N o t e b o o k

2 June 2025

Pre-culture preparation of E.coli DH5𝛂 and E.coli BL21 to make them super-competent

Inoculate 1 colony of E. coli DH5𝛂 into a 10 mL Erlenmeyer flask of LB and 1 colony of E. coli BL21 into another 10 mL Erlenmeyer flask of LB. Shake overnight at 37°C

3 June 2025

Preculture Dilution

Under sterile conditions (Bunsen burner flame)

• 1. Retrieve the E. coli DH5 alpha and BL21 preculture
• 2. Add 1 mL of preculture to a 500 mL Erlenmeyer flask containing 100 mL of LB (separately for DH5 alpha and BL21)
• 3. Incubating for 2 hours at 37°C with shaking at 180 rpm

OD Measurement by Spectrophotometer

Measure the OD of the cells in 1 mL cuvettes to reach an approximate OD600 = 0.5.

Preparation of Supercompetent Cells (DH5 alpha and BL21)

Preparation of Solutions 1 and 2 (used during centrifugation steps) :

Solution 1 (40 mL):

• 1,2 mL KoAc 1 M
• 4 mL MnCl2 0,5 M
• 4 mL Kcl 1 M
• 4 mL CaCl2 0,1 M
• 7,5 mL Glycérol 80%
• 19,3 mL sterile H2O

Solution 2 (40 mL):

• 200 µL MOPS 0,2 M
• 40 µL KCl 1 M
• 3 mL CaCl2 0,1 M
• 750 µL glycérol 80%
• 10µL sterile H2O

• 1. Transfer 80 mL from the Erlenmeyer flasks into two 40 mL Falcon tubes (for DH5 alpha and BL21)
• 2. Centrifuge cells for 10 min at 3500 rpm, 4°C
• 3. Discard the supernatant and resuspend the pellet in 400 µL of Solution 1 (first wash). Combine pellets into one Falcon tube and add 39.6 mL of Solution 1.
• 4. Centrifuge for 5 min at 3500 rpm, 4°C.
• 5. Discard the supernatant and resuspend the pellet in 200 µL of Solution 2 (second wash). Combine pellets into one tube and add 3.8 mL of Solution 2.
• 6. Freeze aliquots (100 µL) in liquid nitrogen, then store at -80°C. Retain one aliquot for supercompetency testing.

Test : Transformation with Supercompetent Cells

Preparation of LB + Ampicillin plates (final concentration : 1µg/µL)

Under sterile conditions (Busen burner flame) :

• 1. Prepare 2 tubes with 20 µL of bacteria
• 2. Add 1 µL of each plasmid (10 ng/µL) in 1 tube
• 3. Incubate on ice for 15 min
• 4. Apply heat shock : 45 sec at 42°C (preheat block in advance)
• 5. Return to ice for 2 min
• 6. Add 1 mL of LB.
• 7. Incubate at 37°C with shaking for 45 min and 1 hour max
• 8. Centrifuge at 8000 rpm for 30 sec
• 9. Remove ~900 µL of supernatant and resuspend the remaining 100 µL by pipetting
• 10. Spread 3µl of bacteria onto half of an LB + ampicillin plate for each plasmid (half DH5 alpha / half BL21) (using a spreader/pasteur pipette)

Contamination Test

Spread 3 µL of E. coli DH5 alpha on half of 3 plates and 3 µL of E. coli BL21 on the other half of each plate (on LB agar + ampicillin, LB agar + kanamycin and LB agar + chloramphenicol)

4 June 2025

Tuesday’s transformation failed; repeating the procedure.

Preparation of Supercompetent Cells (DH5 alpha and BL21)

Follow the same protocol as Monday 03/06/2025

10 June 2025

Preparation of pre-cultures for E. coli DH5𝛂 and E. coli BL21 to make them super-competent

Inoculated 1 colony of E. coli DH5𝛂 into a 5 mL LB Erlenmeyer flask. Incubated overnight at 37°C with shaking.

Inoculated 1 colony of E. coli BL21 into a 5 mL LB Erlenmeyer flask. Incubated overnight at 37°C with shaking.

