optimal for continuing the procedure of CV assay. New cell cultures were made.

    96-well plates for 24 h, 48 h and 72 h incubation were made. However, since the cell cultures could not be diluted to OD = 0.1, the results of the CV assay will not be comparable.
  + Aug

    12

    Incubation of cell cultures for biofilm assay

    **Goal :**

    Preparation for cell adhesion on 96-well plates for 24 h, 48 h and 72 h incubation.

    **Procedure :**

    1. The overnight cultures were resuspended, before dilution until a target OD = 0.1.

    2. The diluted cell cultures were resuspended and added on 96-well plates and incubated for 4 h at 37℃.

    3. After 4 h incubation, the wells were washed with physiological saline (100 μL) and the supernatant was discarded.

    4. New media (100 μL) were added to the plates, and the plates were incubated at 37℃.

    **Results :**
* Week

  33

  Week 33

  + Aug

    13

    Biofilm measurements after 24hrs

    **Procedure:**

    Followed the crystal violet assay:

    Changed medium in the plate for 48 and 72 hrs.

    **Results:**

    Results did not show any clear trend. Excel sheet attached.

    Wells with only medium, not cells, showed too high absorbance measurements, probably because of contamination of the mediums. Decided to autoclave all the mediums again. Will take measurements at 48 hrs, but not at 72 hrs.
  + Aug

    14

    Biofilm measurement after 48 hrs

    **Goal:**

    1. Measure biofilm after 48 hrs.
    2. Prepare RRVT-Biobrick and biobrick backbone
    3. Ligate and transform RRvT- biobrick and biobrick backbone into DH5alpha cells.**Procedure:**

    1. Performed the Crystal Violet Assay procedure for the plate designated for 48hrs and for the plate that initially was designated for 72 hrs measurement.
  + Aug

    15

    Biobrick ligation and transformation

    **Goal:**

    1. Ligate plasmid backbone and RRvT-plasmid and transform into cells.
    2. Due to the lack of colonies from yesterdays transformation the ligation of plasmid backbone and RRvT-plasmid and transformation into E.Coli Dh5α cells was redone.**Procedure:**

    Preparations of plasmid backbone and RRvT-biobrick:

    Made enzyme master mix for plasmid backbone by adding 5μL NEB buffer 2, 0.5 μL EcoRI-HF, 0.5 μL PstI, 0.5 μL BSA and 18.5 μL dH2O to an Eppendorf tube.

    Made enzyme master mix for RRvT-biobrick by adding 5μL NEB buffer 2, 0.5μL EcoRI-HF, 0.5 μL PstI and 19 μL dH2O to an Eppendorf tube. Incubated mix for 20 min at 50℃.

    Digested Plasmid backbone by adding 4 μL linearized backbone (25 ng/μL for 100 ng) and 4 μL of enzyme master mix for plasmid backbone.

    Digested RRvT-biobrick by adding 4 μL of the biobrick and 4 μL of the enzyme master mix for RRvT-biobrick.
    Digested both reactions at 37℃ for 90 min and did heat kill at 80℃ for 20 min.
    Ligation:

    Added 2 μL of digested plasmid backbone.

    Added 2 μL of digested RRvT-biobrick.

    Added 1 μL T4 DNA ligase buffer.

    Added 0.5 μL of T4 DNA ligase.

    Added water to 10 μL.

    Ligated at 16℃ for30min. Did heat kill at 80℃ for 20 min.

    Transformed  *E.Coli*  Dh5α cells with 1-2 μL of product following the protocol for transformation.

    Inoculated .. plates and let them incubate at 37℃ to the next day.+ Aug

      16

      Preparation for biobrick (RRvT) measurement and biofilm assay

      **Goal:**

      1. Inoculate successful transformed cells into LB medium with CM.
      2. Revive TG1 and DH5α cells in LB medium with AMP, CM and 0% glucose. **Procedure:**

      Preparation for biobrick (RRvT) measurement:

      Successful transformed cell colonies from yesterday's incubation were inoculated into LB - medium with CM and incubated at 37°C with shaking.

      Preparation for cell cultures for biofilm assay:

      TG1 and DH5α cells with pgRNA and pdCas9 were revived in LB medium with AMP, CM and 0% glucose. The cell cultures were incubated at 37°C with shaking.
    + Aug

      17

      Preparation for biofilm assay

      **Goal:**

      - Dilute the overnight cultures for biofilm assay.
      - Do Crystal Violet assay after 3 h, 5 h and 8 h of incubation of a 96-well plate.
       **Procedure:**

      The media for biofilm assay were made by adding the following ingredients:

      - LB 0.4% glucose, TET: 10 mL LB, 10µL AMP, 10µL CM, 0.2 mL 20% Glucose, 19.7µL 0.5mg/mL TET
      - LB 0.8% glucose, TET: 10 mL LB, 10µL AMP, 10µL CM, 0.4 mL 20% Glucose, 20µL 0.5mg/mL TET
      - LB 0.4% glucose: 10 mL LB, 10µL AMP, 10µL CM, 0.2 mL 20% Glucose
      - LB 0.8% glucose: 10 mL LB, 10µL AMP, 10µL CM, 0.4 mL 20% Glucose
      - M63B1 0.4% glucose, TET: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.2 mL 20% Glucose, 19.7 µL TET
      - M63B1 0.8% glucose, TET: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.4 mL 20% Glucose, 20 µL TET
      - M63B1 0.4% glucose: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.2 mL 20% Glucose
      - M63B1 0.8% glucose: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.4 mL 20% Glucose

      Biofilm assay:

      - Pipette 3-4 mL overnight cultures to sterile tubes (total 16 tubes).
      - Centrifuge the cultures for 10 min at 2000 rpm at 9°C.
      - Remove the supernatant.
      - Add 3-4 mL media (LB/ M63B1, with 0.4%/ 0.8% glucose, with/ without TET) to 2 tubes each.
      - Resuspend/ vortex the cultures, and measure OD 600.
      - Dilute the cultures until target OD 600 = 0.1 and store the cells on ice until they are added to the wells.
      - Resuspend/ vortex the cultures, add 100 µL culture to each well on the plate.
      - Seal the top of the plate with parafilm, and incubate the plate at 37°C.
      - Do CV assay after 3 h, 5 h, 8 h, 24 h and 48 h incubation.

* Week

  34

  Week 34

  + Aug

    20

    Preparation for 3h, 5h, 8h, 24h, 30h CV and Bactiter-Glo Assay.

    **Goal:**

    Redo CV assay from 17th August to check the validity of previous results.

    Perform Bactiter-Glo Assay parallell to CV Assay (3h, 5h, 8h, 24h, 30h) to examine the amount of living cells in biofilm.

    **Procedure:**

    Crystal Violet Assay: Same procedure as from 17.08.2018.

    Bactiter-Glo Assay

    **Results:**

    The results and measurement settings can be found in the attached files.
  + Aug

    21

    No lab

    No lab.
  + Aug

    22

    No lab

    No lab.
  + Aug

    23

    No lab

    No lab.
  + Aug

    24

    No lab

    No lab.
* Week

  35

  Week 35

  + Aug

    27

    No lab

    No lab.
  + Aug

    28

    No lab

    No lab.
  + Aug

    29

    No lab

    No lab.
  + Aug

    30

    No lab

    No lab.
  + Aug

    31

    No lab

    No lab.
* Week

  36

  Week 36

  + Sept

    3

    No lab

    No lab.
  + Sept

    4

    No lab

    No lab.
  + Sept

    5

    Introduction of site directed mutagenesis on pdCas9 and make medium for V. natrigens

    **Goal:**

    Make LB medium and 10x v2 salts for Vibrio natrigens

    Amplify and introduce site directed mutagenesis in pdCas9

    **Procedure:**

    LB - medium

    10x v2 salts:

    Make the salt solutions after the following recipe:
    - 11.935 g NaCl (204 mM)
    - 0.3134 g KCl (4.2 mM)
    - 4.710 g MgCl2\*6H2O (23.14 mM)
    - 1 L ddH2O

    Site directed mutagenesis:

    Forward and reverse primers for site directed mutagenesis were designed and ordered from Sigma Aldrich.
    Introduce site directed mutagenesis in pgCas9 via PCR after following the protocol from 29. June. 2018.
    The first PCR was not successful. The primer annealing temperature was changed, and the elongation time as well.
  + Sept

    6

    Introduce site directed mutagenesis in pdCas9 and amplify RRvT and pSB1C3e

    **Goal:**
    Introduce site directed mutagenesis in pdCas9
    Amplify RRvT and pSB1C3 via PCR

    **Procedure:**
    Site directed mutagenesis in dCas9:
    pdCas9 was amplified via gradient PCR, where the annealing temperatures were 57.3 ℃, 59.3 ℃, 61.1 ℃ and 64.1℃ with an elongation time for 5 min.
    The samples were stored in the fridge after the PCR was finished.
    Amplification of RRvT and pSB1C3:
    RRvT and pSB1C3 fragments were amplified by running the samples (6 µL FW primer, 6 µL REV primer, 1 µL template, 12.5 µL Takara Hi-Fi PCR premix) on PCR with the following settings: annealing temperature 37 ℃ for 10 s, elongation temperature 72 ℃ for 1 min.
    The samples were stored in the fridge after the PCR was finished.
  + Sept

    7

    Gel purification of PCR products (pdCas9, RRvT and pSB1C3), incubate V. natriegens

    **Goal:**

    Separate the PCR products of RRvT and pSB1C3 on gel, and miniprep the desired fragments on the gel.
    Incubate Vibrio natreigens (V. natriegens).
    Do another round of site directed mutagenesis on pdCas9 via PCR.

    **Procedure:**
    **Gel purification of RRvT and pSB1C3 on gel:**

    5 μL loading dye was added to the PCR products (25.5 μL).
    The PCR products were separated on Gel green (90 V, 40 min).
    The desired fragments were cut out from the gel and collected in eppendorf tubes
    Gel digestion and DNA isolation of RRvT and fragment of pSB1C3:
    The PCR products with the desired length on the gel was isolated by following after the same protocol from 02.07.2018.

    **Inoculation and incubation of V. natriegens:**

    Sample of V. natriegens sent by the iGEM team from Marburg, was inoculated and incubated by following after the protocol provided by the team.

    **Introduce site directed mutagenesis in pdCas9:**

    PCR mix with pdCas9 was prepared after following the same protocol from 29.06.2018.
    Two samples were set on two different programs:
    3 step PCR: DNA denaturation temperature at 98℃, primer annealing temperature at 55 ℃ for 10 s, elongation temperature at 72 ℃ for 7 min.
    2 step PCR: same conditions as 3 step PCR without the primer annealing step.
  + Sept

    8

    Purification of RRvT, separation of PCR product of pdCas9 and measurement of growth curve of V. natriegens (failed)

    **Goal:**
    - Measure the growth curve of V. natrigens
    - Gel purification of RRvT
    - Separte the PCR products of pdCas9 on gel**Procedure:**

    Growth curve of V. natrigens:

    The experiment failed due to dilution in the flasks and not in the cuvettes

    Gel purification of RRvT:

    The PCR product of RRvT was purified on gel, and the DNA was isolated after following the same protocol from 02.07.2018.

    Gel separation of pdCas9 PCR product:

    The PCR products of pdCas9 were separated on both gel red and gel green for imaging and for isolation of the desired fragments by following the same protocol from 02.07.2018.

    See results page for results

    .
  + Sept

    9

    Amplification of RRvT and introduce SDM in pdCas9

    **Goal:**

    Amplify RRvT and introduce SDM in pdCas9 via PCR

    **Procedure:**

    RRvT and introduction of pdCas9 was necessary since the concentration of the products isolated from gel on 08.09.2018 were too low.

    Amplification of RRvT:

    RRvT was amplified via PCR with an annealing temperature 55 ℃ for 10 s and elongation temperature at 72 ℃ for 1 min.

    Introduce SDM in pdCas9:

    Site directed mutagenesis was introduced via PCR with an annealing temperature at 55 ℃ for 10 s and elongation temperature 72 ℃ for 7 min.

    The products were stored in the fridge.
* Week

  37

  Week 37

  + Sept

    10

    Gibson assembly of RRvT and backbon and transformation of SDM pdCas9 and assembled RRvT into competent cells

    **Goal:**

    Gel purification of PCR products
    Gel digestion and isolation of desired DNA fragments
    Assemble RRvT and pSB1C3 via Gibson assembly
    Transform pdCas9 and the assembled RRvT into competent cells

    **Procedure:**

    Gel purification:
    The PCR products were separated on gel green
    RRvT: 25.5 µL DNA, 5.05 µL loading dye
    pdCas9: 25 µL DNA, 5 µL loading dye

    Gel digestion and DNA isolation:

    The desired fragments (RRvT: approx. 2000 bp, pdCas9: approx. 6000 – 7000 bp) were cut out from the gel. The gel was digested by following after the protocol from 02.07.18.

    Transformation of SDM pdCas9 into competent cells:

    6 µL DNA was transformed into competent cells by following after the protocol from 25.06.18.

    Gibson assembly of RRvT and pSB1C3:

    The fragments were assembled together by following the protocol provided from the kit
    2 µL RRvT, 8 µL pSB1C3 (0.03 – 0.2 pmol fragments), 10 µL assembly master mix was incubated in a thermocycler at 50C for 15 min. The sample was stored on ice for transformation.

    Transformation of assembled RRvT and pSB1C3 into competent cells:

    2 µL Gibson assembly product was transformed into competent cells by following after the protocol from 25.06.2018.
  + Sept

    11

    PCR colony screening, isolation and verify successful SDM on pdCas9

    **Goal:**

    - PCR colony screening of RRvT gene
    - Isolate pdCas9 from cell cultures
    - Verify successful SDM on pdCas9 by restriction digest the plasmid

    **Procedure:**

    PCR colony screening:

    None of the colonies were red/ pink coloured like the colonies with RFP.

    PCR colony screening was done to test whether assembly of RRvT and pSB1C3 fragments was successful

    PCR mix was prepared after the following protocol:
    5 µL Takara Hi-Fi PCR premix, 1 µL ddH2O, 6 µL RRvT FWD primer, 6 µL RRvT REV primer
    8 different colonies were chosen and transferred to a new agar plate with CM, before the colony was transferred to a PCR tube with PCR mix.
    The samples ran on a PCR machine with the same settings from day 28 – 06. 09. 2018.

    Plasmid isolation:

    pdCas9 with SDM was isolated from three cell replicates by following the protocol from day 3 – 22. 06. 2018.

    Restriction digest:

    Introduction of SDM in pdCas9 was verified by adding restriction enzyme which originally recognize and cut at a specific site in the plasmid. EcoRI has two restriction sites in pdCas9 before any mutations.

    Mix 2 µL DNA (pdCas9 SDM), 2 µL Cut smart buffer, 15.5 µL ddH2O, 0.5 µL EcoRI Hi-Fi
    Spin down the solutions
    Incubate overnight at 37 ℃
  + Sept

    12

    Gel separation of digested pdCas9 (SDM) and products from PCR colony screening of RRvT, amplification of pdCas9 fragments for Gibson assembly

    **Goal:**

    - Separate the DNA fragments of pdCas9 SDM after restriction digest
    - Visualize the products from PCR colony screening on gel
    - Amplify the different fragments of pdCas9 for Gibson assembly
    - Inoculate colonies with RRvT in LB medium with CM

    **Procedure:**

    Gel electrophoresis:

    Both pdCas9 fragments obtained from restriction digest, and the products from PCR colony screening of RRvT were separated and visualized on gel by mixing the DNA with loading dye

    RRvT: 3 µL DNA, 2 µL dye, 7 µL ddH2O

    pdCas9: add 4 µL loading dye to each samples

    The DNA fragments were separated on gel red.

    Preparation for Gibson assembly of pdCas9:

    Segments for Gibson assembly were obtained by amplify the desired fragments with designed primer pairs via PCR.

    Primers for Gibson assembly (ordered from Sigma Aldrich), were resuspended with ddH2O and diluted to 1 µ
    Dilute pdCas9 SDM – 2 µL DNA (PC1), 8 µL ddH2O (to obtain <26 ng/µL)
    PCR samples (1 µL diluted template (pdCas9 SDM, <26 ng/μL), 6 µL FWD primer, 6 µL REV primer, 12.5 µL Takara HI-FI PCR premix) were incubated in PCR machine with the same settings from day 29 – 07. 09. 2018.

    Inoculate cell colonies with RRvT:

    Colonies with RRvT was inoculated in LB medium with CM from the agar plate, and the cell cultures were incubated overnight at 37 ℃ with shaking.

    Cells were inoculated from the following agar plate.
    Cells with RFP were revived in LB medium with CM.
  + Sept

    13

    Gel purification, isolation and Gibson assemble the PCR products of pdCas9 fragments, transformation of Gibson assembled product, fluorescence test round of RRvT

    **Goal:**

    - Gel purification of the PCR product of pdCas9 fragments
    - Gel digestion and isolation of the pdCas9 fragments
    - Gibson assemble the pdCas9 fragments
    - Transformation of Gibson assembled product into competent cells
    - Test round of RRvT by measuring the fluorescence

    **Procedure:**

    Gel purification of pdCas9 fragments:

    25.5 µL PCR products were mixed with 5.05 µL loading dye, and separated on gel by following after the same procedure from 22.06.2018.

    Gel digest and DNA isolation:

    After gel electrophoresis, the desired pdCas9 fragments were cut out from the gel. The gel was digested and the DNA fragments were isolated by conducting the same protocol from 02.07.2018.

    Gibson assembly of the pdCas9 fragments:

    The pdCas9 fragments, isolated from the gel, were assembled after the following protocol

    Mix 0.60 µL G1 fragment, 3.49 µL G2 fragment, 3.67 µL G3 fragment, 2.24 µL G4 fragment (the volumes were calculated by our instructor) with 10 µL NEBuilder HiFi DNA Assembly Master mix, and the sample was incubated at 50 ℃ for 1 hour.

    The thermocycler was switched off without notice, so the samples stayed at room temperature for an hour before the thermocycler was turned on again…

    Transformation of Gibson assembly product:

    2 µL Gibson assembly product was transformed into DH5α cells by following after the procedure from 25. 06. 2018

    Measure the fluorescence of RRvT:

    The overnight cultures with RRvT, RFP and a negative control was diluted until target absorbance Abs600 = 0.1
    Four replicates of each culture (100 µL) were added to a 96 well microtiter plate.
    The Absorbance and the fluorescence were measured with the following settings:
    150 cycles with 5 min interval
    Emission wavelength: 607 nm
    Exitation wavelength: 584 nm
    Temperature: 37 ℃
    Gain: 22

    **Results:**

    Gel purification of pdCas9 fragments for Gibson assembly:

    The order of the different fragments from left to right: ladder, G1, G2, G3 and G4. The fragments for Gibson assembly should have the length of 641 bp, 481 bp, 2917 bp and 1468 bp, respectively.

    Fluorescence of RRvT:

    The values of the fluorescence were too low when the samples were measured with a gain of 22. The liquid in the wells has been evaporated during the measurement.
  + Sept

    14

    Inoculate successful transformed cells, Restreak V. natriegens, amplify gRNA and dCas9 for insertion into LITMUS28i vector

    **Goal:**

    - Inoculate successful transformed cells
    - Restreak V. natriegens on agar plates
    - Amplify gRNA and dCas9 (without mutation) sequence for insertion into LITMUS28i vector

    **Procedure:**

    Colony picking:

    Successful transformed cells (probably with correct Gibson Assembly product of pdCas9) were inoculated to LB medium with CM. The cell cultures were incubated at 37 ℃ overnight with shaking.

    Re-streak Vibrio natriegens on agar plates:

    Vibrio natriegens was inoculated and was stoke on LA with v2 salt aseptically. The agar plates were incubated at room temperature.

