Difference between revisions of "Team:NTNU Trondheim/Experiments"

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

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℃.

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

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

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

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

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.

Goal:

Procedure:

• 7.1 μL Plasmids
• 0.5 μL PstI
• 0.5 μL BamHI-HF
• 2 μL NEBuffer 1.1
• 10.4 μL ddH2O

Results:

Goal : InterLab Study: Revive bacteria glycerol stock

• 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

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.

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.

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.

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.

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.

• 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.

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.

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.

Crystal Violet Assay: Same procedure as from 17.08.2018.

Bactiter-Glo Assay

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

Make LB medium and 10x v2 salts for Vibrio natrigens

Amplify and introduce site directed mutagenesis in pdCas9

LB - medium

10x v2 salts:

• 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:

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.

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.

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.

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

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.

• Measure the growth curve of V. natrigens
• Gel purification of RRvT
• Separte the PCR products of pdCas9 on gel

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

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.

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

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.

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 ℃

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.

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.

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.

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.

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 ℃.

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.

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...

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 ℃.

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.

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℃.

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

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 ℃.

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.

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.

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

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.

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

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.