11 June 2025

The LB was contaminated, repeated the pre culture

Preparation of solutions for super-competent cell transformation

• MnCl2 0,5M ; 197,9 g/mol
• Kcl 1M ; 74,555 g/mol
• MOPS 0,2M ; 209,26 g/mol
• KoAc 1M ; 98,17 g/mol
• Prepared by dissolving:
• 9,9 g MnCl2 and 7,455g Kcl in 100mL d’H20
• 2,09 g in 50 mL d’H20
• 4,9 g KoAc in 50 mL d’H20

12 June 2025

Preparation of pre-culture for E.coli DH5alpha and E.coli W3110 to make them super-competent:

Inoculated 1 colony of E.coli DH5alpha into a 5mL LB Erlenmeyer flask. Inoculated 1 colony of E.coli W3110 into a separate 5mL LB Erlenmeyer flask. Incubated overnight at 37°C with shaking.

13 June 2025

Dilution of pre-culture

(Performed under sterile conditions using a Bunsen burner flame)

• Added:
• 1 mL of pre-culture into a 500 mL Erlenmeyer flask containing 100 mL LB (for DH5𝛂).
• 250 µL of pre-culture into a 125 mL Erlenmeyer flask containing 25 mL LB (for W3110).
• Incubated with shaking for 2 hours at 37°C.

OD measurement using a spectrophotometer

Measured cell OD in 1 mL cuvettes to target OD₆₀₀ ≈ 0.5.

W3110 : 0,72

DH5 alpha : 0,47

Preparation of Supercompetent Cells DH5 alpha cells

Prepared Solution 1 and Solution 2 for use during centrifugation steps.

40 mL of Solution 1 :

• 1,2 mL KoAc 1 M
• 4 mL MnCl2 0,5 M
• 4 mL Kcl 1 M
• 4 mL CaCl2 0,1 M
• 7,5 mL Glycérol 80%
• 19,3 mL H2O stérile

4 mL of Solution 2 :

• 200 µL MOPS 0,2 M
• 40 µL KCl 1 M
• 3 mL CaCl2 0,1 M
• 750 µL glycérol 80%
• 10µL H2O stérile

Preparation of Supercompetent Cells DH5 alpha cells

• 1. Transferred 80 mL from the Erlenmeyer flask into two 40 mL Falcon tubes (for DH5𝛂).
• 2. Centrifuged cells at 3500 rpm for 10 min at 4°C.
• 3. Discarded supernatant and resuspended pellet in 20 mL Solution 1 per tube (first wash).
• 4. Centrifuged again at 3500 rpm for 10 min at 4°C.
• 5. Discarded supernatant and resuspended pellet in 2 mL Solution 2 (second wash), combining pellets into one tube and adding 3.8 mL Solution 2.
• 6. Aliquoted into 100 µL samples, flash-frozen in liquid nitrogen, and stored at -80°C.
• 7. Kept one aliquot for super-competency testing.

Contamination test

Plated 3 µL of E. coli DH5𝛂 on:

• LB agar + Ampicillin
• LB agar + Kanamycin
• LB agar + Chloramphenicol

For W3110 :

Prepared 10% glycerol solution:

• Vi = 10% (Cf) * 200 mL (Vf) / 80% (Ci) = 25 mL glycérol 80%
• Added H2O to final volume of 200 mL

Preparation of Electrocompetent Cells W3110 cells

Two washes with 35 mL of 10% glycerol (perform on ice)

• 1. Transfer 35 mL from the Erlenmeyer flask into a 50 mL Flacon tube for W3110
• 2. Wait in ice 20 minutes
• 3. Centrifuge at 5000 rpm, 10 min, 4°C
• 4. Discard the supernatant
• 5. Resuspend in 35 mL of 10% glycerol
• 6. Wait in ice 5 minutes
• 7. Centrifuge at 5000 rpm, 10 min, 4°C
• 8. Discard the supernatant
• 9. Resuspend in 35 mL of 10% glycerol
• 10. Centrifuge at 5000 rpm, 10 min, 4°C
• 11. Discard the supernatant
• 12. Resuspend in 250 µL of 10% glycerol

