Team:NTNU Trondheim/Experiments

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:
  
  Separate the PCR product from circular DNA
  
  Isolate the linear DNA (pgRNA with anti-luxS from the gel
  
  Transform the linear DNA into competent cells
  
  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:
  
  Thaw the competent cells on ice (10 min), and pre-chill the eppendorf tubes.
  
  Resuspend the DNA in the wells (provided by iGEM HQ) with dH₂O (10 μL).
  
  Add competent cells (50 μL) to the eppendorf tubes.
  
  Add resuspended DNA (1 μL) to the Eppendorf tubes.
  
  Incubate on ice for 25 min
  
  Heat shock the cells for 45 s at 42℃ in water bath
  
  Incubate on ice for 5 min.
  
  Add LB-medium (950μL) to the tubes
  
  Incubate for 1h at 37℃ with shacker
  
  Pipette each sample (100 μL) to agar plate with CM
  
  Centrifuge the samples for 3 min at 6800g, discard supernatant (800 μL) and resuspend the pellet with the remaining supernatant
  
  Pipette each sample (100 μL) to agar plate with CM
  
  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:
  
  Inoculate colonies of successful transformed competent cells
  
  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:
  
  InterLab - transformation of plasmids into competent cells
  
  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:
  
  Transform both pdCas9 and pgRNA with anti-luxS into competent cells
  
  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:
  
  Inoculate successful double transformed cells (probably carrying pdCas9 and pgRNA with anti-luxS)
  
  InterLab study - measure absorbance and the fluorescence of each cell cultures
  
  Procedure:
  Inoculation of successful double transformed cells
  
  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).
  
  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 ddH2O
  
  The 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:
  
  Measure biofilm after 48 hrs.
  
  Prepare RRVT-Biobrick and biobrick backbone
  
  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:
  
  Ligate plasmid backbone and RRvT-plasmid and transform into cells.
  
  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:
  
  Inoculate successful transformed cells into LB medium with CM.
  
  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
  
  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
  
  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.
- Oct
  
  13
  
  No lab
- Oct
  
  14
  
  Double transformation TG1 and miniprep of pgRNA
  
  Goal:
  
  - Succesfully transformed TG1 cells with the new dCas sequence (with iGEM backbone) and pgRNA
  
  Procedure:
  
  1. Eluted DNA from DH5a-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 uL of pgRNA and 10 uL 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.
- Oct
  
  15
  
  No lab
- Oct
  
  16
  
  Biofilm measurement
  Goal:
  
  Measure biofilm formation after 0, 3, 5, 8, 24 and 30 hours.
  
  Measure metabolic activity after 0, 8 and 24 hours.
  
  Procedure:

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

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

	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

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