    Amplification of gRNA and dCas9:

    The primer pairs of gRNA and dCas9 (ordered from Sigma Aldrich) were diluted with ddH2O, so the concentration of the primers was 100 µM
    The primers were further diluted until target concentration = 1 µM
    PCR mix were prepared after the following recipe:
    pgRNA (0.5 µL, 37.2 ng/ µL), 6 µL FWD primer, 6 µL REV primer, 12.5 µL Takara HiFi PCR premix.
    pdCas9 (0.5 µL, 53.4 ng/ µL), 6 µL FWD primer, 6 µL REV primer, 12.5 µL Takara HiFi PCR premix.
    The gRNA sequence was amplified by running the sample with a PCR machine with an annealing temperature 55 ℃ for 10 s, and an elongation temperature 72 ℃ for 1 min. The dCas9 sequence was amplified with an annealing temperature 55 ℃ for 10 s, and an elongation temperature 72 ℃ for 7 min.
  + Sept

    15

    Isolation, digestion of Gibson assembled pdCas9, gel digestion and isolation of gRNA and dCas9 fragments (for insertion into LITMUS28i), inoculation of V. natriegens

    **Goal:**

    - Isolate Gibson assembled pdCas9 from overnight cultures
    - Test digest the Gibson assembled pdCas9
    - Make glycerol stock of cells with Gibson assembled pdCas9 (pdCas9 mutated)
    - Gel separate the PCR products of gRNA and dCas9 (for insertion into LITMUS28i vector)
    - Gel digest and isolate the gRNA and dCas9 fragments (for insertion into LITMUS28i vector)
    - Inoculate natriegens in LB medium with v2 salts

    **Procedure:**

    Isolation of Gibson assembled pdCas9:

    Overnight cultures were diluted until OD600 ≈9
    pdCas9 was isolated from the cells by following after the miniprep protocol from 22.06.2018.

    Test digest Gibson assembled pdCas9:

    The enzymes EcoRI and XbaI were selected to verify whether the Gibson assembly of pdCas9 was successful or not. For 0.5 µL enzyme, 250 ng DNA is required. The different replicates were prepared by following recipe:

    PC1: 5 µL DNA, 2 µL Buffer, 0.5 µL EcoRI, 0.5 µL XbaI, 12 µL ddH2O

    PC2: 7 µL DNA, 2 µL Buffer, 0.5 µL EcoRI, 0.5 µL XbaI, 10 µL ddH2O

    PC3: 12 µL DNA, 2 µL Buffer, 0.5 µL EcoRI, 0.5 µL XbaI, 5 µL ddH2O

    The samples were incubated at 37 ℃ for 3h, before gel separation.

    Glycerol stock of Gibson assembled pdCas9 (pdCas9 mutated):

    DH5α cells with the Gibson assembled pdCas9 were stored in glycerol stock at -80 ℃ by following after the procedure from day 28.06.2018.

    Gel separation of gRNA and dCas9 (not mutated) fragments (for insertion into LITMUS28i vector):

    5 µL loading dye was added to each sample. The samples ran on gel green at 90 V for 50 min.

    Gel digest and DNA isolation:

    The gRNA and dCas9 fragments were isolated from the gel by conducting the same protocol from 02.07.2018.

    Incoulation of V. natriegens:

    Coloies of V. natriegens were inoculated into LB medium with v2 salt. The cell cultures were incubated at 37 ℃ overnight with shaking.
  + Sept

    16

    Transformation of plasmid with TetR into competent cells, mak glycerol stocks of V. natriegens

    **Goal:**

    - Transform the part BBa\_R0040 (plasmid with TetR) into competent cells
    - Make glycerol stocks of natriegens

    **Procedure:**

    Transformation of BBa\_R004:

    The plasmid was resuspended with 10 µL ddH2O in the well of the plate (provided by iGEM).

    10 µL DNA was transformed into DH5α by following after the protocol from 25.06.2018.

    Glycerol stock of Vibrio natriegens:

    Overnight cultures of V. natriegens was stored in glyrecol stock at -80 ℃.
* Week

  38

  Week 38

  + Sept

    17

    Amplification of LITMUS28i vector, inoculation of successful transformed cells, revive cells with RRvT and RFP

    **Goal:**

    - Amplification of LITMUS28i sequence
    - Inoculate successful transformed cells with part BBa\_R0040 (plasmid with TetR)
    - Revive cells with RRvT and RFP

    **Procedure:**

    Amplification of LITMUS28i sequence:

    - Designed primers (bought from Sigma Aldrich) were resuspended and diluted to 1 μM.
    - The LITMUS28i vector (sponsored by NEB) was diluted to 25 ng/μ
    - The diluted primer pair (6 μL of each primer) for LITMUS28i was mixed with the diluted LITMUS28i vector (1 μL) and Takara HiFi PCR premix (12.5 μL).
    - The LITMUS 28i sequence was amplified after running in a PCR machine with an annealing temperature of 55 ℃ for 10 s and elongation temperature 72 ℃ for 2 min.

    Inoculation of successful transformed cells:

    Successful transformed cells with TetR was inoculated into LB medium with CM. The cell culture was incubated overnight at 37 ℃ with shaking.

    Revive cells with RRvT and RFP:

    Cells stored at -80 ℃ were revived by inoculation into LB medium with CM and incubated overnight at 37 ℃ with shaker.
  + Sept

    18

    Measure the fluorescence of RRvT, separation and isolation of LITMUS28i fragment, Gibson assemble LITMUS28i fragment, dCas9 and gRNA

    **Goal:**

    - Measure the fluorescence of RRvT, RFP and TetR
    - Separate LITMUS28i sequence on gel
    - Gel digest and isolate LITMUS28i segment
    - Gibson assemble LITMUS28i sequence, dCas9 and gRNA with anti-luxS

    **Procedure:**

    Fluorescence measurement:

    - The overnight cell cultures with RRvT, RFP and TetR were diluted to target Abs600 = 0,2.
    - 100 μL of each cell cultures were pipetted to a 96 well plate with 4 replicates. The cellcultres were surrounded with LB medium with CM to reduce evaporation of the cell cultures in the wells. The plate was covered with a lid and the plate was sealed with parafilm.
    - The absorbance and the fluorescence were measured with a five-minute interval for 150 cycles with a target temperature 37 ℃. The gain was set to 40, wave length of absorbance was set to 588 nm, while the excitation and the emission wavelength were set to 584 nm and 607 nm, respectively.

    Gel electrophoresis:

    05 μL loading dye was added to the PCR product of LITMUS28i vector (25 μL).
    The PCR product was separated on gel green, and the desired sequence was cut out from the gel.

    Gel digest and DNA isolation:

    The desired DNA sequence was isolated by digesting the gel by conducting the same procedure from 02.07.2018.

    Gibson assembly:

    LITMUS28i, dCas9 and gRNA with anti-luxS were assembled via Gibson assembly. The amount (pmol) required of the different fragments were calculated by the calculator provided by the homepage of NEB, and by following after the protocol of NEBuilder HiFi DNA Assembly

    LITMUS28i sequence (2295 bp, 68 ng/μL): 0.8 μL
    gRNA (538 bp, 43 ng/μL): 0.59 μL
    dCas9 (5025 bp, 52 ng/ μL): 4.92 μL
    NEBuilder HiFi DNA assembly master mix: 10 μL
    ddH2O: 3.69 μL

    The sample was incubated at 50 ℃ for an hour. The sample was stored in the fridge

    **Results:**

    Fluorescence measurement of RRvT:

    The fluorescence of RRvT and RFP was constant over time, and the value of the fluorescence of RRvT and RFP was lower than the fluorescence measured from the medium...
  + Sept

    19

    Measure growth curve of V. natriegens, transformation of Gibson assembled LITMUS28i, test digest mutated pdCas9

    **Goal:**

    - Measure the growth curve of V. natriegens
    - Transform Gibson assembled LITMUS28i (with unmutated dcas9 and gRNA with anti-luxS) into DH5α cells
    - Test digest the mutated pdCas9 with EcoRI and SpeI

    **Procedure:**

    Growth curve of V. natriegens:

    Overnight cultures were diluted to a target OD600 = 0.05 in 250 mL baffled flasks with a final volume 40 mL.
    The cell cultures were incubated at 37 ℃ with shaking.
    The OD600 of each replicate was measured after 10 min incubation for 5 hours.

    Transformation of Gibson assembled LITMUS28i vector:

    2 μL Gibson assembled LITMUS28i vector that consists unmutated dCas9 and gRNA with anti-luxS was transformed into competent cells after following the same procedure from 25.06.2018.

    Restriction digest the mutated pdCas9:

    The mutated pdCas9 was cut with EcoRI and SpeI by mixing 5 μL DNA (PC1), 2 μL cut smart buffer, 0.5 μL EcoRI, 0.5 μL SpeI and 12 μL dH2O. The reagent was incubated overnight at 37 ℃.
  + Sept

    20

    Gel separation of digested pdCas9 (mutated), preparation of ligation of mutated dCas9 and iGEM backcone

    **Goal:**

    Gel separation of the product from the enzyme digestion of mutated pdCas9

    Digestion of mutated pdCas9 and pSB1C3 (preparation for ligation of dCas9 and pSB1C3).

    **Procedure:**

    Gel electrophoresis:

    The products of enzyme digestion of mutated pdCas9 that has been incubated overnight, were mixed with 3.34 μL loading dye. The DNA segments were separated after running them on gel red at 90 V for 45 min.

    Digestion of mutated pdCas9 and pSB1C3:

    The DNA were digested after the following recipe

    pdCas9: 5 μL DNA (PC1, 50.8 ng/μL), 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 12 μL ddH2O.
    Linear pSB1C3: 10 μL DNA (linear pSB1C3, 25 ng/μL), 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 7 μL ddH2O.

    The samples were incubated at 37 ℃ overnight.
  + Sept

    21

    Note title

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* Week

  39

  Week 39

  + Sept

    24

    Note title

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  + Sept

    25

    No lab

    No lab.
  + Sept

    26

    No lab

    No lab.
  + Sept

    27

    No lab

    No lab.
  + Sept

    28

    Note title

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* Week

  40

  Week 40

  + Oct

    1

    Note title

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  + Oct

    2

    Note title

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  + Oct

    3

    Note title

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  + Oct

    4

    Note title

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  + Oct

    5

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* Week

  41

  Week 41

  + Oct

    8

    Note title

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  + Oct

    9

    No lab

    No lab.
  + Oct

    10

    No lab

    + Oct

      11

      Double transformation with pgRNA and new pdCas9

      **Goal:**

      1. Transformation of pgRNA and pdCas9 into DH5a and TG1-cells.

      **Procedure:**

      Both pdCas9(2) and pgRNA(2) with anti-luxS were simultaneously transformed into competent cells (DH5α) after the following protocol:

      - Thaw competent cells on ice.
      - Add pdCas9(2) (5μL) and pgRNA(2) with anti-luxS (5μL) to the same tube containing competent cells.
      - Incubate the cells on ice for 20min.
      - Heat shock the cells for 45s at 42℃.
      - Incubate the cells on ice for 2min.
      - Add LB- medium (900μL).
      - Incubate the cells for 1.5h (max. 2h) at 37℃ with shaker.
      - Plate the cells (100μL) on agar with AMP and CM
      - Centrifuge the cell culture for 3min at 6800g.
      - Discard the supernatant (200μL)
      - Resuspend the pellet with the remining supernatant in the tube
      - Plate the cells (100μL) on agar with AMP and CM.**Results:**

      Colonies on the plates with DH5a. No colonies on the plates with TG1.
    + Oct

      12

      Note title

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![](https://static.igem.org/mediawiki/2018/8/85/T--NTNU_Trondheim--nbs.png)

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# Team:NTNU Trondheim/Experiments

[![](https://static.igem.org/mediawiki/2018/e/eb/T--NTNU_Trondheim--navlogo.png)](https://2018.igem.org/Team%3ANTNU_Trondheim)

* Team

  [Team Members](https://2018.igem.org/Team%3ANTNU_Trondheim/Team)
  [Collaborations](https://2018.igem.org/Team%3ANTNU_Trondheim/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3ANTNU_Trondheim/Description)
  [Design](https://2018.igem.org/Team%3ANTNU_Trondheim/Design)
  [Model](https://2018.igem.org/Team%3ANTNU_Trondheim/Model)
  [Results](https://2018.igem.org/Team%3ANTNU_Trondheim/Results)
  [Demonstrate](https://2018.igem.org/Team%3ANTNU_Trondheim/Demonstrate)
* Lab

  [Lab Journal](https://2018.igem.org/Team%3ANTNU_Trondheim/Experiments)
  [Protocols](https://2018.igem.org/Team%3ANTNU_Trondheim/Protocols)
  [Measurement](https://2018.igem.org/Team%3ANTNU_Trondheim/Measurement)
  [Safety](https://2018.igem.org/Team%3ANTNU_Trondheim/Safety)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3ANTNU_Trondheim/Parts)
  [New Part](https://2018.igem.org/Team%3ANTNU_Trondheim/Improve)
* [InterLab](https://2018.igem.org/Team%3ANTNU_Trondheim/InterLab)
* Human Practices

  [Integrated Human Practices](https://2018.igem.org/Team%3ANTNU_Trondheim/Human_Practices)
  [Education and Engangement](https://2018.igem.org/Team%3ANTNU_Trondheim/Public_Engagement)
* Acknowledgements

  [Sponsors](https://2018.igem.org/Team%3ANTNU_Trondheim/Sponsors)
  [Attributions](https://2018.igem.org/Team%3ANTNU_Trondheim/Attributions)
* [Awards](https://2018.igem.org/Team%3ANTNU_Trondheim/Awards)

![](https://static.igem.org/mediawiki/2018/9/92/T--NTNU_Trondheim--labjournalheader.png)

![](https://static.igem.org/mediawiki/2018/d/df/T--NTNU_Trondheim--labjournalmobile.png)

In our lab journal we have thoroughly documented what we have done in the laboratory, what our goals each session has been, how we have worked and results from each session. If you want a closer look on our methods/receipies, take a look at our protocol.

* Week

  25

  Week 25

  + Jun

    20

    Our first day in the laboratory!

    **Goal:** Incubate the Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 on agar plates separately with antibiotics.

    1. LA- medium (1.5%) was made.

    2. Two different agar plates were made – one containing ampicillin (AMP) and one containing chloramphenicol (CM).

    **Incubation of bacteria**

    Two Escherichia coli (E. coli) samples which carry pgRNA and pdCas9 plasmids respectively, were ordered from Addgene. E.coli carrying the pgRNA plasmid were plated on agar plates with AMP, while E.coli carrying the pdCas9 plasmid were plated on agar plates with CM. Incubation for 24 h at 37℃.

    **Results:**

    There was growth and single colonies on all the plates.
  + Jun

    21

    Inoculation of E.coli

    **Goal:** Inoculate a colony from each agar plate in LB- medium with antibiotics.

    **Procedure**
    One colony of pgRNA- and pdCas9- bacteria were picked and inoculated in LB-media (25 mL) with 25 µL of the associated antibiotic (AMP or CM). The cell cultures were incubated at 37℃ in a shaking incubator at 204 rpm.
  + Jun

    22

    Isolation and verification of pgRNA and pdCas9

    **Goal:** Isolate and verify that the E.coli carry pgRNA and pdCas9.

    **Plasmid isolation**
    pgRNA and pdCas9 were isolated from the incubated bacteria prepared yesterday (June 21) by following the miniprep protocol.
    **Determine plasmid concentration and purity**
    The concentration of plasmids and the purity of each sample were determined by using Nanodrop.

    **Restriction digest and plasmid verification**
    The presence of pgRNA and pdCas9 in the cell cultures were verified after following the restriction digest protocol and separation of the DNA fragments by gel electrophoresis.
* Week

  26

  Week 26

  + Jun

    25

    Transformation of pgRNA and pdCas9 into competent cells

    **Goal:** Transform pgRNA and pdCas9 into competent cells (E.coli K-12 DH5α).

    **Procedure:**
    The extracted pgRNA and pdCas9 plasmid DNA were transformed into E.coli K-12 DH5α cells.
  + Jun

    26

    Transfer successful transformed cells to LB-medium and preparation of standard curves for the InterLab study

    **Goal:**

    Inoculate successful transformed competent cells to LB-medium, and prepare the standard curves for InterLab study.

    **Procedure:**

    Inoculate colony from agar plates to LB-medium:

    A colony from each agar plate was selected and inoculated to LB- medium with antibiotics. Competent cells with pgRNA were transferred to LB- medium with AMP, while competent cells with pdCas9 were inoculated into LB- medium with CM. The cell cultures were incubated at 37℃ with shaker.

    Preparation of 1xPBS solution for the InterLab-study.

    Preparation of agar plates with CM, LA- and LB-medium.

    Preparation of standard curves for InterLab study:

    Calibration 1: OD 600 reference point – LUDOX protocol

    A conversion factor to transform absorbance data from plate reader into OD600 was obtained in spectrometer by measuring four replicates of LUDOX CL-X and ddH2O. The data were imported into Excel sheet.

    Calibration 2: Particle standard curve – Microsphere protocol

    The solution of silica beads provided in the kit from iGEM HQ, was vortexed before dilution with water (96 μL Silica beads in 904 μL dd H2O). A serial dilution of microspheres was obtained by adding 100 μL dd H2O into 4x11 wells (E2, F2, G2, H2…E12, F12, G12, H12) on a 96 well plate. Microsphere stock solution (200 μL) was pipetted into E1 and 100 μL of the solution was transferred to E2 and mixed well before transferring 100 μL to E3. The procedure repeated until 100 μL solution was pipetted into E11, and 100 μL was transferred to liquid waste. The dilution series was repeated for row F, G and H. The absorbance of the samples was measured by a plate reader with shaker at 24℃. The data were exported to Excel sheet.
  + Jun

    27

    Isolation of pgRNA and pdCas9 from compentent cells (DH5α) and preparation of stock solution for calibration 3 for InterLab study

    **Goal:**

    Isolate plasmids from competent cells (DH5α), and prepare the fluorecein stock solution for InterLab study.

    **Procedure:**

    Dilution of cell cultures:

    Each cell cultures (successful transformed competent cells from day 5 – 26.06.2018) (2.5 mL) were transferred into fresh LB-medium (22.5 mL) with CM (22.5 μL). The new cultures were incubated for about 2 hours at 37℃ with shaking .

    Measurement of the cell density:

    The cell density of the new cultures was measured at OD 600, after calibration with water (blank sample).

    Plasmid isolation and determination of concentration:

    The plasmids were isolated from the competent cell after the protocol of ZR plasmid miniprep kit (see lab journal from day 3 - 22. June. 2018).

    The concentration of the plasmids was determined by using Nanodrop.

    Restriction digest:

    The plasmids from each culture (5 μL) were digested by mixing with 10xbuffer (2 μL), distilled water (12.5 μL) and restriction enzyme BspHI (0.5 μL). The solution was centrifuged down before the samples were incubated over night at 37℃.
  + Jun

    28

    Plasmid verification and storing the cell cultures

    **Goal:**

    Verify that the successfully transformed DH5α cells carry pgRNA or pdCas9, and store the cell cultures for future use.

    **Procedure:**

    Plasmid verification:

    The digested plasmid samples from yesterday’s incubation (lab 6 – 27. June.2018) were separated by gel electrophoresis.

    Bacteria glycerol stocks:

    The cell cultures made from 27. June were stored in a glycerol stock (500 µL cell culture and 500 µL 50% glycerol) in the freezer at -80℃.

    **Results:**

    Plasmid verification:

    L, pR1, pR2, pC1 and pC2 are the DNA-fragments from ladder solution, the two samples of pgRNA and pdCas9, respectively. From **Figure 1** the two fragments with 1.5 kb and 1.0 kb were found from the fragments of pgRNA after digestion by BspHI. The fragment with 100 bp from the digested pgRNA is not visible on the gel, probably due to masking of the dye colour at the bottom of the gel. Two bands from the digested pdCas9 with 2.7 kb and 4 kb were also detected. By comparing the results og gel elecrophoresis obtained from today's experiment with the results from day 3 (22. June. 2018), one can clearly see that the bands with the expected length from successful transformed competent cells, are much clearer. Hence, this also indicate the samples from the successful transformed DH5α cells are purer, which is also true by comparing the results from Nanodrop (see day 6 - 27. June. 2018, and day 3 - 22. June. 2018).
  + Jun

    29

    Insertion of anti-luxS via PCR and fluorescence standard curve for InterLab study

    **Goal:**

    Insert anti-luxS in pgRNA plasmid, and generate fluorescence standard curve for the InterLab study.

    **Procedure:**

    Insertion of anti-luxS gene and amplification of pgRNA with PCR.

    InterLab – Calibration 3

    A fluorescence standard curve was generated from the data obtained after measuring the fluorescence of the serial dilution of fluorescein.