Electroporation of pKOBEG Vector into W3110

Prepare two 1.5 mL Eppendorf tubes :

• 1.
  • CONTROL : 60 µL W3110 culture in 10% glycerol
  • pKOBEG : 60 µL W3110 culture in 10% glycerol + 1 µL pKOBEG vector
• 2. Incubate on ice for 15 min
• 3. Transfer the 60 µL mixtures into electroporation cuvettes
• 4. Electroporate using the appropriate machine
• 5. Immediately add 900 µL of LB broth to each cuvette (control and vector) and resuspend
• 6. Transfer into 1.5 mL Eppendorfs tubes
• 7. Shake at 30°C, 750 rpm, 1 hour
• 8. Centrifuge 5000 rpm, 5 min
• 9. Remove 800 µL of supernatant
• 10. Resuspend the pellet in the remaining 100 µL
• 11. Streak into LB agar + chloramphenicol plates for selection

16 June 2025

Preparation of E.coli DH5𝛂 and E.coli W3110 precultures to make them super-competent.

Inoculated 1 colony of E.coli DH5𝛂 into a 5mL LB Erlenmeyer flask.

Inoculated 1 colony of E.coli W3110 into a 5mL LB Erlenmeyer flask.

Shake overnight at 37°C.

17 June 2025

Preparation of antibiotic petri dishesMake a stock of 10 plates !

Cm : (chloramphenicol) :

• Working concentration Ci = 30 µL/mL
• 1,3 µL/mL culture.
• 1 plate = 20 mL for 10 plates = 200 mL final volume
• 1,3 * 200 = 260 µL Cm in 200 mL de LB agar

Km : (kanamycin) :

• Working concentration Ci = 10,5 µL/mL
• 1 plate = 20 mL for 5 * 20 = 100 µL Km in 20 mL de LB agar → only made 1 plate.

Amp : Ampicillin

• Working concentration Ci = 25,4 µL/mL
• 1 plate = 20 mL, for 100 plates = 200 mL final volume
• 4 * 200 = 800 µL Amp in 200 mL de LB agar

Preculture Dilution

Follow the same procedure as made the 13 june

OD Measurement

Preparation of Competent Cells

• 1. Transfer 80 mL from Erlenmeyer flask into two 40 mL Flacon tubes for DH5 alpha.
• 2. Centrifugate cells at 3500 rpm à for 10 minute 4°C
• 3. The supernatant is discarded and the pellet is resuspended in 20 mL of solution 1 for each of two tubes ( first wash). The pellet is resuspended gently using two sticks to avoid damaging it.
• 4. Centrifugate again at 3500rpm for 10 minutes at 4°C.
• 5. The supernatant is discarded and the pellet is resuspended in 2 mL of Solution 2 (second wash). The pellets are then combined into a single flask, and 3.8 mL of Solution 2 is added.
• 6. The mixture is left to rest for 1 hour on ice.
• 7. The solutions are aliquoted into 100 µL portions, flash-frozen in liquid nitrogen and stored at -80°C. One aliquot is kept for the supercompetency test

Contamination Test 6 µL of E. coli DH5 alpha bacteria were spread on each plate :

• LB agar Ampicillin
• LB agar Kanamycin
• LB agar Chloramphenicol

Preparation of Supercompetent DH5 alpha CellsPreparation of Petri plates with LB + Kanamycin (1 µG/µL final concentration)

• 1. Prepare 1 tube with 100 µL of bacteria
• 2. Add 1 µl of plasmid pRSF (10ng/µl) into a tube (one tube per plasmid)
• 3. Incubate the tubes on ice for 15 min.
• 4. Apply heat shock : 42°C for 45 sec (pre-warm the heat block in advance)
• 5. Return the tubes to ice for 2 min
• 6. Add 1 mL of LB medium
• 7. Incubate the tubes at 37°C with shaking for 45 min to 1 hour (max)
• 8. Centrifuge at 8000 rpm for 30 sec
• 9. Remove ~900 µL of supernatant and resuspend the remaining 100 µL by pipetting
• 10. Spread 6µL of bacteria onto an LB + kanamycin plate (one plate per plasmid) using a spreader/pasteur pipette.

18 June 2025

Dilution of pre-cultureFollow the same procedure as described on Tuesday, 03/06 but this time with E.coli CC118

OD Measurement by Spectrophotometer

22 July 2025

1st Hydrogel Synthesis (Test) → Hydrogel Marbles

Hydrogel Preparation (1.5% Alginate Solution):

• 1. Weigh 150 mg of sodium alginate powder and transfer it into a Falcon tube.
• 2. Add 10 mL of distilled water and vortex thoroughly until a homogeneous viscous solution is obtained.

Gelation:

• 3. For a 1% CaCl₂ solution (prepared from a 1 M stock) into a clean glass container.
• 4. Using a pipette, carefully drop the alginate solution into the CaCl₂ bath drop by drop, allowing each droplet to solidify into a mini hydrogel sphere (this will be our hydrogel marbles) via ionic crosslinking.

Results:

Preculture W3110 non-motile pBAD33 and W3110ΔompT pBAD33 + arabinose to test the motility of the strains.

• Inoculate 1 colony of the strain into 5 mL LB + 0.1% arabinose (with cloraphenicol) in a sterile 25 mL flask. Shake overnight at 37°C

23 July 2025

Magic Box

We transferred 500 µL of our pBAD33 + W3110 non-motile preculture and 500 µL of our pBAD33 + W3110ΔompT preculture into two eppendorf tubes, then centrifuged them in order to obtain cell pellets. We took a look at our pellets at the magic box to check the GFP expression → we decided to redo our precultures with a 0.2% arabinose induction to obtain better results during our tests

Preculture pBAD33 + W3110 non-motile + arabinose :

Inoculate 1 colony of non-motile E. coli W3110 (pBAD33) into 20 mL LB + 0.2% arabinose (with cloraphenicol) in a sterile 100 mL flask. Shake overnight at 37°C

Preculture pBAD33 + W3110ΔompT + arabinose

Inoculate 1 colony of E. coli W3110ΔompT (pBAD33) into 20 mL LB + 02% arabinose (with cloraphenicol) in a sterile 100 mL flask. Shake overnight at 37°C

Magic BoxWe transferred 1mL of our pBAD33 + W3110 non-motile preculture and 1mL of our pBAD33 + W3110ΔompT preculture into two eppendorf tubes, then centrifuged them in order to obtain cell pellets. We took a look at our pellets at the magic box to check the GFP expression → our 0.2% arabinose induced precultures expressed better our GFP protein.

Hydrogel bacterial escape test

1. Bacterial pellet preparation:

• Materials: Overnight w3110 non motile + pBAD33 preculture and W3110 ΔompT + pBAD33 preculture, LB + Cloraphenicol broth, Spectrophotometer cuvettes, Centrifuge, Spectrophotometer, 1 mL Eppendorf tubes, Vortex
• 1. Using LB as a blank, measure OD of overnight cultures.
• 2. Dilute the overnight cultures with LB (+antibiotic) until the value of its OD600 is equal 1.0 (Final volume 900 µL)
• NOTE: Consider preparing a LB + Cloramphenicol (CM) solution to use in each step we need LB.
• 3. Prepare 2 Eppendorf tubes, one containing 900 µL OD 1.0 W3110 non motile + pBAD33 bacterial culture and the other one containing 900 µL OD 1.0 W3110ΔompT + pBAD33 bacterial culture.
• 4. Centrifuge the cultures at 4,000 rpm for 10 min
• 5. Remove supernatant and obtain bacterial pellets

2. Bacterial encapsulation:

• Materials: Bacterial pellets, Alginate, 1% CaCl2 solution, DI water, Analytical balance, Mixing spatula, 2 mL Eppendorf tubes, 14mL Falcon tubes
• 6. Weight hydrogel reagent: 96 mg alginate
• 7. Dissolve the bacterial pellets into 6,4 mL of DI water. For practicity, start by dissolving the pellets with 1mL of DI water then transfer the solution to a 14mL falcon tube containing 5,4mL of di water.
• 8. Dissolve 96 mg alginate in the mixture. Mix until alginate is fully dissolved to obtain a 1.5% bacterial alginate hydrogel
• 9. Carefully pipette 200 µL of the alginate mix in order to create 200µL hydrogel drops and add a 1% CaCl2 solution to obtain hydrogel marbles