    The fluorescence was measured with the following settings:

    Temperature: 24℃

    Gain (manual): 56

    |  | Wave length [nm] | Band width [nm] |
    | --- | --- | --- |
    | Excitation | 494 | 4 |
    | Emission | 525 | 20 |

    **Results:**

    InterLab – Calibration 3:

    Figure 1 shows the florescence standard curve in the concentration interval [0, 2.5 μM], with a R2 value of 0,9956. The model of the fluorescence standard curve is:
* Week

  27

  Week 27

  + Jul

    2

    Separation, isolation and transformation of linear DNA (pgRNA with anti-luxS)

    **Goal:**

    1. Separate the PCR product from circular DNA
    2. Isolate the linear DNA (pgRNA with anti-luxS from the gel
    3. Transform the linear DNA into competent cells
    4. InterLab study - transformation of plasmids to competent cells
    **Procedure:**

    Separation of linear DNA from circular DNA:

    The leaner DNA obtained from PCR (from day 8 – 29. June. 2018), was separated from the circular DNA (original plasmids) after running the sample (25 μL) mixed with loading dye (5 μl) on gel electrophoresis (gelGreen) simultaneously with a ladder (1 μL gene ruler + 1 μL loading dye + 4 μL dH2O) for 50 min at 90 V. The sample and the ladder were prepared by the gel electrophoresis protocol.

    Gel digestion and DNA isolation:

    The agarose was digested and the DNA was isolated from the sample following the gel purification protocol.

    Determine the DNA concentration and purity:

    The concentration and the purity of the isolated DNA was determined by using Nanodrop.

    Transformation of PCR product to competent cells:
    The PCR product (5 μL) was transformed to competent cells after following the transformation protocol.

    InterLab – transformation of plasmids to competent cells:

    The plasmids listed on the protocol, was transformed into competent cells (DH5α) after the following protocol:

    1. Thaw the competent cells on ice (10 min), and pre-chill the eppendorf tubes.
    2. Resuspend the DNA in the wells (provided by iGEM HQ) with dH2O (10 μL).
    3. Add competent cells (50 μL) to the eppendorf tubes.
    4. Add resuspended DNA (1 μL) to the Eppendorf tubes.
    5. Incubate on ice for 25 min
    6. Heat shock the cells for 45 s at 42℃ in water bath
    7. Incubate on ice for 5 min.
    8. Add LB-medium (950μL) to the tubes
    9. Incubate for 1h at 37℃ with shacker
    10. Pipette each sample (100 μL) to agar plate with CM
    11. Centrifuge the samples for 3 min at 6800g, discard supernatant (800 μL) and resuspend the pellet with the remaining supernatant
    12. Pipette each sample (100 μL) to agar plate with CM
    13. Incubate overnight at 37℃.
    **Results:**

    Separation of linear DNA from circular DNA:

    **Figure 1** shows the image of the gel where the linearized DNA was separated from the circular DNA. The linearized DNA is approximately 2 kb. The linearized DNA fragment was then cut out from the gel and stored in eppendorf tube.

    Note! Due to UV-light exposure, the DNA may mutate. In this case, this may affect the later steps of our project. The worse scenario, mutation of anti-luxS. Hence our CSRPRi system may not recognize luxS in the bacteria. Biofilm production will not be inhibited…

    Determine the DNA concentration and purity:

    **Table 1 shows the results of the linear pgRNA (2) from PCR (probably with anti-luxS gene).**

    **Table 1:** The concentration and the relative purity of the pgRNA (2) from PCR.

    | Concentration [ng/μL] | 260 nm/280 nm | 260 nm/230 nm |
    | --- | --- | --- |
    | 52.8 | 1.90 | 0.27 |

    From the 260/230 ratio, the sample has a relatively a small amount of nucleic acids. Probably, it is contaminated by the agarose from the gel electrophoresis. On the other hand, the 260/280 ratio is ok, indicating that the sample contains a relatively larger amount of RNA than DNA.
    InterLab – transformation of plasmids to competent cells:

    No visible colonies were detected on agar plates, except the positive and negative control cells.
  + Jul

    3

    Inoculation of successful transformed competent cells and InterLab study

    **Goal:**

    1. Inoculate colonies of successful transformed competent cells
    2. InterLab study - transformation of plasmids to competent cells
    **Procedure:**

    InterLab – transformation of plasmids to competent cells:

    No visible cell colonies were formed on the agar plates that were incubated on day 9 - 02. Junly. 2018. Only the positive and negative control cells formed colonies.

    Therefore, the remaining plasmids (6 μL) were transformed to the competent cells and plated on agar based on the same protocol from day 2. Jul. 2018. During the incubation at 37℃ with shaker, the incubation time was extended to 2 h. The cell cultures were centrifuged, and 700 μL supernatant was discarded, and the pellets were resuspended with the remaining supernatant. The cell cultures were then plated on agar plated with CM, and incubated overnight at 37℃.

    Inoculation of successful transformed competent cells:

    Colonies of successful transformed competent cells with pgRNA (probably containing anti-luxS) were inoculated from the agar plates (from day 9 – 02. July. 2018) to LB-medium with AMP. The cell cultures were incubated with shaker overnight at 37℃.
  + Jul

    4

    Verification of successful inserted anti-luxS in pgRNA

    **Goal:**

    1. InterLab - transformation of plasmids into competent cells
    2. Verification of succsesful insertion of anti-luxS in pgRNA(2)
    **Procedure:**

    InterLab – Transformation of plasmids to competent cells

    There has been a mistake of numbering the plates with plasmids provided by iGEM HQ. Therefore, we did not get any cell colonies from the previous two transformations…

    The plasmids (1.5 μL) from the right wells were transformed to competent cells by following the transformation protocol from day – 02. July. 2018, after resuspension of the plasmids in the wells.

    Cell density:

    The cell cultures with pgRNA(2) (probably with anti-luxS), inoculated from day 10 – 04. July. 2018, were diluted 10 times by following the same dilution procedure from day 6 – 27. June. 2018. The cells were incubated with shaker for 1h before measuring at OD 600.

    Plasmid isolation:

    The plasmids from the two cell cultures were isolated after the same mini-prep protocol from day 3 – 22. June. 2018. The concentration and the purity of the isolated plasmids were measured by Nanodrop.

    Restriction digest and gel elecrophoresis:

    Insertion of anti-luxS gene was verified after the protocol of restriction digest and gel electrophoresis (see the protocols from day 3 – 22. June. 2018). The restriction enzyme PstI was chosen, and the expected length of the plasmid fragments are 490 bp and 2094 bp.

    Note! PstI has a cut site at the anti-luxS gene in the plasmid.

    Bacteria glycerol stock:

    Two samples of the diluted cell cultures (made at this morning), were stored in 50% glycerol solution at -80℃ (the same green box with the other successful transformed cells with pgRNA (without anti-luxS) or cells with pdCas9 plasmids).

    **Results:**

    InterLab - Transformation:

    Colonies were formed on agar plates with CM.

    Cell density:

    Table 1: Absorbance of the cell cultures with pgRNA(2) after 1 h incubation at 37℃ with shaker.

    | Culture | OD 600 |
    | --- | --- |
    | pR2 (1) | 0.954 |
    | pR2 (2) | 0.998 |

    Plasmid isolation:
    Table 2: Concentration and the relative purity of isolated pgRNA(2) from the two cell cultures.

    |  | Concentration (ng/μL) | 260 nm/280 nm | 260 nm/230 nm |
    | --- | --- | --- | --- |
    | pR2(1) | 37.2 | 2.06 | 4.29 |
    | pR2(2) | 123.3 | 1.77 | 1.01 |
  + Jul

    5

    Double transformation and InterLab study - Inoculation

    **Goal:**

    1. Transform both pdCas9 and pgRNA with anti-luxS into competent cells
    2. InterLab study - inoculate colonies in LB-medium
    **Procedure:**

    Double transformation of pdCas9 and pgRNA with anti-luxS:

    Both pdCas9(2) and pgRNA(2) with anti-luxS (isolated from day 11 – 05. July. 2018) were simultaneously transformed into competent cells (DH5α). The amount of added pdCas9(2) (5 μL) and pgRNA(2) with anti-luxS was 5 µL per/plasmid, and it was added to the same tube containing competent cells. The cells were plated on agar with both AMP and CM. A couple of steps were added at the end, and the purpose was to increase the cell concentration plated on agar, and probably increase the possibility of colony formation. The cell culture was therefore centrifuged for 3 minutes at 6800g, and the supernatant discarded. The pellet was then resuspended and cells were plated.

    InterLab – Inoculation of colonies

    A colony from each agar plate was inoculated in LB-medium (5 mL) with CM (5 μL). The cell cultures were incubated overnight at 37℃ with shaker.
  + Jul

    6

    Inoculation of successful double transformed cells, interLab study - Cell measurement

    **Goal:**

    1. Inoculate successful double transformed cells (probably carrying pdCas9 and pgRNA with anti-luxS)
    2. InterLab study - measure absorbance and the fluorescence of each cell cultures
    **Procedure:**

    Inoculation of successful double transformed cells

    1. A colony from each agar plates with AMP and CM (made from day 13 – 05. July 2018) was picked and inoculated in LB medium (20 mL) with AMP (25 μL) and CM (25 μL).
    2. The cell cultures were incubated at 37℃ with shaker.
    InterLab – Cell measurement

    We were not able to measure the cell cultures today, since the TECAN device was under reparation.

    Each cell cultures were stored with 50% glycerol (see day 7 – 28. June. 2018) at -80℃.
  + Jul

    7

    Verification of pdCas9 and pgRNA with anti-luxS after double tranformation

    **Goal:**

    Verify that both pdCas9 and pgRNA with anti-luxS are tranformed into DH5α cells

    **Procedure:**

    Dilution of cell culture:

    Each cell cultures, inoculated and incubated from day 13 – 07. July. 2018, were diluted (1mL) with LB medium with antibiotics until the OD 600 reached 1.9.

    **Plasmid verification:**

    Plasmid isolation:

    The plasmids were isolated from each cell cultures after following the mini-prep plasmid protocol from day 3 – 22. June. 2018. The concentration and the purity of the samples were determined by Nanodrop.

    Restriction digest and gel electrophoresis:

    The plasmids in each sample were digested with restriction enzymes BamHI-HF and PstI with the following ingredients and amounts:

    - 7.1 μL Plasmids
    - 0.5 μL PstI
    - 0.5 μL BamHI-HF
    - 2 μL NEBuffer 1.1
    - 10.4 μL ddH2OThe samples were incubated for 1.5 h at 37℃.

    Note! NEBbuffer 1.1 was chosen instead of Cut smart since the activity of PstI in Cut smart was 50%.

    The activity of the enzymes in NEBbuffer 1.1:

    BamHI-HF: 100%

    PstI:75%

    After the plasmids have been degraded by restriction enzymes, the fragments were separated by gel electrophoresis.

    **Results:**

    **Plasmid verification:**

    Plasmid isolation:

    | Sample | Concentration [ng/μL] | 260 nm/280 nm | 260 nm/230 nm |
    | --- | --- | --- | --- |
    | 1 | 219.9 | 2.01 | 1.82 |
    | 2 | 35.3 | 2.03 | 3.86 |

    Restriction digest and gel electrophoresis:

    The bands on the gel, corresponded with the expected fragments (6705bp, 2094bp, 465bp, 25bp) from digestion of the plasmids by PstI and BamHI-HF. Therefore, we concluded that both of pdCas9 and pgRNA with anti-luxS have successfully been transformed into DH5α cells.
* Week

  28

  Week 28

  + Jul

    9

    No lab
  + Jul

    10

    No lab
  + Jul

    11

    No lab
  + Jul

    12

    InterLab Study: Revive bacteria glycerol stock

    **Goal:**

    InterLab Study: Revive bacteria glycerol stock

    **Procedure:**

    The frozen bacteria glycerol stock made from 6th of July were thawed on ice. Each culture were inoculated in LB medium with CM (5 mL), and incubated over night at 37°C.
  + Jul

    13

    InterLab Study: CFU/mL/OD calculations

    **Goal:**

    Count colony forming units on plates spread the day before.

    **Procedure:**

    Counted CFU on all 36 plates incubated 13. July.

    **Results:**

    The colonies on agar plates, from yesterday’s incubation, were counted and registered:

    | CFU | Dilution 3 | Dilution 4 | Dilution 5 |
    | --- | --- | --- | --- |
    | 1.1 | 195 | 6 | 1 |
    | 1.2 | 185 | 2 | 0 |
    | 1.3 203 | 15 | 3 | |
    | 2.1 | 222 | 19 | 2 |
    | 2.2 | 38 | 22 | 1 |
    | 2.4? | 52 | 15 | 1 |
    | 3.1 | 113 | 56 | 3 |
    | 3.2 | 112 | 12 | 2 |
    | 3.3 | 127 | 25 | 2 |
    | 4.1 | 79 | 4 | 0 |
    | 4.2 | 75 | 10 | 1 |
    | 4.3 | 107 | 3 | 1 |

    where 1 and 2 are positive controls, 3 and 4 are negative controls. (= 2.1 means the second dilution of the second positive control culture)
  + Jul

    14

    InterLab – CFU/mL/OD calculation

    **Goal:**

    Counting colonies on each agar plate, and calculate CFU/mL in starting samples with an OD600 = 0.1.

    **Procedure:**

    The colonies on the agar plates, prepared from yesterday’s incubation, were counted and registered.

    **Results:**

    **Table 1:** The average values of counted colonies from two cultures of positive and negative controls with different final dilution factor (dilution 1 = 8x104, dilution 2 = 8x105, dilution 3 = 8x106).

    |  | Positive control | Negative control |
    | --- | --- | --- |
    | Culture 1, dilution 1 | 194.3 | 117.3 |
    | Culture 1, dilution 2 | 7.7 | 31.0 |
    | Culture 1, dilution 3 | 1.3 | 2.3 |
    | Culture 2, dilution 1 | 104.0 | 87.0 |
    | Culture 2, dilution 2 | 18.7 | 5.7 |
    | Culture 2, dilution 3 | 1.3 | 0.7 |
* Week

  29

  Week 29

  + Jul

    16

    Transformation of the RFP-biobrick (test)

    **Goal:**

    Transform the RFP-biobrick (BBa\_J04450) into competent DH5α-cells.

     **Procedure :**

    Before doing the transformation the biobrick was resuspended with 10 µL dH2O in the plate (position 23O, plate 7). The cells with and without the biobrick (negative control) were both incubated and plated after the transformation. The negative control was plated on LA-petri dishes while the transformed cells was plated on LA-petri dishes with CM.

     **Results:**

    There was only one colony on each plate
  + Jul

    17

    Colony picking biobrick (test) and transformation

    **Goal :**

    1. Get colonies of the transformed DH5α (biobrick)
    2. Get colonies on the negative control plate

    **Procedure:**

    1. Due to the lack of colonies on the negative control plates, new plates was streaked.

    2. New LA-plates without antibiotics were made.

    3. Colonypicking the two colonies from the plates with the transformed bacteria in to liquid medium.

    4. The transformation of bacteria with the biobrick plasmid was redone on four plates because of the lack of growth on the plates (see 16.07.18).

    **Results:**

    There were more colonies on the four new plates, and they were light red as well (About 20 colonies).
    The new negative control plates had colonies as well.
  + Jul

    18

    RFP biobrick fluorescens measurement (test)

    **Goal:**

    1. To get a descriptive curve for the rise in fluorescens (transformed DH5α)

    2. Find out how often the measurements need to be done.

    **Procedure:**

    The second batch of plates from transformation and the negative control:

    1. Colonypicking and exchange to liquid medium

    Measurements of the first batch (Tecan):

    1. Measured the OD of the blank (LB and CM), and measured the OD of the two cultures.

    2. For the next measurements we diluted the cultures by 1:100 in separate tubes.

    3. Incubation of the tubes

    4. The first measurement was done in the Tecan, and repeated for every 30 minutes and then 1 hour (because of minor changes in values)

    5. The measurements was set overnight as well using the following script: FluorescenceLong. The data was set to 588 nm absorbance, 584 nm excitation, 607 nm emission, 37°C, 40 gain, 150 kinetic cycles, 300s shaking (orbital 3 mm amplitude).

    **Results:**

    The second batch from transformation has turned red. From the measurements of the test batch we have an idea of the increase in fluorescence intensity and the change in absorbance over time. This can be used to determine the timing of the second batch with the negative control as well.
  + Jul

    19

    Biobrick: Preparations, Fluoresence microscopy

    **Goal:**

    Prepare for measurements tomorrow.

    Learn about fluorescence microscopy

    **Procedure:**

    Preparations:

    Made LA-petri dishes with CM.
    Diluted cultures of the biobrick Bba\_J04450 and negative control and incubated them at 37°C overnight.

    Demonstration and training in fluorescence microscopy:

    Astrid Bjørkøy showed us how to use the fluorescence microscope, and we tried it out by taking photos of cultures of the Bba\_J04450 biobrick. She also told us how to count cells by adjusting a photo's threshold and calculate based on image size.

    **Results:**

    The biobrick Bba\_J004450 was very bright. We used excitation 561 nm and emission ca 625-680 nm as the biobrick was too bright at emission 607 nm. Consider using another laser further away from excitation point (584 nm) for the real photos of the biobrick, and the improved biobrick.
  + Jul

    20

    Biobrick: Red Fluorescent Protein measurement (FAILED)

    **Goal:**

    Measure the expression of the Bba\_J04450 biobrick in transformed dH5α cells. This is done by measuring fluorescence.

    **Procedure:**

    1. Incubated dH5α, J04450 transformed cells were dilluted 1:100 with LB

    2. 200 µL, 5 replicates of LB, LB with Chloramphenicol and 5 dH5α dillutions made from different batches were applied to a 96 well plate

    3. Tecan plate reader was used to measure absorbance at 588 nm and fluorescence at excitation 584 nm and emission 607 nm with the following parameters:

    - Temperature: 37,0°C, varying from 36,5°C to 37,5°C
    - Kinetic cycles: 750
    - Gain: 40
    - Z position: 18055 µM
    - Shaking: 300 s
    - Orbital shaking amplitude: 3 mm

    **Results:**

    The wells dried out, presumably due to us choosing not to use a lid on the plate, and the measurements were therefore aborted by the machine.
* Week

  30

  Week 30

  + Jul

    23

    Make super competent cells of the TG1

    **Goal:**

    Make supercompetent cells of the E.coli - TG1 strain

    **Procedure:**

    DAY 1:

    - Made Psi media, transformation buffer 1 and transformation buffer 2.

    - Made an E.coli TG1 culture in a small flask with 10 mL Psi- medium. Inoculated culture at the end of the day and left the flask for incubating while shaking and at 37°C overnight.
  + Jul

    24

    Make supercompetent cells of the TG1, day 2

    **Goal:**

    Make supercompetent cells of the TG1

    **procedure:**

    This was done from an overnight culture of TG1-cells, see protocol.

    **Results:**

    There was no growth on the plates with CM and AMP after the transformation (25. July. 2018), so the conclusion was that the cells were not supercompetent. The protocol was then updated and optimized for another trial.
  + Jul

    25

    Miniprep and transformation of TG1-cells

    **Goal:**

    1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene.

    2. Make LA-plates with both CM and AMP antibiotics

    **Procedure:**

    pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmids were isolated and extracted by the miniprep protocol. Transformation of the TG1-cells with the extracted plasmids was done, and the bacteria were incubated.

    **Results:** No bacterial growth has been observed.
  + Jul

    26

    Preparations for biofilm measurements

    **Goal:**

    1. Make media for the biofilm measurements (LB and M63B1)

    2. Prepare TG1, TG2 buffers and Psi medium for supercompetent cells.

    **Procedure:**

    1. Made three versions of the two media with pH-values at 4.5, 7.2 and 9.5. The media was then autoclaved and sugar will be added before use of the media.

    2. Made TG1, TG2 buffers and Psi medium.
  + Jul

    27

    Make supercompetent cells of TG1, second attempt

    **Goal:**

    Make supercompetent cells of TG1 by following an improved protocol.

    **Procedure:**

    See Protocols for details.

    **Results:**

    Transformation successful
* Week

  31

  Week 31

  + Jul

    30

    Transformation of TG1-cells and flow cytometry test

    **Goal:**

    1. Transformation of TG1-cells with DNA that includes pdCas9 and pgRNA with antiluxS-gene.

    2. Learn and conduct a flow cytometry test of the E.Coli DH5a that contains the Bba\_J04450-biobrick and a negative control. The goal was also to optimize a protocol for the biobrick measurements.

    **Procedure:**

    pgRNA with antiluxS and pdCas9 has already been transformed into E.coli DH5α. The plasmids were isolated and extracted by the miniprep protocol, and the cells transformed and incubated. For the transformation, 5 µL of the miniprepped plasmids were used instead of 2 µL.