3. Leakage Test

• Materials: 2 mL Eppendorf tubes, Spectrophotometer, Incubator set to 37°C, LB + Chloramphenicol broth, Bacterial hydrogel marbles
• 10. Add 1 bacterial hydrogel marble to the bottom an eppendorf tube (repeat the procedure 3 times for each of the two pellets and name the tubes 1,2,3)
• 11. Place the tubes in the incubator at 37°C for 20 min.
• 12. Add 1200 µL of LB+CM to tubes number 1
• 13. For tubes number 2, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 5.6 mL LB+CM. Transfer back 1,4mL of the new mix into tube number 2
• 14. For tubes number 3, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 8 mL LB+CM. Transfer back 2 mL of the new mix into tube number 3.
• NOTE: Consider the working concentration of chloramphenicol and dilute the stock accordingly into the LB
• 15. Measure OD after filling the tubes. Record the results, then recharge each tube with the sample measured and leave to incubate at 37 degrees.
• 16. Repeat the OD measuring process after 30 min, 1 hour, 1.5 hours, etc

30 July 2025

Hydrogel bacterial escape test

1. Bacterial pellet preparation:

• Materials: Overnight w3110 non motile + pBAD33 preculture and W3110 ΔompT + pBAD33 preculture, LB + Cloraphenicol broth, Spectrophotometer cuvettes, Centrifuge, Spectrophotometer, 1 mL Eppendorf tubes, Vortex
• 1. Using LB as a blank, measure OD of overnight cultures.
• 2. Dilute the overnight cultures with LB (+antibiotic) until the value of its OD600 is equal 1.0 (Final volume 900 µL)
• NOTE: Consider preparing a LB + Cloramphenicol (CM) solution to use in each step we need LB.
• 3. Prepare 2 Eppendorf tubes, one containing 900 µL OD 1.0 W3110 non motile + pBAD33 bacterial culture and the other one containing 900 µL OD 1.0 W3110ΔompT + pBAD33 bacterial culture.
• 4. Centrifuge the cultures at 4,000 rpm for 10 min
• 5. Remove supernatant and obtain bacterial pellets

2. Bacterial encapsulation:

• Materials: Bacterial pellets, Alginate, 1% CaCl2 solution, DI water, Analytical balance, Mixing spatula, 2 mL Eppendorf tubes, 14mL Falcon tubes
• 6. Weight hydrogel reagent: 96 mg alginate
• 7. Dissolve the bacterial pellets into 6,4 mL of DI water. For practicity, start by dissolving the pellets with 1mL of DI water then transfer the solution to a 14mL falcon tube containing 5,4mL of di water.
• 8. Dissolve 96 mg alginate in the mixture. Mix until alginate is fully dissolved to obtain a 1.5% bacterial alginate hydrogel
• 9. Carefully pipette 200 µL of the alginate mix in order to create 200µL hydrogel drops and add a 1% CaCl2 solution to obtain hydrogel marbles

3. Leakage Test

• Materials: 2 mL Eppendorf tubes, Spectrophotometer, Incubator set to 37°C, LB + Chloramphenicol broth, Bacterial hydrogel marbles
• 10. Add 1 bacterial hydrogel marble to the bottom an eppendorf tube (repeat the procedure 3 times for each of the two pellets and name the tubes 1,2,3)
• 11. Place the tubes in the incubator at 37°C for 20 min.
• 12. Add 1200 µL of LB+CM to tubes number 1
• 13. For tubes number 2, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 5.6 mL LB+CM. Transfer back 1,4mL of the new mix into tube number 2
• 14. For tubes number 3, transfer the bacterial hydrogel marble to a 14mL falcon tube and break the pellet from one of the Eppendorf tubes then vortex the broken pellet with 8 mL LB+CM. Transfer back 2 mL of the new mix into tube number 3.
• NOTE: Consider the working concentration of chloramphenicol and dilute the stock accordingly into the LB
• 15. Measure OD after filling the tubes. Record the results, then recharge each tube with the sample measured and leave to incubate at 37 degrees.
• 16. Repeat the OD measuring process after 30 min, 1 hour, 1.5 hours, etc

Results:

31 July 2025

Hydrogel bacterial escape test

Follow the same protocol as Wednesday 30/07/2025. Consider preparing a stock solution of approximately 50 mL of LB supplemented with antibiotic in an Erlenmeyer flask, to facilitate LB handling during the protocol.