    **Results:**

    Noticeable bacterial growth was observed on all plates, with no apparent contamination.
  + Jul

    31

    TG1 colonypicking

    **Goal:**

    1. Make cultures out of plates made on the 30th of July, prepare for test digest.

    2. Make DH5α cultures for later use in biobrick measurements.

    **Procedure:**

    Colonypicking into five beakers with 25 mL LB, 25uL Amp and 25 µL CM. Incubation at 37°C, 225 RPM overnight. New LB-media was made.
  + Aug

    1

    Miniprep, Nanodrop, Test digest

    Miniprep, Nanodrop and Test digest was done.

    + Aug

      2

      Verification of transformation of TG1 and preparation of media for biofilm measurements

      **Goal:**

      1. Confirm successful transformation of TG1 with pgRNA and PdCas9 with anti-luxS.

      2. Prepare media for biofilm measurements

      3. Inoculate TG1 in newly made media

      **Procedure:**

      Verification of the plasmids was done by miniprep, nanodrop, test digest and gel electrophoresis. Different amounts of glucose was added to the respective media.
    + Aug

      3

      Measure OD + change medium

      OD600 was measured and medium changed.
* Week

  32

  Week 32

  + Aug

    6

    E.Coli TG1 biofilm measurement

    The two E.Coli TG1 96-well plates made yesterday (06.08.2018) underwent the Crystal Violet Assay protocol (see Protocols), and the biofilm produced was measured by measuring absorbance at 590 nm with Tecan Infinite 200 Pro plate reader.
  + Aug

    7

    Incubate DH5α and TG1 in chosen media for biofilm measurements.

    **Goal:**

    - Incubate DH5α and TG1 (with pgRNA and pdCas) in selected media (LB and M63B1, pH 7.2, 0.4%/0.8% Glucose)
    - Make Tetracycline stock solution (5.0 mg/ml)

     **Procedure:**

    Incubated the frozen DH5α-stocks in LB (with CM and AMP) for a few hours. Inoculated into the chosen media in eppendorf tubes. Incubated TG1 into the chosen media in eppendorf tubes overnight.
    Made 5mg/mL Tetracyclin-solution (inducer). Note: light sensitive.

    Protocol:

    - Tetracyclin-HCl(powder): 54 mg

    - Ethanol: 7 mL

    - dH2O: 3 mL

    After being incubated for some time tetracyclin solution was added to the eppendorf tubes do induce the transcription of Cas9, and the culture tubes were further incubated.
  + Aug

    8

    Incubation of cell cultures for biofilm assay and preparation of M63B1 medium

    **Goal :**

    1. Incubate cell cultures on 96-well plates for biofilm assay

    2. Make M63B1 medium

    **Procedure :**

    Preparation for biofilm assay:

    1. Overnight cultures (with TET) was diluted until OD600 = 0.1

    2. Each diluted culture (100μL) was added on 96-well plate and incubated for 4 h at 37℃

    3. After 4 h incubation, the non-adhered cells were removed by washing the wells with physiological saline and the supernatant was discarded.

    4. New medium (100 μL) was added to each well and the plate was incubated for 24 h at 37℃.

    Note! The media that were added to the wells right before 24 h incubation did not contain TET

    M63B1 medium:

    M63B1 medium was made after following the protocol from 26. July. 2018
  + Aug

    9

    Preparation of media with TET, and incubation of cell cultures on 96-well plate for biofilm assay

    **Goal :**

    1. Preparation of media with tetracycline (2 μM)

    2. Incubate cell cultures with and without tetracycline (TET) on a 96-well plate for the 24 h biofilm assay.

    3. Measure the absorbance of CV- assay with and without TET, made from yesterdays’ incubation.

    4. Prepare TG1 and dH5α cell cultures in growth medium (LB or M631B) with or without TET (2 μM).

    **Procedure :**

    Preparation of media with TET (2 μM):

    1. Tetracycline (TET) (50 μL, 0.5 mg/mL) was added to a 25mL LB medium with 0.4% glucose, AMP and CM. The same amount of tetracycline was added to LB medium with 0.8% glucose, AMP and CM

    2. For the final M63B1 medium (0.5 L) with MgSO4\*7H2O, AMP and CM and 0.4% or 0.8% glucose, TET (1mL, 0.5 mg/mL) was added.

    Preparation of 96-wells plate for 24h biofilm assay:

    1. Overnight cultures were diluted with or without TET (2 μM) containing media (LB or M63B1) until a target OD600 = 0.1.

    2. Each diluted culture (100 μL) were incubated on a 96-wells plate for 20 h at 37℃.

    Absorbance measurement of CV-assay:

    1. The supernatant was discarded from a 24h incubation of cell cultures on a 96-wells plate (prepared from 08. August. 2018) and rinsed with PS (100μL).

    2. The biofilm was fixed by adding methanol (100 μL, 99%) and waited for 15min. The supernatant was discarded, and the plate was airdried.

    3. CV-solution (100 μL, 0.1%) was added to each well and coloured the biofilm for 20 min. The excess CV was washed away with dH2O
    4. Acetic acid (150 μL, 33%) was added to the each well.

    5. The absorbance was measured at Abs 590 nm
  + Aug

    10

    Preparation of overnight cultures for biofilm assay, and storage of TG1 cells with pgRNA and pdCas9

    **Goal :**

    1. Prepare overnight cultures in LB (0.4% or 0.8% glucose, with AMP and CM) or M63B1 (0.4% or 0.8% glucose, with AMP and CM) with or without TET.

    2. Wash the 96-well plate (after 20 h adhesion - prepared from 09.08.2018) with physiological saline, add new media and incubate the plate.

    3. Make bacteria glycerol stocks of TG1 cells with pgRNA and pdCas9.

    **Procedure :**

    Preparation of overnight culture:

    TG1 and dH5α were incubated in eppendorf tubes with different media (LB or M63B1 with glucose (0.4% or 0.8%), AMP and CM) with or without TET (2 μM) for overnight at 37℃ with shaking.

    Preparation for biofilm assay:

    1. After 20 h adhesion (plate prepared from 09. August. 2018), the non-adhered cells were removed by washing the wells with physiological saline (PS) and the supernatant was discarded.

    2. New medium (100 μL) was added to each well and the plate was incubated for 24 h at 37℃.

    Bacteria glycerol stock preparation:

    TG1 cells with pgRNA and pdCas9 (500 μL) was stored with glycerol (500 μL, 50%) at -80℃.
  + Aug

    11

    Preparation of overnight cultures for biofilm assay

    **Goal :**

    Prepare 96-well for cell adhesion and CV-assay

    **Procedure :**

    The OD of some of the overnight cultures was not optimal for continuing the procedure of CV assay. New cell cultures were made.

    96-well plates for 24 h, 48 h and 72 h incubation were made. However, since the cell cultures could not be diluted to OD = 0.1, the results of the CV assay will not be comparable.
  + Aug

    12

    Incubation of cell cultures for biofilm assay

    **Goal :**

    Preparation for cell adhesion on 96-well plates for 24 h, 48 h and 72 h incubation.

    **Procedure :**

    1. The overnight cultures were resuspended, before dilution until a target OD = 0.1.

    2. The diluted cell cultures were resuspended and added on 96-well plates and incubated for 4 h at 37℃.

    3. After 4 h incubation, the wells were washed with physiological saline (100 μL) and the supernatant was discarded.

    4. New media (100 μL) were added to the plates, and the plates were incubated at 37℃.

    **Results :**
* Week

  33

  Week 33

  + Aug

    13

    Biofilm measurements after 24hrs

    **Procedure:**
    Crystal violet assay

    Changed medium in the plate for 48 and 72 hrs.

    **Results:**

    Results did not show any clear trend. Excel sheet attached.

    Wells with only medium, not cells, showed too high absorbance measurements, probably because of contamination of the mediums. Decided to autoclave all the mediums again. Will take measurements at 48 hrs, but not at 72 hrs.
  + Aug

    14

    Biofilm measurement after 48 hrs

    **Goal:**

    1. Measure biofilm after 48 hrs.
    2. Prepare RRVT-Biobrick and biobrick backbone
    3. Ligate and transform RRvT- biobrick and biobrick backbone into DH5alpha cells.**Procedure:**

    1. Performed the Crystal Violet Assay procedure for the plate designated for 48hrs and for the plate that initially was designated for 72 hrs measurement.
  + Aug

    15

    Biobrick ligation and transformation

    **Goal:**

    Biobrick ligation and transformation

    1. Ligate plasmid backbone and RRvT-plasmid and transform into cells.
    2. Due to the lack of colonies from yesterdays transformation the ligation of plasmid backbone and RRvT-plasmid and transformation into E.Coli Dh5α cells was redone.**Procedure:**

    Preparations of plasmid backbone and RRvT-biobrick:

    Made enzyme master mix for plasmid backbone by adding 5μL NEB buffer 2, 0.5 μL EcoRI-HF, 0.5 μL PstI, 0.5 μL BSA and 18.5 μL dH2O to an Eppendorf tube.

    Made enzyme master mix for RRvT-biobrick by adding 5μL NEB buffer 2, 0.5μL EcoRI-HF, 0.5 μL PstI and 19 μL dH2O to an Eppendorf tube. Incubated mix for 20 min at 50℃.

    Digested Plasmid backbone by adding 4 μL linearized backbone (25 ng/μL for 100 ng) and 4 μL of enzyme master mix for plasmid backbone.

    Digested RRvT-biobrick by adding 4 μL of the biobrick and 4 μL of the enzyme master mix for RRvT-biobrick.
    Digested both reactions at 37℃ for 90 min and did heat kill at 80℃ for 20 min.
    Ligation:

    Added 2 μL of digested plasmid backbone.

    Added 2 μL of digested RRvT-biobrick.

    Added 1 μL T4 DNA ligase buffer.

    Added 0.5 μL of T4 DNA ligase.

    Added water to 10 μL.

    Ligated at 16℃ for30min. Did heat kill at 80℃ for 20 min.

    Transformed  *E.Coli*  Dh5α cells with 1-2 μL of product following the protocol for transformation.

    Inoculated .. plates and let them incubate at 37℃ to the next day.+ Aug

      16

      Preparation for biobrick (RRvT) measurement and biofilm assay

      **Goal:**

      1. Inoculate successful transformed cells into LB medium with CM.
      2. Revive TG1 and DH5α cells in LB medium with AMP, CM and 0% glucose. **Procedure:**

      Preparation for biobrick (RRvT) measurement:

      Successful transformed cell colonies from yesterday's incubation were inoculated into LB - medium with CM and incubated at 37°C with shaking.

      Preparation for cell cultures for biofilm assay:

      TG1 and DH5α cells with pgRNA and pdCas9 were revived in LB medium with AMP, CM and 0% glucose. The cell cultures were incubated at 37°C with shaking.
    + Aug

      17

      Preparation for biofilm assay

      **Goal:**

      - Dilute the overnight cultures for biofilm assay.
      - Do Crystal Violet assay after 3 h, 5 h and 8 h of incubation of a 96-well plate.
       **Procedure:**

      The media for biofilm assay were made by adding the following ingredients:

      - LB 0.4% glucose, TET: 10 mL LB, 10µL AMP, 10µL CM, 0.2 mL 20% Glucose, 19.7µL 0.5mg/mL TET
      - LB 0.8% glucose, TET: 10 mL LB, 10µL AMP, 10µL CM, 0.4 mL 20% Glucose, 20µL 0.5mg/mL TET
      - LB 0.4% glucose: 10 mL LB, 10µL AMP, 10µL CM, 0.2 mL 20% Glucose
      - LB 0.8% glucose: 10 mL LB, 10µL AMP, 10µL CM, 0.4 mL 20% Glucose
      - M63B1 0.4% glucose, TET: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.2 mL 20% Glucose, 19.7 µL TET
      - M63B1 0.8% glucose, TET: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.4 mL 20% Glucose, 20 µL TET
      - M63B1 0.4% glucose: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.2 mL 20% Glucose
      - M63B1 0.8% glucose: 10 mL M63B1, 10µL AMP, 10µL CM, 10 µL 1M MgSO4\*7H2O, 0.4 mL 20% Glucose

      Biofilm assay:

      - Pipette 3-4 mL overnight cultures to sterile tubes (total 16 tubes).
      - Centrifuge the cultures for 10 min at 2000 rpm at 9°C.
      - Remove the supernatant.
      - Add 3-4 mL media (LB/ M63B1, with 0.4%/ 0.8% glucose, with/ without TET) to 2 tubes each.
      - Resuspend/ vortex the cultures, and measure OD 600.
      - Dilute the cultures until target OD 600 = 0.1 and store the cells on ice until they are added to the wells.
      - Resuspend/ vortex the cultures, add 100 µL culture to each well on the plate.
      - Seal the top of the plate with parafilm, and incubate the plate at 37°C.
      - Do CV assay after 3 h, 5 h, 8 h, 24 h and 48 h incubation.

* Week

  34

  Week 34

  + Aug

    20

    Preparation for 3h, 5h, 8h, 24h, 30h CV and Bactiter-Glo Assay.

    **Goal:**

    Redo CV assay from 17th August to check the validity of previous results.

    Perform Bactiter-Glo Assay parallell to CV Assay (3h, 5h, 8h, 24h, 30h) to examine the amount of living cells in biofilm.

    **Procedure:**

    Crystal Violet Assay: Same procedure as from 17.08.2018.

    Bactiter-Glo Assay
  + Aug

    21

    No lab

    No lab.
  + Aug

    22

    No lab

    No lab.
  + Aug

    23

    No lab

    No lab.
  + Aug

    24

    No lab

    No lab.
* Week

  35

  Week 35

  + Aug

    27

    No lab

    No lab.
  + Aug

    28

    No lab

    No lab.
  + Aug

    29

    No lab

    No lab.
  + Aug

    30

    No lab

    No lab.
  + Aug

    31

    No lab

    No lab.
* Week

  36

  Week 36

  + Sept

    3

    No lab

    No lab.
  + Sept

    4

    No lab

    No lab.
  + Sept

    5

    Introduction of site directed mutagenesis on pdCas9 and make medium for V. natrigens

    **Goal:**

    Make LB medium and 10x v2 salts for Vibrio natrigens

    Amplify and introduce site directed mutagenesis in pdCas9

    **Procedure:**

    LB - medium

    10x v2 salts:

    Make the salt solutions after the following recipe:
    - 11.935 g NaCl (204 mM)
    - 0.3134 g KCl (4.2 mM)
    - 4.710 g MgCl2\*6H2O (23.14 mM)
    - 1 L ddH2O

    Site directed mutagenesis:

    Forward and reverse primers for site directed mutagenesis were designed and ordered from Sigma Aldrich.
    Introduce site directed mutagenesis in pgCas9 via PCR after following the protocol from 29. June. 2018.
    The first PCR was not successful. The primer annealing temperature was changed, and the elongation time as well.
  + Sept

    6

    Introduce site directed mutagenesis in pdCas9 and amplify RRvT and pSB1C3e

    **Goal:**
    Introduce site directed mutagenesis in pdCas9
    Amplify RRvT and pSB1C3 via PCR

    **Procedure:**
    Site directed mutagenesis in dCas9:
    pdCas9 was amplified via gradient PCR, where the annealing temperatures were 57.3 ℃, 59.3 ℃, 61.1 ℃ and 64.1℃ with an elongation time for 5 min.
    The samples were stored in the fridge after the PCR was finished.
    Amplification of RRvT and pSB1C3:
    RRvT and pSB1C3 fragments were amplified by running the samples (6 µL FW primer, 6 µL REV primer, 1 µL template, 12.5 µL Takara Hi-Fi PCR premix) on PCR with the following settings: annealing temperature 37 ℃ for 10 s, elongation temperature 72 ℃ for 1 min.
    The samples were stored in the fridge after the PCR was finished.
  + Sept

    7

    Gel purification of PCR products (pdCas9, RRvT and pSB1C3), incubate V. natriegens

    **Goal:**

    Separate the PCR products of RRvT and pSB1C3 on gel, and miniprep the desired fragments on the gel.
    Incubate Vibrio natreigens (V. natriegens).
    Do another round of site directed mutagenesis on pdCas9 via PCR.

    **Procedure:**
    **Gel purification of RRvT and pSB1C3 on gel:**

    5 μL loading dye was added to the PCR products (25.5 μL).
    The PCR products were separated on Gel green (90 V, 40 min).
    The desired fragments were cut out from the gel and collected in eppendorf tubes
    Gel digestion and DNA isolation of RRvT and fragment of pSB1C3:
    The PCR products with the desired length on the gel was isolated by following after the same protocol from 02.07.2018.

    **Inoculation and incubation of V. natriegens:**

    Sample of V. natriegens sent by the iGEM team from Marburg, was inoculated and incubated by following after the protocol provided by the team.

    **Introduce site directed mutagenesis in pdCas9:**

    PCR mix with pdCas9 was prepared after following the same protocol from 29.06.2018.
    Two samples were set on two different programs:
    3 step PCR: DNA denaturation temperature at 98℃, primer annealing temperature at 55 ℃ for 10 s, elongation temperature at 72 ℃ for 7 min.
    2 step PCR: same conditions as 3 step PCR without the primer annealing step.
  + Sept

    8

    Purification of RRvT, separation of PCR product of pdCas9 and measurement of growth curve of V. natriegens (failed)

    **Goal:**
    - Measure the growth curve of V. natrigens
    - Gel purification of RRvT
    - Separte the PCR products of pdCas9 on gel**Procedure:**

    Growth curve of V. natrigens:

    The experiment failed due to dilution in the flasks and not in the cuvettes

    Gel purification of RRvT:

    The PCR product of RRvT was purified on gel, and the DNA was isolated after following the same protocol from 02.07.2018.

    Gel separation of pdCas9 PCR product:

    The PCR products of pdCas9 were separated on both gel red and gel green for imaging and for isolation of the desired fragments by following the same protocol from 02.07.2018.

    See results page for results

    .
  + Sept

    9

    Amplification of RRvT and introduce SDM in pdCas9

    **Goal:**

    Amplify RRvT and introduce SDM in pdCas9 via PCR

    **Procedure:**

    RRvT and introduction of pdCas9 was necessary since the concentration of the products isolated from gel on 08.09.2018 were too low.

    Amplification of RRvT:

    RRvT was amplified via PCR with an annealing temperature 55 ℃ for 10 s and elongation temperature at 72 ℃ for 1 min.

    Introduce SDM in pdCas9:

    Site directed mutagenesis was introduced via PCR with an annealing temperature at 55 ℃ for 10 s and elongation temperature 72 ℃ for 7 min.

    The products were stored in the fridge.
* Week

  37

  Week 37

  + Sept

    10

    Gibson assembly of RRvT and backbon and transformation of SDM pdCas9 and assembled RRvT into competent cells

    **Goal:**

    Gel purification of PCR products
    Gel digestion and isolation of desired DNA fragments
    Assemble RRvT and pSB1C3 via Gibson assembly
    Transform pdCas9 and the assembled RRvT into competent cells

    **Procedure:**

    Gel purification:
    The PCR products were separated on gel green
    RRvT: 25.5 µL DNA, 5.05 µL loading dye
    pdCas9: 25 µL DNA, 5 µL loading dye

    Gel digestion and DNA isolation:

    The desired fragments (RRvT: approx. 2000 bp, pdCas9: approx. 6000 – 7000 bp) were cut out from the gel. The gel was digested by following after the protocol from 02.07.18.

    Transformation of SDM pdCas9 into competent cells:

    6 µL DNA was transformed into competent cells by following after the protocol from 25.06.18.

    Gibson assembly of RRvT and pSB1C3:

    The fragments were assembled together by following the protocol provided from the kit
    2 µL RRvT, 8 µL pSB1C3 (0.03 – 0.2 pmol fragments), 10 µL assembly master mix was incubated in a thermocycler at 50C for 15 min. The sample was stored on ice for transformation.

    Transformation of assembled RRvT and pSB1C3 into competent cells:

    2 µL Gibson assembly product was transformed into competent cells by following after the protocol from 25.06.2018.
  + Sept

    11

    PCR colony screening, isolation and verify successful SDM on pdCas9

    **Goal:**

    - PCR colony screening of RRvT gene
    - Isolate pdCas9 from cell cultures
    - Verify successful SDM on pdCas9 by restriction digest the plasmid

    **Procedure:**

    PCR colony screening:

    None of the colonies were red/ pink coloured like the colonies with RFP.

    PCR colony screening was done to test whether assembly of RRvT and pSB1C3 fragments was successful

    PCR mix was prepared after the following protocol:
    5 µL Takara Hi-Fi PCR premix, 1 µL ddH2O, 6 µL RRvT FWD primer, 6 µL RRvT REV primer
    8 different colonies were chosen and transferred to a new agar plate with CM, before the colony was transferred to a PCR tube with PCR mix.
    The samples ran on a PCR machine with the same settings from day 28 – 06. 09. 2018.

    Plasmid isolation:

    pdCas9 with SDM was isolated from three cell replicates by following the protocol from day 3 – 22. 06. 2018.

    Restriction digest:

    Introduction of SDM in pdCas9 was verified by adding restriction enzyme which originally recognize and cut at a specific site in the plasmid. EcoRI has two restriction sites in pdCas9 before any mutations.

    Mix 2 µL DNA (pdCas9 SDM), 2 µL Cut smart buffer, 15.5 µL ddH2O, 0.5 µL EcoRI Hi-Fi
    Spin down the solutions
    Incubate overnight at 37 ℃
  + Sept

    12

    Gel separation of digested pdCas9 (SDM) and products from PCR colony screening of RRvT, amplification of pdCas9 fragments for Gibson assembly

    **Goal:**

    - Separate the DNA fragments of pdCas9 SDM after restriction digest
    - Visualize the products from PCR colony screening on gel
    - Amplify the different fragments of pdCas9 for Gibson assembly
    - Inoculate colonies with RRvT in LB medium with CM

    **Procedure:**

    Gel electrophoresis:

    Both pdCas9 fragments obtained from restriction digest, and the products from PCR colony screening of RRvT were separated and visualized on gel by mixing the DNA with loading dye

    RRvT: 3 µL DNA, 2 µL dye, 7 µL ddH2O

    pdCas9: add 4 µL loading dye to each samples

    The DNA fragments were separated on gel red.

    Preparation for Gibson assembly of pdCas9:

    Segments for Gibson assembly were obtained by amplify the desired fragments with designed primer pairs via PCR.

    Primers for Gibson assembly (ordered from Sigma Aldrich), were resuspended with ddH2O and diluted to 1 µ
    Dilute pdCas9 SDM – 2 µL DNA (PC1), 8 µL ddH2O (to obtain <26 ng/µL)
    PCR samples (1 µL diluted template (pdCas9 SDM, <26 ng/μL), 6 µL FWD primer, 6 µL REV primer, 12.5 µL Takara HI-FI PCR premix) were incubated in PCR machine with the same settings from day 29 – 07. 09. 2018.

    Inoculate cell colonies with RRvT:

    Colonies with RRvT was inoculated in LB medium with CM from the agar plate, and the cell cultures were incubated overnight at 37 ℃ with shaking.

    Cells were inoculated from the following agar plate.
    Cells with RFP were revived in LB medium with CM.
  + Sept

    13

    Gel purification, isolation and Gibson assemble the PCR products of pdCas9 fragments, transformation of Gibson assembled product, fluorescence test round of RRvT

    **Goal:**

    - Gel purification of the PCR product of pdCas9 fragments
    - Gel digestion and isolation of the pdCas9 fragments
    - Gibson assemble the pdCas9 fragments
    - Transformation of Gibson assembled product into competent cells
    - Test round of RRvT by measuring the fluorescence

    **Procedure:**

    Gel purification of pdCas9 fragments:

    25.5 µL PCR products were mixed with 5.05 µL loading dye, and separated on gel by following after the same procedure from 22.06.2018.

    Gel digest and DNA isolation:

    After gel electrophoresis, the desired pdCas9 fragments were cut out from the gel. The gel was digested and the DNA fragments were isolated by conducting the same protocol from 02.07.2018.

    Gibson assembly of the pdCas9 fragments:

    The pdCas9 fragments, isolated from the gel, were assembled after the following protocol

    Mix 0.60 µL G1 fragment, 3.49 µL G2 fragment, 3.67 µL G3 fragment, 2.24 µL G4 fragment (the volumes were calculated by our instructor) with 10 µL NEBuilder HiFi DNA Assembly Master mix, and the sample was incubated at 50 ℃ for 1 hour.

    The thermocycler was switched off without notice, so the samples stayed at room temperature for an hour before the thermocycler was turned on again…

    Transformation of Gibson assembly product:

    2 µL Gibson assembly product was transformed into DH5α cells by following after the procedure from 25. 06. 2018

    Measure the fluorescence of RRvT:

    The overnight cultures with RRvT, RFP and a negative control was diluted until target absorbance Abs600 = 0.1
    Four replicates of each culture (100 µL) were added to a 96 well microtiter plate.
    The Absorbance and the fluorescence were measured with the following settings:
    150 cycles with 5 min interval
    Emission wavelength: 607 nm
    Exitation wavelength: 584 nm
    Temperature: 37 ℃
    Gain: 22

    **Results:**

    Gel purification of pdCas9 fragments for Gibson assembly:

    The order of the different fragments from left to right: ladder, G1, G2, G3 and G4. The fragments for Gibson assembly should have the length of 641 bp, 481 bp, 2917 bp and 1468 bp, respectively.

    Fluorescence of RRvT:

    The values of the fluorescence were too low when the samples were measured with a gain of 22. The liquid in the wells has been evaporated during the measurement.
  + Sept

    14

    Inoculate successful transformed cells, Restreak V. natriegens, amplify gRNA and dCas9 for insertion into LITMUS28i vector

    **Goal:**

    - Inoculate successful transformed cells
    - Restreak V. natriegens on agar plates
    - Amplify gRNA and dCas9 (without mutation) sequence for insertion into LITMUS28i vector

    **Procedure:**

    Colony picking:

    Successful transformed cells (probably with correct Gibson Assembly product of pdCas9) were inoculated to LB medium with CM. The cell cultures were incubated at 37 ℃ overnight with shaking.

    Re-streak Vibrio natriegens on agar plates:

    Vibrio natriegens was inoculated and was stoke on LA with v2 salt aseptically. The agar plates were incubated at room temperature.

    Amplification of gRNA and dCas9:

    The primer pairs of gRNA and dCas9 (ordered from Sigma Aldrich) were diluted with ddH2O, so the concentration of the primers was 100 µM
    The primers were further diluted until target concentration = 1 µM
    PCR mix were prepared after the following recipe:
    pgRNA (0.5 µL, 37.2 ng/ µL), 6 µL FWD primer, 6 µL REV primer, 12.5 µL Takara HiFi PCR premix.
    pdCas9 (0.5 µL, 53.4 ng/ µL), 6 µL FWD primer, 6 µL REV primer, 12.5 µL Takara HiFi PCR premix.
    The gRNA sequence was amplified by running the sample with a PCR machine with an annealing temperature 55 ℃ for 10 s, and an elongation temperature 72 ℃ for 1 min. The dCas9 sequence was amplified with an annealing temperature 55 ℃ for 10 s, and an elongation temperature 72 ℃ for 7 min.
  + Sept

    15

    Isolation, digestion of Gibson assembled pdCas9, gel digestion and isolation of gRNA and dCas9 fragments (for insertion into LITMUS28i), inoculation of V. natriegens

    **Goal:**

    - Isolate Gibson assembled pdCas9 from overnight cultures
    - Test digest the Gibson assembled pdCas9
    - Make glycerol stock of cells with Gibson assembled pdCas9 (pdCas9 mutated)
    - Gel separate the PCR products of gRNA and dCas9 (for insertion into LITMUS28i vector)
    - Gel digest and isolate the gRNA and dCas9 fragments (for insertion into LITMUS28i vector)
    - Inoculate natriegens in LB medium with v2 salts

    **Procedure:**

    Isolation of Gibson assembled pdCas9:

    Overnight cultures were diluted until OD600 ≈9
    pdCas9 was isolated from the cells by following after the miniprep protocol from 22.06.2018.

    Test digest Gibson assembled pdCas9:

    The enzymes EcoRI and XbaI were selected to verify whether the Gibson assembly of pdCas9 was successful or not. For 0.5 µL enzyme, 250 ng DNA is required. The different replicates were prepared by following recipe:

    PC1: 5 µL DNA, 2 µL Buffer, 0.5 µL EcoRI, 0.5 µL XbaI, 12 µL ddH2O

    PC2: 7 µL DNA, 2 µL Buffer, 0.5 µL EcoRI, 0.5 µL XbaI, 10 µL ddH2O

    PC3: 12 µL DNA, 2 µL Buffer, 0.5 µL EcoRI, 0.5 µL XbaI, 5 µL ddH2O

    The samples were incubated at 37 ℃ for 3h, before gel separation.

    Glycerol stock of Gibson assembled pdCas9 (pdCas9 mutated):

    DH5α cells with the Gibson assembled pdCas9 were stored in glycerol stock at -80 ℃ by following after the procedure from day 28.06.2018.

    Gel separation of gRNA and dCas9 (not mutated) fragments (for insertion into LITMUS28i vector):

    5 µL loading dye was added to each sample. The samples ran on gel green at 90 V for 50 min.

    Gel digest and DNA isolation:

    The gRNA and dCas9 fragments were isolated from the gel by conducting the same protocol from 02.07.2018.

    Incoulation of V. natriegens:

    Coloies of V. natriegens were inoculated into LB medium with v2 salt. The cell cultures were incubated at 37 ℃ overnight with shaking.
  + Sept

    16

    Transformation of plasmid with TetR into competent cells, mak glycerol stocks of V. natriegens

    **Goal:**

    - Transform the part BBa\_R0040 (plasmid with TetR) into competent cells
    - Make glycerol stocks of natriegens

    **Procedure:**

    Transformation of BBa\_R004:

    The plasmid was resuspended with 10 µL ddH2O in the well of the plate (provided by iGEM).

    10 µL DNA was transformed into DH5α by following after the protocol from 25.06.2018.

    Glycerol stock of Vibrio natriegens:

    Overnight cultures of V. natriegens was stored in glyrecol stock at -80 ℃.
* Week

  38

  Week 38

  + Sept

    17

    Amplification of LITMUS28i vector, inoculation of successful transformed cells, revive cells with RRvT and RFP

    **Goal:**

    - Amplification of LITMUS28i sequence
    - Inoculate successful transformed cells with part BBa\_R0040 (plasmid with TetR)
    - Revive cells with RRvT and RFP

    **Procedure:**

    Amplification of LITMUS28i sequence:

    - Designed primers (bought from Sigma Aldrich) were resuspended and diluted to 1 μM.
    - The LITMUS28i vector (sponsored by NEB) was diluted to 25 ng/μ
    - The diluted primer pair (6 μL of each primer) for LITMUS28i was mixed with the diluted LITMUS28i vector (1 μL) and Takara HiFi PCR premix (12.5 μL).
    - The LITMUS 28i sequence was amplified after running in a PCR machine with an annealing temperature of 55 ℃ for 10 s and elongation temperature 72 ℃ for 2 min.

    Inoculation of successful transformed cells:

    Successful transformed cells with TetR was inoculated into LB medium with CM. The cell culture was incubated overnight at 37 ℃ with shaking.

    Revive cells with RRvT and RFP:

    Cells stored at -80 ℃ were revived by inoculation into LB medium with CM and incubated overnight at 37 ℃ with shaker.
  + Sept

    18

    Measure the fluorescence of RRvT, separation and isolation of LITMUS28i fragment, Gibson assemble LITMUS28i fragment, dCas9 and gRNA

    **Goal:**

    - Measure the fluorescence of RRvT, RFP and TetR
    - Separate LITMUS28i sequence on gel
    - Gel digest and isolate LITMUS28i segment
    - Gibson assemble LITMUS28i sequence, dCas9 and gRNA with anti-luxS

    **Procedure:**

    Fluorescence measurement:

    - The overnight cell cultures with RRvT, RFP and TetR were diluted to target Abs600 = 0,2.
    - 100 μL of each cell cultures were pipetted to a 96 well plate with 4 replicates. The cellcultres were surrounded with LB medium with CM to reduce evaporation of the cell cultures in the wells. The plate was covered with a lid and the plate was sealed with parafilm.
    - The absorbance and the fluorescence were measured with a five-minute interval for 150 cycles with a target temperature 37 ℃. The gain was set to 40, wave length of absorbance was set to 588 nm, while the excitation and the emission wavelength were set to 584 nm and 607 nm, respectively.

    Gel electrophoresis:

    05 μL loading dye was added to the PCR product of LITMUS28i vector (25 μL).
    The PCR product was separated on gel green, and the desired sequence was cut out from the gel.

    Gel digest and DNA isolation:

    The desired DNA sequence was isolated by digesting the gel by conducting the same procedure from 02.07.2018.

    Gibson assembly:

    LITMUS28i, dCas9 and gRNA with anti-luxS were assembled via Gibson assembly. The amount (pmol) required of the different fragments were calculated by the calculator provided by the homepage of NEB, and by following after the protocol of NEBuilder HiFi DNA Assembly

    LITMUS28i sequence (2295 bp, 68 ng/μL): 0.8 μL
    gRNA (538 bp, 43 ng/μL): 0.59 μL
    dCas9 (5025 bp, 52 ng/ μL): 4.92 μL
    NEBuilder HiFi DNA assembly master mix: 10 μL
    ddH2O: 3.69 μL

    The sample was incubated at 50 ℃ for an hour. The sample was stored in the fridge

    **Results:**

    Fluorescence measurement of RRvT:

    The fluorescence of RRvT and RFP was constant over time, and the value of the fluorescence of RRvT and RFP was lower than the fluorescence measured from the medium...
  + Sept

    19

    Measure growth curve of V. natriegens, transformation of Gibson assembled LITMUS28i, test digest mutated pdCas9

    **Goal:**

    - Measure the growth curve of V. natriegens
    - Transform Gibson assembled LITMUS28i (with unmutated dcas9 and gRNA with anti-luxS) into DH5α cells
    - Test digest the mutated pdCas9 with EcoRI and SpeI

    **Procedure:**

    Growth curve of V. natriegens:

    Overnight cultures were diluted to a target OD600 = 0.05 in 250 mL baffled flasks with a final volume 40 mL.
    The cell cultures were incubated at 37 ℃ with shaking.
    The OD600 of each replicate was measured after 10 min incubation for 5 hours.

    Transformation of Gibson assembled LITMUS28i vector:

    2 μL Gibson assembled LITMUS28i vector that consists unmutated dCas9 and gRNA with anti-luxS was transformed into competent cells after following the same procedure from 25.06.2018.

    Restriction digest the mutated pdCas9:

    The mutated pdCas9 was cut with EcoRI and SpeI by mixing 5 μL DNA (PC1), 2 μL cut smart buffer, 0.5 μL EcoRI, 0.5 μL SpeI and 12 μL dH2O. The reagent was incubated overnight at 37 ℃.
  + Sept

    20

    Gel separation of digested pdCas9 (mutated), preparation of ligation of mutated dCas9 and iGEM backcone

    **Goal:**

    Gel separation of the product from the enzyme digestion of mutated pdCas9

    Digestion of mutated pdCas9 and pSB1C3 (preparation for ligation of dCas9 and pSB1C3).

    **Procedure:**

    Gel electrophoresis:

    The products of enzyme digestion of mutated pdCas9 that has been incubated overnight, were mixed with 3.34 μL loading dye. The DNA segments were separated after running them on gel red at 90 V for 45 min.

    Digestion of mutated pdCas9 and pSB1C3:

    The DNA were digested after the following recipe

    pdCas9: 5 μL DNA (PC1, 50.8 ng/μL), 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 12 μL ddH2O.
    Linear pSB1C3: 10 μL DNA (linear pSB1C3, 25 ng/μL), 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 7 μL ddH2O.

    The samples were incubated at 37 ℃ overnight.
  + Sept

    21

    Gel separation and DNA isolation of digested pSB1C3 and mutated pdCas9, isolation of Gibson assembled LITMUS28i vector from overnight cultures

    **Goal:**

    - Gel separate the overnight product of enzymatic digested mutated pdCas9 and linear pSB1C3.
    - Gel digestion and isolate the desired DNA fragments of mutated and enzymatic digested pdCas9 and pSB1C3.
    - Isolate Gibson assembled LITMUS28i vector (with unmutated dCas9 and gRNA) from overnight cultures
    - Test digest the Gibson assembled LITMUS28i vector, and separate on gel

    **Procedure:**

    Gel separation of fragments of mutated pdCas9 and linear pSB1C3:

    3.34 μL loading dye was added to each sample, and the fragment were separated on gel green at 90 V for 45 min.

    The fragments with the desired length (dCas9: approx. 5000bp, backbone: approx. 2000bp) were cut out from the gel and collect in separate tubes.

    Gel digestion and DNA isolation:

    The DNA fragments of mutated pdCas9 and pSB1C3 were isolated from the gel by following after the protocol form 02.07.2018.

    Plasmid isolation of Gibson assembled LITMUS28i vector:

    The Gibson assembled LITMUS28i vector was isolated from two overnight cultures (L1 and L2) by following after the same protocol from 22.07.2018.

    Test digest the Gibson assembled LITMUS28i vector:

    The vectors were digested with two different enzyme pairs.

    The samples cut with AlwNI and EcoRI were incubated at 37 ℃ for 2 hours, while the samples cut with PstI and EcoRI were incubated for 1 hour.

    Gel separation of enzymatic digested L1 and L2:

    3.34 μL loading dye was added to each sample, the DNA fragments were visualized by running them on gel red at 90 V for 45 min.

    **Results:**

    Successfully transformed cells with LITMUS28i (probably with dCas9 and gRNA with anti-luxS)
    The concentrations of the fragments of mutated pdCas9 and pSB1C3 were too low (negative values)…
    L2 may be the correct Gibson assembled phagemid. However, there is an extra segment with a length approx. 6000 bp. Probably, the enzymatic activity of AlwNI is slower compared to EcoRI and PstI, hence some fragments were not cut by AlwNI. In order to verify this, another sample of L2 was cut with AlwNI and EcoRI and incubated overnight at 37℃.
  + Sept

    22

    Gel separation of digested mutated pdCas9, pSB1C3 and Gibson assembled LITMUS28i, DNA isolateion of digested pdCas9 and pSB1C3

    **Goal:**

    - Gel separation of fragments from enzymatic digested mutated pdCas9, linear pSB1C3 and Gibson assembled LITMUS28i vector.
    - Gel digestion and isolation of the desired DNA fragments of pdCas9 and linear pSB1C3.
    - Incubate new samples of mutated pdCas9 and linear pSB1C3 were incubate overnight with restriction enzymes.
    - Revive competent cells with mutated pdCas9 (PC1)

    **Procedure:**

    Gel separation:

    Fragments of mutated pdCas9 and pSB1C3 (digested with EcoRI and PstI overnight) were separated on gel green. The desired segments were cut out and isolated from the gel by following after the same procedures from 02.07.2018.

    Fragments of L2 (incubated from yesterday – 21.09.2018) were digested with AlwNI and EcoRI, separated and visualized on gel red.

    Revive competent cells with mutated pdCas9:

    Cells with PC1 stored at -80 ℃ were inoculated in LB medium with CM and incubated overnight at 37 ℃ with shaking.

    We were running out of plasmids with mutated pdCas9, so it’s necessary to isolate the them from the cells again…

    Restriction digest mutated pdCas9 (PC2) and linear pSB1C3:

    PC2: 10 μL DNA, 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 7 μL ddH2O

    pSB1C3: 10 μL DNA, 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 7 μL ddH2O

    The samples were incubated overnight at 37 ℃

    **Results:**

    The concentrations of the desired fragments of pdCas9 and pSB1C3 were too low to be detected. New samples of mutated pdCas9 and pSB1C3 were incubated with EcoRI and PstI overnight

    .
  + Sept

    23

    Ligation of mutated pdCas9 and pSB1C3, plasmid isolation, digestion of mutated pdCas9 and pSB1C3

    **Goal:**

    - Ligation of enzymatic digested pSB1C3 and mutated pdCas9
    - Plasmid isolation from overnight cultures
    - Digest the mutated pdCas9 and pSB1C3 with restriction enzymes

    **Procedure:**

    Ligation of DNA fragments:

    According to the protocol for ligation of DNA fragments, the amount of the fragments was calculated by using the NEBiocalculator.

    We assume that all of the DNA is completely cut by the enzymes, so we have 400 ng dCas9 and 250 ng digested backbone

    The concentration of PC2 is 40 ng/μL and 10 μL pdCas9 was cut with enzymes, while the concentration of linear pSB1C3 was 25 ng/ μL and 10 μL was digested.

    If we would like to have 25 ng backbone in the reaction solution, we should have approx. 181.3 ng dCas9 according to NEBiocalculator with the ratio of 3:1 for insert and backbone.

    The theoretical concentration of the digested pdCas9 is 20 ng/ μL (total 400 ng plasmid was cut, and the final volume in the restriction digest tube was 20 μL), and 12.5 ng/ μL for the digested pSB1C3 (total 250 ng linear pSB1C3 was cut, and the final volume in the restriction digest tube was 20 μL).

    Thus, the volume of the different compounds for ligation is

    - 2 μL T4 DNA ligase buffer
    - 1 μL T4 DNA ligase
    - 2 μL Backbone
    - 10 μL dCas9
    - 5 μL ddH2O
    - Total volume: 20 μL

    The sample was incubated at 16 ℃ overnight

    P.S. The fragments were not separated on gel before ligation (after so many times of failure to isolate DNA from gel…), wrong insert or re-ligation of pdCas9 fragments may occur…

    Plasmid isolation from competent cells:

    The plasmid with mutated pdCas9 was isolated from two cell cultures by following the same procedure from 02.07.2018.

    Enzymatic digestion of mutated psCas9 and linear pSB1C3:

    PCI: 17 μL DNA, 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI

    Dried linearized pSB1C3 was resuspended with 10 μL ddH2O to obtain the final concentration 100 ng/ μL

    pSB1C3: 9 μL DNA (100 ng/ μL), 2 μL 2.1 buffer, 0.5 μL EcoRI, 0.5 μL PstI, 8 μL ddH2O

    The samples were incubated overnight at 37 ℃.
* Week

  39

  Week 39

  + Sept

    24

    Gel separation and DNA isolation of digested pSB1C3 and mutated dCas9, transformation of ligation product, amplification of mutated pdCas9 and pSB1C3

    **Goal:**

    - Separation of DNA fragments on gel
    - Gel digestion and DNA isolation of dCas9 and pSB1C3
    - Transformation of ligation product
    - Amplify mutated dCas9 and linear pSB1C3

    **Procedure:**

    Gel electrophoresis:

    Enzymatically digested pdCas9 and pSB1C3 (prepared from yesterday - 23.09.2018) were separated on gel green at 90 V for 45 min.

    Gel digestion and DNA isolation:

    The desired fragments of dCas9 and backbone were cut out from the gel. The DNA fragments were further isolated by following after the same procedure fromn02.07.2018.

    Transformation of ligation product into competent cells:

    Heat inactivate the ligase at 65 ℃ for 20 min
    Transform 5 μL of ligation product into competent cells by following the protocol from 25.06.2018
    P.S. The fragments were not separated on gel before ligation (after so many times of failure to isolate DNA from gel…), wrong insert or re-ligation of pdCas9 fragments may occur… so inoculate more colonies and store one of the plates sealed with parafilm in the fridge.

    Amplify mutated pdCas9 and pSB1C3:

    PCR mix for amplification of pdCas9 and pSB1C3 were prepared by mixing:

    pdCas9: 0.5 μL template (PCII, 65.8 ng/ μL), 6 μL FWD GA1 primer, 6 μL REV GA3 primer, 12.5 μL Takara HiFi PCR premix.

    This sample was amplified in a PCR machine with an annealing temperature at 55 ℃ for 10 s, and an elongation temperature at 72 ℃ for 7 min.

    pSB1C3: 1 μL template (linear pSB1C3, 25 ng/ μL), 6 μL FWD primer, 6 μL REV primer, 12.5 μL Takara HiFi PCR premix.

    The sample of pSB1C3 ran in a PCR machine with an annealing temperature at 55 ℃ for 10s, and an elongation temperature 72 ℃ for 2 min.

    Nanodrop - mutated dCas9 and backbone:

    The concentrations of enzymatically digested pdCas9 and pSB1C3 were too low to be detected (negative values).

    Therefore, the mutated pdCas9 and pSB1C3 were amplified via PCR to increase the amount. Further we plan on cutting the PCR product with restriction enzymes over two nights, before separation on gel and isolate the desired fragments. This may increase the concentration of the products after gel digestion. Probably, heat inactivation of the restriction enzymes after gel digestion and DNA isolation may be a good idea (most of the enzymes are separated from the DNA fragments on gel), before the ligation step.
  + Sept

    25

    Restriction digest of PCR-amplified mutdCas9 and pSB1C3; Media and agarose gel preparation; Inoculation

    **Goal:**

    - Restriction digest of PCR-amplified mutated dCas9 and pSB1C3
    - Make LB and M63B1 medium
    - Make 0.8% agarose GelRed and GelGreen
    - Inoculation of LB medium with transformed bacteria

    **Procedure:**

    Restriction digest of mutated dCas9 and pSB1C3:

    PCR-amplified dCas9 and pSB1C3 were digested in separate tubes. The restriction mixtures were made by combining:

    - 17 µL PCR-amplification product (mutated dCas9 or pSB1C3)
    - 2 µL NEB 2.1 buffer
    - 0.5 µL EcoRI-HF
    - 0.5 µL PstI
    - The restriction mixtures were incubated at 37 °C for two days.

    Media preparation

    Both, LB and M63B1 medium were prepared as described previously (\_\_\_\_). The pH was adjusted to 7.1-7.2 by adding NaOH. The media were autoclaved and stored for later use.

    Agarose gel preparation

    For preparation of 200 mL of both GelGreen and GelRed 0.8% agarose gel, 1.6 g agarose was added to 200 mL 1xTAE. The solution was heated in a microwave until the agarose was completely dissolved (approximately 3 min at maximum heat). The gel was cooled down before 10 µL of dye (GelGreen or GelRed) was added.

    Colony picking and inoculation

    Four colonies from agar plates with bacteria transformed with possibly ligated dCas9 and pSB1C3 were picked and inoculated in 25 mL LB medium containing 25 µL chloramphenicol. The cultures were incubated at 37 °C with shaking (225 rpm) until the next day.
  + Sept

    26

    Miniprep and nanodrop of yesterday's cultures

    **Goal:**

    Miniprep yesterday’s bacterial cultures

    Measure quantity and quality of miniprep product (Nanodrop)

    **Procedure:**

    The OD600 value of the bacterial cultures from yesterday were measured. The cultures were diluted to a final OD600 value of about 1, and miniprepped following the ZR Plasmid Miniprep protocol. The isolated plasmids were nanodropped and stored at 4 °C till next day.
  + Sept

    27

    \*\*Gel separation and purification of PCR-ampl. dCas9 and pSB1C3; Restriction digest of isolated plasmids from Sept. 25th-cultures

    **Goal:**

    - Gel separation and purification of digested PCR-amplified dCas9 and pSB1C3
    - Measure the quality and quantity of the purified product (Nanodrop)
    - Restriction digest of yesterday’s miniprep product

    **Procedure:**

    Gel separation and purification of digested PCR-amplified dCas9 and pSB1C3

    4 μL loading dye was added to the digested PCR-amplified dCas9 and pSB1C3 backbone and the total volume of the samples (24 μL) were run on a 0.8% agarose gel containing GelGreen. The bands at approximately 5000 bp and 2000 bp for dCas9 and pSB1C3, respectively, were excised from the gel. The plasmid fragments were further purified following the ZymocleanTM Gel DNA Recovery protocol, using 480 μL and 210 μL ADB buffer for dCas9 and pSB1C3 backbone, respectively. The concentration and quality of the products were measured using the Nanodrop.

    Restriction digest of plamids isolated from colonies

    Yesterday’s miniprepped plasmids from the bacterial cultures with potentially correctly ligated dCas9 into the pSB1C3 backbone were nanodropped and digested with EcoRI-HF and PstI at 37 °C for about 2 hours. The products was ran on a 0.8% agarose gel containing GelRed.

    - 7.5 μL dH2O
    - 0.5 μL EcoRI-HF
    - 0.5 μL PstI
    - 2.0 μL NEB 2.1 buffer
    - 10 μL miniprepped plasmids
  + Sept

    28

    Gel showed failed ligation of mut dCas9 and backbone, started new ligation process

    **Goal:**

    Analyze ligated mutdCas9 and backbone

    **Procedure:**

    Gel electrophoresis of enzyme digested mutated dCas9:

    Ran tube 1, 2, 3 and 4 on gel electrophoresis.

    Ligation of mutated dCas9 and iGEM-backbone:

    Started ligation process of mutated dCas9 and iGEM-backbone from samples collected in the freezer.

    How we did it:

    - Added (in order) 2uL T4 DNA ligase buffer, 3uL backbone, 9,5 uL mutated dCas9, 4,5 uL ddH2O and 1uL T4 DNA ligase (thawed in room temperature, no contact with hands)
    - mixed gently by pipetting up and down
    - microfuged tubes at 16^C, 10 rpm in one minute.
    - incubated at 16\*C over night

    Watered out DNA fragments from idt:

    - Both DNA fragments from idt were watered out with 40 uL ddH2O till a concentration of 25ng/uL. stored in fridge.

    Next step will be: PCR amplification->run gel and isolate -> nanodrop to find concentration -> Gibson assembly with primers (same primers as with RRvT biobrick)

    **Results:**

    Gel electrophoresis:

    Had only two bands on gel, which meant that the backbone and the mutated dCas9 was not ligated correctly.
  + Sept

    29

    Ligation of PCR amplified dCas9 and pSB1C3; Restriction digest of dCas9-fragments ordered from IDT

    **Goal:**

    Ligation of PCR-amplified dCas9 and pSB1C3

    Restriction digest and ligation of dCas9 ordered from IDT in two separate parts

    **Procedure:**

    Ligation of PCR-amplified dCas9 and pSB1C3

    The gel purified dCas9 and pSB1C3 backbone were heated at 80 °C for 20 min in order to heat kill any restriction enzymes still present in the samples. The ligation mixture was prepared as follows:

    - 2 μL T4 DNA ligase buffer
    - 1 μL T4 DNA ligase
    - 3 μL backbone
    - 9 μL dCas9 (Note! Not fully 9 μL was added)
    - 5 μL ddH2O
    - The fragments were incubated overnight at 16 ºC

    Restriction cutting and ligation of dCas9 ordered from IDT in two separate parts

    The two dCas9 fragments were restriction digested in separate tubes with BtgI. The recipe for the restriction mixtures is given below. Each fragment was handled similarly.

    - 20 μL dCas9 (diluted yesterday)
    - 1 μL BtgI
    - 5 μL cutsmart buffer
    - 24 μL dH2O

    The fragments were digested at 37 ºC for about 2 hours and the restriction enzymes were then heat killed at 80 ºC for 20 min. Finally, the separate dCas9 fragments were combined in ligation mixtures with varying concentrations of dCas9 and T4 DNA ligase buffer:

    - 2 μL | 2 μL | 2.5 μL T4 DNA ligase buffer
    - 1 μL T4 DNA ligase (same for all)
    - 5 μL | 10 μL | 15 μL dCas9 fragment 1
    - 5 μL | 10 μL | 15 μL dCas9 fragment 2

    The ligation reaction was run overnight at 16 ºC.
  + Sept

    30

    \*\*Transformation of PCR-amplified dCas9 and pSB1C3; Verification of ligated dCas9 fragments from IDT

    **Goal:**

    Transformation of PCR-amplified dCas9 and pSB1C3 into competent DH5α cells.

    Verification of ligated dCas9 fragments from IDT

    **Procedure:**

    Transformation of PCR-amplified dCas9 and pSB1C3 into competent DH5a cells

    Prior to the transformation, the T4 DNA ligase was heat killed at 80 ºC for 20 min. 5 μL of the ligation mixtures prepared September 28th and 29th was added to separate tubes of thawed competent DH5α cells. The cells were incubated on ice for 20 min, heat shocked at 42 ºC for 45 sek, 950 μL LB was added and the cells were further incubated for 1.5 hour at 37 ºC with shaking (225 rpm). 100 μL of the cell suspensions were added to agar plates containing chloramphenicol. The suspensions were then centrifuged at 6800 g for 4 min, and about 800 μL of supernatants were discharged. The cell pellets were resuspended in the remaining medium and plated.

    Verification of ligated dCas9 fragments

    The overnight ligation mixtures with the dCas9 fragments from IDT were heated at 80 ºC for 20 min. The total volume for each of the samples were run at 85 V for 45 min on an 0.8% agarose gel containing GelGreen. Fragments at 2000 bp and 3000 bp was excised from the gel and purified following the ZymocleanTM Gel DNA Recovery protocol. The recovered fragments were stored -20 ºC in case of further usage.
* Week

  40

  Week 40

  + Oct

    1

    Single colony picking and inoculation (Bacteria with potentially correctly ligated PCR-amplifed dCas9 and pSB1C3)

    **Goal:**

    Check colony formation on yesterday’s prepared agar plates

    If colonies, pick a few single colonies and make pure bacterial cultures

    **Procedure:**

    Colonies were visible on all 7 agar plates. 7 colonies were picked and inoculated in 25 mL LB with 25 μL chloramphenicol. The cultures were incubated overnight at 37 °C with shaking (225 rpm).
  + Oct

    2

    Failed ligation of dCas9 and pSB13

    **Goal:**

    Verify successful ligation of mutated dCas9 and iGEM-backbone by:

    - Isolating plasmids from overnight cultures
    - Digest the plasmids containing mutated pdCas9 ligated with pSB1C3.
    - Verify with gel electrophoresis

    **Procedure:**

    Yesterday 7 colonies were picked and incubated in culture medium overnight. Today, plasmids from each culture were isolated using the miniprep protocol.

    The concentrations in each parallel were found using Nanodrop.
    After the plasmids were isolated they were digested using the BamH1 and Pst1 restriction enzymes (we used 3.5uL plasmids). If we have the correct plasmids, then there will be two fragments, one at 6181bp and one at 893bp.

    Gel electrophoresis is performed using GelRed and standard protocol.

    The remaining plasmids from the plasmid isolation (not enzyme digested) are stored in the freezer in the phage-lab. They are in the brown case and are marked "dCas9 +bb 02.10.18".

    **Results:**

    Gel electrophoresis showed only one band at about 1000bp. Cannot explain why (only backbone should be at 2000bp)
  + Oct

    3

    No lab

    No lab
  + Oct

    4

    Transformation of biobricks; Making of bacterial cultures (mutdCas9, RRvT and mRFP1)

    **Goal:**

    Transform biobrick BBa\_K592009 (plate 1, well 19E) and BBa\_K592010 (plate 6, well 17G) into competent DH5α cells

    Make bacterial cultures of mutated dCas9 (with Addgene backbone), RRvT and mRFP1

    **Procedure:**

    Transformation

    10 μL ddH2O was used to resuspend the biobricks. For the transformation, 1.5 μL of the biobrick resuspensions were added to competent DH5α cells. The transformation was performed as described in our protocol section with slight modification: 1.5 μL of the biobrick resuspensions were added to competent DH5α cells, and 950 μL LB added prior to a 1.5 hour incubation time. The cells were plated on agar plates with chloramphenicol as the selective marker.

    Make bacterial cultures

    Bacterial cultures with mutated dCas9 (with Addgene backbone), RRvT and mRFP1 was made by inoculating previously transformed stored bacterial stocks in 25 mL LB medium containing 25 μL chloramphenicol. The cultures were incubated at 37 °C with shaking.
  + Oct

    5

    Miniprep and restriction cutting of biobrick blue+dcas

    **Goal:**

    - To get enough DNA for the backbone (blue) and dcas seperately to be ready for ligation.

    **Procedure:**

    1. 2 colonies of blue biobrick were picked and inoculated in LB+CM for 6h until OD-0.3. Checked the OD of dCas culture and diluted to approximately 1,4.

    2. Did a miniprep from the inoculated culture of blue biobrick(3 paralelles) + dcas(2 paralelles per culture)

    3. Test digest with restriction enzymes Pst1 (0.5 ul) and EcoR1-HF (0,5 ul), with NEBuffer 2.1 (2 ul) and water (see table) overnight at 37 degrees.
  + Oct

    6

    Gel electrophoresis and purification of digested mutdCas9 and biobricks; Repeated miniprep and digestion of biobricks

    **Goal:**

    - Gel elecrohoresis of yesterday’s digested mutated dCas9 and biobricks
    - If visible, gel purification of bands at appr. 2000 for the biobricks (= pSB1C3 backbone) and 5000 bp for dCas9
    - Measure quantity and quality of purified product (Nanodrop)

    **Procedure:**

    Gel electrophoresis, purification and nanodrop

    The restriction enzymes in each sample was heat killed at 80 °C for 20 min. 4 μL loading dye was added to the samples and the total volumes of each sample (24 μL) were run at 80 V for 45 min on a 0.8% agarose gel containing GelGreen. Since neither of the biobrick replicates (B1 and B2) gave any visible bands, only the 5000 bp bands for two of the mutated dCas9 replicates (1.1 and 1.2) was excised from the gel.

    The gel purification was conducted following the ZymocleanTM Gel DNA Recovery protocol, using 216 μL and 252 μL ADB buffer for dCas9 sample 1.1 and 1.2, respectively. The quantity and quality of purified product using Nanodrop.

    Addition to the planned goal: Miniprep of BBa\_K592009 and BBa\_K592010 transformed bacterial cultures

    Since the biobricks did not give any visible bands on the gel, bacterial cultures with cells transformed with BBa\_K592009 and BBa\_K592010 were miniprepped again. The cultures were diluted to a final OD600 value of about 1, and then two of the cultures containing BBa\_K592009 and one culture containing BBa\_K592010 was miniprepped following the ZR Plasmid MiniprepTM protocol. The quantity and quality of the miniprep products were measured using the Nanodrop. The isolated biobricks were then digested by combining:

    - 15 μL sample (appr. 1500 ng, for all three samples)
    - 0.5 μL EcoRI-HF
    - 0.5 μL PstI
    - 2 μL NEB 2.1 buffer
    - 2 μL ddH2O

    The restriction mixtures were incubated overnight at 37 °C with shaking (225 rpm).
  + Oct

    7

    Gel purification of digested biobricks; Ligation of mutdCas9 and digested biobrick backbone; Transformation of ligation mixture

    **Goal:**

    - Gel purification of overnight digested biobricks
    - Measure quantity and quality of the purified digested biobricks
    - Ligation of mutated dCas9 and digested biobricks
    - Transformation of ligation mixture into competent DH5α cells

    **Procedure:**

    Gel purification of overnight digested biobricks

    The overnight digested biobricks were heated at 80 °C for 20 min. Loading dye was added and the samples were run on a 0.8% agarose gel containing GelGreen. The bands at approximately 2000 bp was excised from the gel, and the fragments was recovered following the ZymocleanTM Gel DNA Recovery protocol. The quantity and quality of the purified digested biobricks were measured using the Nanodrop.

    Ligation of mutated dCas9 and digested biobricks

    The mutated dCas9 samples, which were gel purified yesterday, were ligated with the isolated pSB1C3 backbone of the biobricks. Two ligation mixtures was prepared using dCas9 1.1 and the backbone from BBa\_K592009 (sample B2) and dCas9 1.2 and the backbone from BBa\_K592010 (sample G2):

    - 2 μL T4 DNA ligase buffer
    - 5 μL T4 DNA ligase
    - 16 μL dCas9 1.1 (40.0 ng) | 16 μL dCas9 1.2 (33.6 ng)
    - 1 μL pSB1C3 from BBa\_ (40.1 ng) | 1 μL pSB1C3 from BBa\_ (32.5 ng)
    - 5 μL ddH2O
    - The ligation mixtures were incubated at 37 C for 2 hours.

    Transformation of ligation mixture

    10 μL of each ligation mixture was used in the transformation of competent DH5α cells. Also, 950 μL of LB was added prior to a 1.5 hours incubation period. Otherwise, the transformation was conducted as described in our protocol section.
* Week

  41

  Week 41

  + Oct

    8

    No lab

    No lab.
  + Oct

    9

    Miniprep and shipment preparation of our biobricks: mutdCas9-pSB1C3 and RRvT

    **Goal:**

    Dilute bacterial cultures with correctly inserted dCas9 into pSB1C3 and cultures containing RRvT

    Miniprep plasmids from bacterial cultures

    Measure quantify and quality of miniprep product (Nanodrop)

    Plate and air dry plasmids

    **Procedure:**

    The bacterial culture with correctly inserted dCas9 (verified yesterday by colony PCR) and a culture containg RRvT were diluted to an OD600 of about 0.2. The cultures were incubated at 37 °C with shaking (225 rpm) until the OD600 reached a value of approximately 1. The plasmids were isolated following the ZR Plasmid MiniprepTM protocol and the quantity and quality of the products were measured by the Nanodrop. The miniprepped products were then diluted to a final concentration of 50 ng/μL for each of the samples. 10 μL diluted mutated dCas9-pSB1C3 plasmid and 10 μL diluted RRvT-pSB1C3 plasmid was plated on the 96-well plate to be shipped to iGEM Headquarters. The samples were left to air dry in the fume hood until the next day.
  + Oct

    10

    No lab

    + Oct

      11

      Double transformation with pgRNA and new pdCas9

      **Goal:**

      1. Transformation of pgRNA and pdCas9 into DH5a and TG1-cells.

      **Procedure:**

      Both pdCas9(2) and pgRNA(2) with anti-luxS were simultaneously transformed into competent cells (DH5α) after the following protocol:

      - Thaw competent cells on ice.
      - Add pdCas9(2) (5μL) and pgRNA(2) with anti-luxS (5μL) to the same tube containing competent cells.
      - Incubate the cells on ice for 20min.
      - Heat shock the cells for 45s at 42℃.
      - Incubate the cells on ice for 2min.
      - Add LB- medium (900μL).
      - Incubate the cells for 1.5h (max. 2h) at 37℃ with shaker.
      - Plate the cells (100μL) on agar with AMP and CM
      - Centrifuge the cell culture for 3min at 6800g.
      - Discard the supernatant (200μL)
      - Resuspend the pellet with the remining supernatant in the tube
      - Plate the cells (100μL) on agar with AMP and CM.

      **Results:**

      Colonies on the plates with DH5a. No colonies on the plates with TG1.
    + Oct

      12

      (Failed) Double transformation with pgRNA and new pdCas9 into TG1-cells.

      **Goal:**

      1. Transform pgRNA and new pdCas9 into TG1-cells.

      **Procedure:**

      Both pdCas9(2) and pgRNA(2) with anti-luxS were simultaneously transformed into competent cells (TG1) after the following protocol:

      - Thaw competent cells on ice.
      - Add pdCas9(2) (5μL) and pgRNA(2) with anti-luxS (5μL) to the same tube containing competent cells.
      - Incubate the cells on ice for 20min.
      - Heat shock the cells for 45s at 42℃.
      - Incubate the cells on ice for 2min.
      - Add LB- medium (900μL).
      - Incubate the cells for 1.5h (max. 2h) at 37℃ with shaker.
      - Plate the cells (100μL) on agar with AMP and CM (make minimum 5 plates)
      - Centrifuge the cell culture for 3min at 6800g.
      - Discard the supernatant (200μL)
      - Resuspend the pellet with the remining supernatant in the tube
      - Plate the cells (100μL) on agar with AMP and CM.

      **Results:**

      Bacteria colonies did not appear on agar plates the following day.
    + Oct

      13

      (Failed) Double transformation with pgRNA and new pdCas9 into TG1-cells.

      **Goal:**

      1. Transform pgRNA and new pdCas9 into TG1-cells.

      **Procedure:**

      Both pdCas9(2) and pgRNA(2) with anti-luxS were simultaneously transformed into competent cells (TG1) after the following protocol:

      - Thaw competent cells on ice.
      - Add pdCas9(2) (5μL) and pgRNA(2) with anti-luxS (5μL) to the same tube containing competent cells.
      - Incubate the cells on ice for 20min.
      - Heat shock the cells for 45s at 42℃.
      - Incubate the cells on ice for 2min.
      - Add LB- medium (900μL).
      - Incubate the cells for 1.5h (max. 2h) at 37℃ with shaker.
      - Plate the cells (100μL) on agar with AMP and CM (make minimum 5 plates)
      - Centrifuge the cell culture for 3min at 6800g.
      - Discard the supernatant (200μL)
      - Resuspend the pellet with the remining supernatant in the tube
      - Plate the cells (100μL) on agar with AMP and CM.

      **Results:**

      Bacteria colonies did not appear on agar plates the following day.
    + Oct

      14

      Double transformation TG1 and miniprep of pgRNA

      **Goal:**

      - Transformation of TG1 cells with the new dCas sequence (with iGEM backbone) and pgRNA

      **Procedure:**

      1. Eluted DNA from DH5α-cells with only pgRNA by following the protocol for ZR miniprep. 4 tubes of miniprepped pgRNA was made and stored in the fridge.

      2. Did a double transformation of TG1-cells with both pgRNA and dCas by following the same protocol as the day befor, but changing the volume of DNA added and the growing time before plating. Two paralelles of our supercompetent TG1-cells and two paralelles of TG1 (felles) were made. 10 μL of pgRNA and 10 μL of dCas was added to each of them, and they were growing for 3 hours before plating on LA-petri dishes with CM and AMP.

      **Results**

      Transformation was successfull.

      Nanodrop results from the miniprep of cells with pgRNA:

      |  | ng/ul | A260/A280 | A260/A230 |
      | --- | --- | --- | --- |
      | 1 | 41.8 | 1.65 | 0.85 |
      | 2 | 115.5 | 1.61 | 0.72 |
      | 3 | 43.8 | 1.91 | 1.87 |
      | 4 | 40.1 | 1.69 | 0.93 |

      Succesful transformation, all the plates from the samples containing our supercompetent cells had colonies.
* Week

  42

  Week 42

  + Oct

    15

    Single colony pick, preparation of cell activity measurement

    **Goal:**

    Find out if the cell growth were affected by the prescence of tetracycline

    **Procedure**

    Colonies of successfully transformed TGI and DH5α were picked and thrown in beakers with LB + CM + AMP, and incubated at 37 °C and 220 RPM shaking.

    Cells containing the RRvT and mRFP biobrick were incubated in their respectful beakers with LB + CM at 37 °C and 220 RPM shaking. These were incubated overnight.
  + Oct

    16

    Biobrick measurement

    **Goal:**

    - Measure fluorescence of the RRvT and mRFP1 biobrick.

    **Procedure:**

    Cultures of RRvT and mRFP1 were diluted until OD=0.1. Then absorbance and fluorescence were measured by the TECAN plate reader. They were placed in wells in the following order:

    |  | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
    | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- |
    | A | RRvT 0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT 1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG | LB-media |
    | B | RRvT 0mM IPTG |  | RRvT 0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG | LB-media |
    | C | RRvT 0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG | LB-media |
    | D | RRvT 0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG | LB-media |
    | E | RRvT 0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG |  |
    | F | RRvT  0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG |  |
    | G | RRvT  0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG |  |
    | H | RRvT  0mM IPTG |  | RRvT  0.5 mM IPTG |  | RRvT  1.0 mM IPTG |  | mRFP 0mM IPTG |  | mRFP 0.5 mM IPTG |  | mRFP 1.0 mM IPTG |  |

    When 0.8 OD600 was reached, IPTG was added in the wells (except the wells for negative control) at a concentration of 0, 0.5 or 1.0 mM according to the table above.

    **Results:**

    The two biobricks showed similar properties when it comes to fluorescence. We found out that it wasn't optimal to use a transparent 96 well plate, and were lucky enough to borrow some black 96 well plates. Decided to repeat the experiment the next day using the black plates.
  + Oct

    17

    BioBrick measurement and metabolic cell activity measurement.

    **Goal:**

    - Measure absorbance and fluoresence for the RRvT and RFP biobricks.
    - Measure metabolic activity after 0 and 8 hours.

    **Procedure:**

    The biobricks (RRvT and RFP) was measured again with the same procedure as yesterday, but with a black 96 well plate.

    See Protocols for general media preparation and XTT procedure.

    Measurements of OD600 were taken and results presented in the table below.

    |  | OD600 (Post 6x dilution) |
    | --- | --- |
    | LB (0.4% Glucose) | 0.100 |
    | LB (0.8% Glucose) | 0.096 |
    | LB (0.4% Glucose TET) | 0.102 |
    | LB (0.8% Glucose TET) | 0.100 |
    | M63B1 (0.4% Glucose) | 0.093 |
    | M63B1 (0.8% Glucose) | 0.097 |
    | M63B1 (0.4% Glucose, TET) | 0.109 |
    | M63B1 (0.8% Glucose, TET) | 0.108 |

    Vitamine B was added to the M63B1 medium before inoculation to the 96 well plate in the following way:
    0.9 ul of stock solution (90 ml, 0.9108 grams) was mixed in a bottle of M63B1 (900 ml).

    0.9 mL MgSO4 and 18 mL glucose were also added to the M63B1 medium.

    The media were mixed according to the table beneath.

    | Media | Content |
    | --- | --- |
    | LB, 0.4 % glucose TET | 20 mL LB, 20 µL AMP, 20 µL CM, 0.4 mL 20% Glucose, 39.4 µL 0.5 mg/mL TET |
    | LB, 0.8 % glucose TET | 20 mL LB, 20 µL AMP, 20 µL CM, 0.8 mL 20% Glucose, 60 µL 0.5 mg/mL TET |
    | LB, 0.4 % glucose | 20 mL LB, 20 µL AMP, 20 µL CM, 0.4 mL 20% Glucose |
    | LB, 0.8 % glucose | 20 mL LB, 20 µL AMP, 20 µL CM, 0.8 mL 20% Glucose |
    | M63B1, 0.4 % glucose TET | 20 mL M63B1, 20 µL AMP, 20 µL CM, 20 µL 1M MgSO4\*7H2O, 0.4 mL 20% Glucose, 39.4 0.5 mg/mL µL TET |
    | M63B1, 0.8 % glucose TET | 20 mL M63B1, 20 µL AMP, 20 µL CM, 20 µL 1M MgSO4\*7H2O, 0.8 mL 20% Glucose, 40 µL 0.5 mg/mL TET |
    | M63B1, 0.4 % glucose | 20 mL M63B1, 20 µL AMP, 20 µL CM, 20 µL 1M MgSO4\*7H2O, 0.4 mL 20% Glucose |
    | M63B1, 0.8 % glucose | 20 mL M63B1, 20 µL AMP, 20 µL CM, 20 µL 1M MgSO4\*7H2O, 0.8 mL 20% Glucose |

    Then, the metabolic activity was measured by following the XTT protocol.

    **Results:**

    - The cells showed no reduced activity because of tetracycline being present.

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# Team:NTNU Trondheim/Results

[![](https://static.igem.org/mediawiki/2018/e/eb/T--NTNU_Trondheim--navlogo.png)](https://2018.igem.org/Team%3ANTNU_Trondheim)

* Team

  [Team Members](https://2018.igem.org/Team%3ANTNU_Trondheim/Team)
  [Collaborations](https://2018.igem.org/Team%3ANTNU_Trondheim/Collaborations)
* Project

  [Description](https://2018.igem.org/Team%3ANTNU_Trondheim/Description)
  [Design](https://2018.igem.org/Team%3ANTNU_Trondheim/Design)
  [Model](https://2018.igem.org/Team%3ANTNU_Trondheim/Model)
  [Results](https://2018.igem.org/Team%3ANTNU_Trondheim/Results)
  [Demonstrate](https://2018.igem.org/Team%3ANTNU_Trondheim/Demonstrate)
* Lab

  [Lab Journal](https://2018.igem.org/Team%3ANTNU_Trondheim/Experiments)
  [Protocols](https://2018.igem.org/Team%3ANTNU_Trondheim/Protocols)
  [Measurement](https://2018.igem.org/Team%3ANTNU_Trondheim/Measurement)
  [Safety](https://2018.igem.org/Team%3ANTNU_Trondheim/Safety)
* Parts

  [Parts Overview](https://2018.igem.org/Team%3ANTNU_Trondheim/Parts)
  [New Part](https://2018.igem.org/Team%3ANTNU_Trondheim/Improve)
* [InterLab](https://2018.igem.org/Team%3ANTNU_Trondheim/InterLab)
* Human Practices

  [Integrated Human Practices](https://2018.igem.org/Team%3ANTNU_Trondheim/Human_Practices)
  [Education and Engangement](https://2018.igem.org/Team%3ANTNU_Trondheim/Public_Engagement)
* Acknowledgements

  [Sponsors](https://2018.igem.org/Team%3ANTNU_Trondheim/Sponsors)
  [Attributions](https://2018.igem.org/Team%3ANTNU_Trondheim/Attributions)
* [Awards](https://2018.igem.org/Team%3ANTNU_Trondheim/Awards)

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Here is an overview of all the results we have gathered from all biofilm assays. At the end of the page is a summary of the results. For a thorough discussion and analysis of the results, go to our [Demonstrate](https://2018.igem.org/Team%3ANTNU_Trondheim/Demonstrate) page.

## Main results

* Isolation of pdCas9 and pgRNA from  *E. coli*  Top10 cells

  The plasmids were ordered from Addgene and delivered in *E. coli* Top10 cells. We started by plating the cells on agar plates containing the selective antibiotics for the specific plasmids – Chloramphenicol for selection of pdCas9 and ampicillin for pgRNA. The plates were incubated overnight, and single colonies were picked and inoculated in LB medium with the respective antibiotics. We isolated the plasmids and checked the quantity and quality using the NanoDrop. The results from the NanoDrop are given in **Table 1**. Since the 260/230-ratio is less than 1.80, it may indicate the presence of contaminants which absorbs strongly at 230 nm. Nevertheless, both the concentrations and the 260/280-ratios indicate that there was enough DNA to continue the process for both replicates of pdCas9 and pgRNA.

  **Table 1:** Nucleic acid concentration, 260/280- and 260/230-ratios of plasmids (pgRNA and pdCas9) isolated from cell cultures of *E. coli* Top10 cells ordered from Addgene. Two replicates were made from separate cell cultures.

  | **Sample (replicate)** | **Concentration [ng/µL]** | **260/280-ratio** | **260/230-ratio** |
  | pgRNA (1) | 62.3 | 1.86 | 1.41 |
  | pgRNA (2) | 68.1 | 1.84 | 1.26 |
  | pgCas9 (1) | 20.1 | 1.96 | 1.41 |
  | pgCas9 (2) | 52.1 | 1.79 | 1.05 |

  The isolated plasmids were digested with the restriction enzyme BspHI and the plasmid fragments were run on an agarose gel alongside GeneRuler 1 kbp ladder. A digestion tool in the software Benchling was used to predict the fragment lengths and gel patterns for each of the plasmids after cutting with BspHI. The pdCas9 was expected to be cut into two fragments with lengths of 2741 and 3964 bp, and the pgRNA was expected to be cut into three fragments with lengths of 105, 1008 and 1451 bp. As seen in **Figure 1** and **Figure 2**, both replicates for pgRNA seem to have bands with the expected lengths. The band at approximately 100 bp seemed to be only vaguely visible, possibly due to the fragment’s short length, which makes it bind a low amount of dye. For pdCas9 only replicate 2 had the correct bands.

  ![](https://static.igem.org/mediawiki/2018/7/7f/T--NTNU_Trondheim--pdCas9_pgRNA_Map.svg)

  **Figure 1:** The cut sites for BspHI in pgRNA and pdCas9, showing the origin of the expected fragments and their lengths.

  ![](https://static.igem.org/mediawiki/2018/d/d0/T--NTNU_Trondheim--Gel_image%281%29_Plasmids_from_Top10.jpg)

  **Figure 2:** Gel imaging of two replicates of both pgRNA (abbr. pR1 and pR2) and pdCas9 (abbr. pC1 and pC2) isolated from *E. coli* Top10 cells after digestion with the restriction enzyme BspHI. The samples are run alongside GeneRuler 1 kbp ladder (λ).
* Plasmid transformation into competent  *E. coli*  DH5α cells

  The isolated pdCas9 and pgRNA from the *E. coli* Top10 cells were separately transformed into competent *E. coli* DH5α cells. The transformed cells were streak plated and single colonies were picked and inoculated the next day. The OD600 values of the overnight cultures were approximately 1 (pdCas9: 1.23; pgRNA: 0.88). The cell cultures were miniprepped and nanodropped. The NanoDrop results, given in **Table 2**, implied that both pgRNA replicates and pdCas9 replicate 2 might have been RNA contaminated.

  **Table 2:** Nucleic acid concentration, 260/280- and 260/230-ratios of plasmids (pgRNA and pdCas9) isolated from *E. coli* DH5α cells transformants. Two replicates were prepared from separate cell cultures.

  | **Sample (replicate)** | **Concentration [ng/µL]** | **260/280-ratio** | **260/230-ratio** |
  | pgRNA (1) | 60.2 | 2.09 | 2.26 |
  | pgRNA (2) | 44.2 | 2.18 | 2.84 |
  | pgCas9 (1) | 194.1 | 1.56 | 0.73 |
  | pgCas9 (2) | 53.4 | 2.25 | 3.53 |

  Test digest was performed on the isolated plasmids using the restriction enzyme BspHI. The gel imaging is shown in **Figure 3**. Both plasmids gave the expected number of bands and fragment lengths (pgRNA: 105, 1008 and 1451 bp; pdCas9: 2741 and 3964 bp). This observation verified that the transformation had been a success. Interestingly, replicate 1 of dCas9 seemed to give the right restriction pattern this time (for comparison see Figure 1). Nevertheless, we still decided to only continue using replicate 2 of this plasmid since the reason for the dissimilarities was uncertain.

  ![](https://static.igem.org/mediawiki/2018/4/43/T--NTNU_Trondheim--Gel_image_28.06.png)

  **Figure 3:** Gel imaging of two replicates of both pgRNA (pR1 and pR2) and pdCas9 (pC1 and pC2) isolated from transformed *E. coli* DH5α cells after digestion with the restriction enzyme BspHI. The samples were run alongside GeneRuler 1 kbp ladder (λ).
* Insertion of anti-*luxS*   into pgRNA by Overhang Extension PCR

  ![](https://static.igem.org/mediawiki/2018/d/d6/T--NTNU_Trondheim--Gel02.07.png)

  **Figure 4:** Gel separation of PCR products after performing overhang extension PCR to insert the anti-luxS sequence into pgRNA (pR2). The samples were run alongside GeneRuler 1 kbp ladder (λ). Marked with a yellow dotted box is the fragment excised from the gel.

  Overhang Extension PCR was performed to insert the 20 bp anti-*luxS* tracker sequence into the pgRNA. This sequence is complementary to a specific location near the TBS in the *luxS* gene, which is a gene involved in the production of the quorum sensing molecule Autoinducer-2. Primers with the anti-luxS overhang were designed using the software Benchling (see [Design](https://2018.igem.org/Team%3ANTNU_Trondheim/Design) for more details about Overhang Extension PCR). The PCR products were run on an agarose gel to separate the linearized plasmid with the inserted of anti-*luxS* from the original circular pgRNA without anti-*luxS*. **Figure 4** shows the gel separation of the PCR products. The strong band at 2500 bp corresponds to the linearized plasmid. The circular configuration of the original pgRNA lacking the anti-*luxS*, makes it coil up and migrate further in the gel than the linearized plasmid due to less resistance. The original pgRNA can be seen at approximately 1500 bp.

  The linearized plasmid at 2500 bp was excised from the gel, purified and nanodropped. The concentration was 52.8 ng/µL and the 260/280- and the 260/230-ratio was 1.90 and 0.27, respectively. All though the DNA concentration and 260/280nm-ratio were acceptable, the low 260/230-ratio indicated that the recovered plasmids probably were contaminated. The contamination might be remnants of substances in the PCR-mix, agarose gel and/or chemicals used during the purification step.

  The linearized pgRNA with the inserted anti-*luxS* sequence was transformed into *E. coli* DH5α cells. Prior to performing test digest, the OD600 of the cell cultures were measured and the plasmids were miniprepped and the isolates were nanodropped. The results are listed in **Table 3**. Both replicates seem to have some impurities. Replicate 1 seems to have some RNA impurities, while replicate 2 seems to have contaminants that absorb at 230 nm. Still, both replicates were deemed good for further use.

  **Table 3:** OD600 values, nucleic acid concentration, 260/280- and 260/230-ratios of two replicates of pgRNA (pR2) isolated from *E. coli* DH5α cells.

  | **Sample (replicate)** | **OD600** | **Concentration [ng/µL]** | **260/280-ratio** | **260/230-ratio** |
  | pR2 (1) | 0.95 | 37.2 | 2.06 | 4.29 |
  | pR2 (2) | 1.00 | 123.2 | 1.77 | 1.01 |

  The transformation was verified by test digest with PstI. PstI was chosen because it has a restriction cut site in the anti-*luxS* sequence. The band pattern obtained after gel electrophoresis corresponded with the expected lengths of the plasmid fragments of 490 bp and 2094 bp (see **Figure 5**). The transformation with pgRNA and insertion of the anti-*luxS* sequence was considered successful.

  ![](https://static.igem.org/mediawiki/2018/f/fa/T--NTNU_Trondheim--Gel_image_F4.png)

  **Figure 5:** (Left) The cut sites for PstI in pgRNA with the anti-luxS sequence, indicating the expected fragment lengths. (Right) Gel imaging of two replicates of pgRNA (pR2) isolated from DH5α E. coli cells after digestion with the restriction enzyme PstI. The samples were run alongside GeneRuler 1 kbp ladder (λ).
* Double transformation of  *E. coli*   DH5α and TG1 cells

  The pdCas9 and the pgRNA with anti-*luxS* were double transformed into competent *E. coli* DH5α and TG1 cells and plated on agar plates with both chloramphenicol and ampicillin. These cell strains were selected due to their different capacities to produce biofilm – DH5α being a poor biofilm producer, whereas TG1 is known to produce an abundancy of biofilm components if the conditions allow. Cultures of each of the transformed strains were miniprepped and the isolated plasmids were nanodropped and digested with PstI and BamHI-HF.

  The NanoDrop results for the isolated plasmids from DH5α and TG1 are given in **Table 4**. For DH5α the 260/280-ratio was higher than 2.00 for both replicates, which may indicate that our isolates might have contained RNA impurities. The 260/230-ratio for the first replicate seemed acceptable, but the ratio for replicate 2 seemed oddly high. This gives reasons to believe that this sample was contaminated or was highly diluted. In addition, bobbles were detected when measuring this replicate. This might have interfered with the result giving a higher ratio than the actual value. The plasmids isolated from TG1 were all deemed good for further use, based on their near optimal ratios.

  **Table 4:** Nucleic acid concentration, 260/280- and 260/230-ratios of pgRNA with the anti-luxS (pR2L) and pdCas9 (pC2) isolated from double transformed E. coli DH5α and TG1 cells. Two replicates were prepared from separate DH5α cell cultures and five replicates were prepared from separate TG1 cell cultures.| **Sample (replicate)** | **Concentration [ng/µL]** | **260/280-ratio** | **260/230-ratio** |
  | DH5α, pR2L + pC2 (1) | 219.9 | 2.01 | 1.82 |
  | DH5α, pR2L + pC2 (2) | 35.3 | 2.03 | 3.86 |
  | TG1, pR2L + pC2 (1) | 24.7 | 1.88 | 2.34 |
  | TG1, pR2L + pC2 (2) | 23.0 | 1.87 | 2.18 |
  | TG1, pR2L + pC2 (3) | 28.3 | 1.86 | 2.34 |
  | TG1, pR2L + pC2 (4) | 29.4 | 1.76 | 1.34 |
  | TG1, pR2L + pC2 (5) | 26.9 | 1.74 | 2.19 |

  The fragments of the digested pgRNA with anti-luxS and pdCas9 were separated by gel electrophoresis. According to the digestion tool in Benchling, the expected fragment lengths were 6705bp, 2094bp, 465bp and 25bp when cutting both plasmids with PstI and BamHI-HF. As shown in **Figure 6**, bands were visible at approximately 7000, 2000 and 500 bp for both DH5α and TG1. We did not expect to see the 25 bp on the gel due to the fragment’s short length and consequently low dyeability. Since bands were visible at the expected lengths, we concluded that both pdCas9 and pgRNA with anti-luxS had successfully been transformed into both E. coli strains.

  ![](https://static.igem.org/mediawiki/2018/4/4e/T--NTNU_Trondheim--Double_transformation.png)

  **Figure 6:** Gel imaging of two replicates of PstI- and BamHI-digested pgRNA with anti-*luxS* (pR2L) and pdCas9 (pC2) isolated from double transformed (left) *E. coli* DH5α and (right)  *E. coli* TG1 cells. The samples were run alongside GeneRuler 1 kbp ladder (λ).
* Testing of biofilm promoting media

  We had been informed by Kåre Bergh and Sven Even Borgos (see [Human Practices](https://2018.igem.org/Team%3ANTNU_Trondheim/Human_Practices) for more information about these interviews) that the bacteria’s ability to form biofilms is highly dependent on their growth conditions, and that they are especially sensitive to glucose concentrations and pH levels. We wanted to test our CRISPRi system in a medium that promotes biofilm formation because it would make it easier to see a noticeable effect if the system works as expected.

  In order to investigate which medium gives the highest degree of biofilm formation, we made nine variants of LB and M63B1 medium varying in pH values (4.5, 7.2 and 9.2) and glucose concentrations (0%, 0.4% and 0.8%). *E. coli* TG1 cells were inoculated in each medium and incubated. **Table 5** shows the measured OD600 values for the overnight TG1 cultures. We observed a higher planktonic growth in LB than in M63B1. This is consistent with the fact that M63B1 is a minimal medium with a limited amount of nutrition, mimicking natural conditions. LB on the other hand, is a nutrition rich medium which provides good conditions for a rapid and numerous cell growth. Generally, the highest growth in both LB and M63B1 were observed in media with a pH value of 7.2 supplemented with glucose, either 0.4% and 0.8%, with only slight differences between the two.

  **Table 5:** Measured OD600 values in overnight *E. coli* TG1 cell cultures grown in LB and M63B1 media with different pH values (4.5, 7.2 and 9.2) and supplemented with different glucose concentrations (0%, 0.4% and 0.8%).| **pH, glucose** | **OD600 LB** | **OD600 M63B1** |
  | 4.5, 0% | 1.790 | 0.000 |
  | 4.5, 0.4% | 0.023 | 0.002 |
  | 4.5, 0.8% | 0.600 | 0.001 |
  | 7.2, 0% | 1.697 | 0.002 |
  | 7.2, 0.4% | 1.200 | 0.201 |
  | 7.2, 0.8% | 1.255 | 0.228 |
  | 9.2, 0% | 0.006 | 0.000 |
  | 9.2, 0.4% | 0.005 | 0.002 |
  | 9.2, 0.8% | 1.366 | 0.002 |

  To measure the biofilm formation capabilities of the TG1 cells in the different media, we plated each culture on microtiter plates and incubated them for 24 hours. The amount of biofilm adhered to the walls of the wells were then measured by a crystal violet assay. The amount of produced biofilm in each medium is illustrated in **Figure 7**. Even though M63B1 gave a low degree of planktonic growth compared with LB (see Table 5), the M63B1 seemed to have a strong biofilm promoting effect in media with pH 7.2 and glucose concentrations of 0.4% and 0.8%. The biofilm formation in LB followed the same trend as the planktonic growth. We concluded that the best biofilm conditions were in M63B1 with a pH value of 7.2 and a glucose concentration of either 0.4% or 0.8%. For comparison purposes we also decided to continue doing measurements in LB medium having the equivalent pH value and glucose concentrations as the optimal biofilm promoting M63B1 media. All the other media were omitted.

  ![](https://static.igem.org/mediawiki/2018/6/63/T--NTNU_Trondheim--Media_testing.png)

  **Figure 7:** Testing of biofilm promoting media by crystal violet staining of biofilm adhered to the walls of the well of a microtiter plate. Prior to the assay, cultures of *E. coli* TG1 cells had been incubated in the wells at 37 °C for 24 hours in LB and M63B1 media differing in pH value (4.5, 7.2 and 9.2) and glucose concentration (0%, 0.4% and 0.8%). Eight replicates were made for each treatment. The absorbance was measured at 590 nm.
* Crystal violet assay for quantitative biofilm measurements

  The next step was to test if we were able to reduce biofilm formation in bacteria using our CRISPRi system targeting the *luxS* gene in *E. coli*. We inoculated the chosen LB and M63B1 (pH 7.2 and glucose concentrations of 0.4% and 0.8%) with *E. coli* DH5α and TG1 cells transformed with pdCas9 and pgRNA with the anti-*luxS* sequence. After an overnight incubation, the cultures were diluted and plated on microtiter plates. Tetracycline was added to the media of our test groups, since it is the inducer for the transcription of dCas9. Our control groups lacked this addition.
  While doing the crystal violet assay, we observed that there was some variation between replicates. We therefore decided to run it several times to refine the protocol to measure biofilm formation using crystal violet. In total we ran 4 assays. For each run we did some slight changes to the protocol. The following outlines the modifications made to the original protocol and the results we obtained for each run. For more information about the final experimental setup for our biofilm measurements, please visit [Measurement](https://2018.igem.org/Team%3ANTNU_Trondheim/Measurement).

  As an extra feature to the biofilm assay, we did a cell viability test to check the condition of our cells when expressing the CRISPRi system to knock out the biofilm production. This test allowed us to confirm that our cells showed reduced biofilm production and were healthy at the same time. We used the  **"Cell Proliferation Kit II (XXT)"** to investigate the health of the cells after 0h and 8h incubation with activated CRISPRi sytem.

  ![](https://static.igem.org/mediawiki/2018/4/41/T--NTNU_Trondheim--x.jpeg)

  1. **Crystal Violet assay after 24 hours of incubation**

     The procedure we followed during the first crystal violet assay was essentially the same as described in “Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates” [1]. This procedure was suggested to us by Sven Even Borgos from SINTEF. The protocol included a wash step after 4 hours of adhesion and a single absorbance measurement after 24 hours of incubation. The quantification of biofilm formation in the different media are shown in **Figure 8**. The biofilm formation seemed to be slightly reduced in M63B1 media for both *E. coli* strains. In the LB media, the biofilm formation of the strains did not seem to be affected by the CRISPRi system. This was seen by the relatively similar absorbance values between the test groups and the control groups. Surprisingly, the measured absorbance values were high for both strains in the LB media. This was not expected since TG1 theoretically should produce a higher amount of biofilm than DH5α cells. Overall, the standard deviations were within an interval that made it difficult to draw any clear conclusions from these results.

     ![](https://static.igem.org/mediawiki/2018/1/15/T--NTNU_Trondheim--CV1_Results.png)

     **Figure 8:** Testing of biofilm-promoting media by crystal violet staining of biofilm adhered to the walls of the well of a microtiter plate. Prior to the assay, cultures of *E. coli* TG1 cells had been incubated in the wells at 37 °C for 24 hours in LB and M63B1 media differing in pH value (4.5, 7.2 and 9.2) and glucose concentration (0%, 0.4% and 0.8%). pH of 7.2 and glucose concentration of 0.4% and 0.8% showed the most biofilm formation and were selected as biofilm-promoting media  [(Test results)](https://2018.igem.org/Team%3ANTNU_Trondheim/Measurement). Eight replicates were made for each treatment. The absorbance was measured at 590 nm.
  2. **Crystal violet assay after 24, 48 and 72 hours of incubation time**

     For the second crystal violet assay we intended to include measurements after 48 and 72 hours of incubations. The results did not show any clear trend. After the 24 hours measurement it was observed that the control wells with only medium had too high absorbance values, which might indicate that the media were contaminated. Therefore, we decided to only do measurements after 48 hours of incubation time and not after 72 hours since this measurement probably would not give any valuable results.
     During the incubation period we discovered that the media evaporated from the wells, leaving only a small volume left for the cells to continue their growth. The evaporation might lead to increased cell death. If so, these dead cell could precipitate and possibly stick to the deposited biofilm in the wells. Since crystal violet binds to all material attached to the walls of the wells, this potentially high cell death due to evaporation might have given higher absorbance values than expected. Therefore, it would be valuable to also do measurements of the cell viability in addition to the crystal violet assay.
  3. **Crystal violet assay after 3, 5, 8, 24 and 30 hours of incubation. Removing wash step after 4 hours of adhesion and sealing of microtiter plates with parafilm.**

     After the previous crystal violet assay, we decided to eliminate the wash step after 4 hours of adhesion to reduce the chance of getting the samples contaminated. To account for the observed evaporation of media from the wells, we sealed the microtiter plates with parafilm. Input from our model showing the development of biofilm formation over time made us also include measurements after 3, 5 and 8 hours of incubation (see [Model](https://2018.igem.org/Team%3ANTNU_Trondheim/Model) for more information). In addition to this, we did measurements after 24 and 30 hours of incubation. The results after the crystal violet staining of biofilm can be seen in **Figure 9**. TG1 cells containing the CRISPRi system incubated in M63B1 media seemed to have a significantly reduced biofilm formation compared with the control groups. The same was also true for the DH5a cells incubated in M63B1 media, but the reduction compared with the control groups were not as remarkable. For both strains incubated in LB media it was not observed any particularly lowered biofilm formation. The reason for this might be due to LB medium being a poor biofilm promoting medium in general.

     ![](https://static.igem.org/mediawiki/2018/c/ce/T--NTNU_Trondheim--resultsbilde.png)

     **Figure 9:** Quantitative measurements of the biofilm formation over time for *E. coli* TG1 and DH5α cells grown in either M63B1 or LB medium (pH 7.2, glucose concentration 0.4% or 0.8%) with an activated CRISPRi system (light and dark blue lines and points) and an inactivated CRISPRi system (yellow and red lines and points). Four replicates were made for each combination of cell type and medium. Crystal violet staining and absorbance measurements at 590 nm were conducted after 3, 5, 8, 24 and 30 hours of incubation. The standard deviations for each treatment are shown with error bars. The standard deviations for each treatment is shown with error bars.

  4. **Crystal violet assay conducted after 3, 5, 8, 24 and 30 hours of incubation (repetition of previous protocol)**

     Based on the somewhat promising results from the previous crystal violet assay we decided to repeat the same procedure to see if the results were repeatable. As illustrated in **Figure 10** the biofilm formation was lowered once again for the TG1 cells incubated in M63B1 media. For the other samples the biofilm formation seemed the be higher than what was observed previously. The variability between the replicates were also slightly higher than during the previous run, which might indicate that these measurements were poorer than before. Further refinements to our protocol for biofilm formation might be needed.

     ![](https://static.igem.org/mediawiki/2018/4/4c/T--NTNU_Trondheim--CV3_Results.png)

     **Figure 10:** Repetition of previous protocol. Quantitative measurements of the biofilm formation over time for *E. coli* TG1 and DH5α cells grown in either M63B1 or LB medium (pH 7.2; glucose concentration 0.4% or 0.8%) with an activated CRISPRi system (light and dark blue lines and points) and an inactivated CRISPRi system (yellow and red lines and points). Four replicates were made for each combination of cell type and medium. Crystal violet staining and absorbance measurements at 590 nm were conducted after 3, 5, 8, 24 and 30 hours of incubation. The standard deviations for each treatment are shown with error bars.
  Source:
  + [1] Comparison of multiple methods for quantification of microbial biofilms grown in microtiter plates.
    J Microbiol Methods. 2008 Feb;72(2):157-65. Epub 2007 Nov 21.
* Our biobrick: Mutation of the dCas9 sequence and assembly into pSB1C3

  The plasmid of dCas9 ordered from Addgene contained three illegal restriction sites according to the criteria for BioBrick RFC10. Therefore, it was necessary to remove these restriction sites via site directed mutagenesis (SDM) and Gibson assembly   **without changing the amino acid sequence of dCas9**  . In addition, standard prefix and suffix sequences were also inserted into pdCas9 for ligation with iGEM backbone.
  The original pdCas9 (bought from Addgene) contained two restriction sites recognized by EcoRI and one by XbaI, which do not fulfil the criteria for BioBrick RFC10. In order to remove all of the unwanted restriction sites, from the original pdCas9, specific primers were designed for introduction of SDM in pdCas9, and amplification of fragments of pdCas9 for Gibson assembly. First, one of the restriction sites, recognized by EcoRI, was removed by using PCR to introduce SDM in the plasmid. After the required PCR product was obtained and transformed into competent cells, the mutated pdCas9 was tested with restriction enzyme EcoRI for verification of a successful mutation in the plasmid. Afterwards, the fragments of the successful SDM pdCas9 was amplified via PCR with designed primers for removal of the last two restriction sites, recognized by EcoRI and XbaI, and insertion of standard prefix and suffix. The required fragments from PCR were isolated from gel electrophoresis and assembled together via Gibson assembly. The Gibson assembled pdCas9 was verified by using restriction enzymes. **Figure 11** shows the results after digestion with the enzymes EcoRI and XbaI, while **Figure 12** shows the fragments obtained after digestion with EcoRI and SpeI. The mutated pdCas9 with prefix and suffix, was assembled with pSB1C3. Correctly assembled plasmid with mutated dCas9 was verified via PCR colony screening **(Figure 13)** (for a more detailed description of the procedure, please read our lab journal –  [(Link)](https://2018.igem.org/Team%3ANTNU_Trondheim/Experiments).
  ![](https://static.igem.org/mediawiki/2018/f/f7/T--NTNU_Trondheim--dCas9gelel.png)

  **Figure 11:** Gel electrophoresis of 1 kb ladder (λ), and three replicates of mutated pdCas9 with prefix and suffix (PC1, PC2, PC3) after digestion with EcoRI and XbaI.

  "
  ![](https://static.igem.org/mediawiki/2018/3/38/T--NTNU_Trondheim--dCas9gelel2.png)

  **Figure 12:** Gel electrophoresis of ladder (λ) and mutated pdCas9 (PC1) after digestion with EcoRI and SpeI.

  ![](https://static.igem.org/mediawiki/2018/4/43/T--NTNU_Trondheim--dCas9gelel3.png)

  **Figure 13:** Gel electrophoresis of 1 kb ladder (λ), and the products from PCR colony screening for correct assemble of mutated dCas9 and pSB1C3 (G1.2).

  **Conclusion**

  If all of the three unwanted restriction sites in pdCas9 are removed, and the mutated plasmid are digested with the enzymes EcoRI and XbaI, the length of the predicted fragments should be 5744 bp, 989 bp and 15 bp, respectively. On the other hand, the length of the expected fragments of a successfully mutated pdCas9 digested with EcoRI and SpeI should be 5014 bp, 919 bp and 815 bp, respectively. By comparing the results obtained from gel electrophoresis of the mutated pdCas9 after digestion with the enzyme pairs, EcoRI and XbaI **(Figure 11)**, and EcoRI and SpeI **(Figure 12)**, with the fragments predicted by Benchling, the three unwanted restriction sites in pdCas9 were successfully removed and the standard prefix and suffix were inserted into the plasmid. Colonies that potentially carried pSB1C3 with mutated dCas9 were confirmed from PCR screening by obtaining a DNA fragment with a length approximately 5000 bp, which corresponds to the length of the mutated dCas9.

## Summary

The results tell us that we successfully managed to isolate the pdCas9 and pgRNA, inserted the anti-*luxS* gene to the plasmids and transformed them into compotent *E. coli* DH5α cells. We have also tested the CRISPRi system, and the results show that the biofilm production from *E.coli* TG1 cells was greatly reduced. Furthermore, the unwanted restriction sites were successfully removed, and the mutated dCas9 was assembled into pSB1C3 plasmid backbone and the biobrick was submitted to the iGEM HQ.

![](https://static.igem.org/mediawiki/2018/8/85/T--NTNU_Trondheim--nbs.png)

![](https://static.igem.org/mediawiki/2018/2/2a/T--UiOslo_Norway--CDL.png)

![](https://static.igem.org/mediawiki/2018/1/1a/T--NTNU_Trondheim--vectron.png)

![](https://static.igem.org/mediawiki/2018/f/f1/T--NTNU_Trondheim--ntnubio.jpg)

![](https://static.igem.org/mediawiki/2018/3/30/T--NTNU_Trondheim--promega1.png)

![](https://static.igem.org/mediawiki/2018/4/40/T--NTNU_Trondheim--scania.png)

![](https://static.igem.org/mediawiki/2018/a/a7/T--NTNU_Trondheim--tekna.png)

![](https://static.igem.org/mediawiki/2018/c/cf/T--NTNU_Trondheim--bayer.png)

![](https://static.igem.org/mediawiki/2018/c/ca/T--NTNU_Trondheim--ie.png)

![](https://static.igem.org/mediawiki/2018/1/1c/T--NTNU_Trondheim--biorender.png)

![](https://static.igem.org/mediawiki/2018/4/4d/T--NTNU_Trondheim--enova.png)

![](https://static.igem.org/mediawiki/2018/9/9b/T--NTNU_Trondheim--ntnuhealth.png)

![](https://static.igem.org/mediawiki/2018/e/e4/T--NTNU_Trondheim--snapgene.png)

![](https://static.igem.org/mediawiki/2018/e/e9/T--NTNU_Trondheim--merck2.png)

![](https://static.igem.org/mediawiki/2018/a/a0/T--NTNU_Trondheim--neb.png)

![](https://static.igem.org/mediawiki/2018/0/00/T--NTNU_Trondheim--ntnubiotech.png)

![](https://static.igem.org/mediawiki/2018/b/b4/T--NTNU_Trondheim--wacker.png)

![](https://static.igem.org/mediawiki/2018/5/56/T--NTNU_Trondheim--adravisa_liten.jpg)

![](https://static.igem.org/mediawiki/2018/4/43/T--NTNU_Trondheim--ntnunv.png)