Résultats: Results interpretation: Although we did not observe a significant difference between our intact and mechanically disrupted hydrogel marbles, as initially expected, we did notice a clear difference between motile and non-motile strains. The OD of our motile E. coli W3110ΔompT cells increased significantly more than that of the non-motile W3110 strain. This suggests that, in order to effectively contain bacteria within the hydrogel matrix, it may be necessary to eliminate motility in the final cell line. A possible strategy could involve deleting flagellar genes such as fliC.

07 August 2025

Hydrogel bacterial survival test

Bacterial pellet preparation:

• Follow the same protocol as Wednesday 30/07/2025

Bacterial encapsulation:

• Follow the same protocol as Wednesday 30/07/2025 and Wash the marbles 3 times with a 1XPBS solution before first incubation at 37 degrees.

Survival Test

• 1. Prepare 36 sterile eppendorf tubes and name them as followed:

• References: 1, 2, 3 corresponds to the triplicate of each cell sample mX corresponds to W3110 non motile + pBAD33 Δ corresponds to W3110 ΔompT + pBAD33 t = time of measurement
• 2. Sample preparation: For each mX tube, add one 200 µL non-motile hydrogel marble. For each Δ tube, add one 200 µL ΔompT hydrogel marble. Add 1200 µL of sterile 1X PBS solution to each tube. Prepare an additional control tube containing only PBS. Incubate all tubes at 37 °C, except for the t=0 tubes, which will be processed immediately.
• 3. Processing t=0 tubes: Under sterile conditions, mechanically disrupt (crash) the hydrogel marble in each t=0 tube. Centrifuge for 3 min at 3000 rpm, then collect 110 µL of supernatant from each tube and transfer to column A (rows 12A–7A) of a sterile 96-well ELISA plate, following the layout diagram.

• 4. Serial dilution: Add 90 µL of sterile 1X PBS to all remaining wells of the same row (B to H). Perform 7 serial 1:10 dilutions by transferring 10 µL from column A to column B, mixing well, and repeating sequentially until column H. This achieves a final dilution factor of 10⁻⁷ for each sample.

• 5. Bacterial culture spotting: collect 4µL of each of the samples on the well plate and spot them onto a LB + CM (Cloramphenicol) + arabinose agar plate. (draw out a grid on the petri dish beforehand in order to facilitate the spotting, and try to maintain the same layout as the ELISA well plate for consistency)
• 6. Subsequent Time Points: After 30 minutes of incubation of the rest of the eppendorf tubes at 37 °C,process the t=30 tubes as described above (crush, centrifuge, transfer supernatant, serial dilution, spotting). Repeat the same procedure for tubes t=1h after 1 hour of incubation, t=3h after 3 hours, and so on.
• 7. Incubation: Incubate the spotted agar plates overnight (ON) at 37 °C.

Résultats: Identification des sites d'interaction clés pour la liaison.

08 August 2025

Hydrogel bacterial survival test results

Only the t=30 min and t=1h samples grew. We think it is due to a problem during the manipulation of the samples, and we will redo our test next week

12 August 2025

Hydrogel bacterial survival test

Follow the same protocol as Wednesday 30/07/2025. Consider preparing a stock solution of approximately 50 mL of LB supplemented with antibiotic in an Erlenmeyer flask, to facilitate LB handling during the protocol.

References: 1, 2, 3 corresponds to the triplicate of each cell sample, xM corresponds to W3110 non motile + pBAD33, Δ corresponds to W3110 ΔompT + pBAD33, * corresponds to the tubes in which the marbles have been crushed, t = time

Résultats: Results interpretation: We observed growth for all samples except for the t=0h samples, which is expected since they were processed immediately after encapsulation. The growth of both motile and non-motile strains over time indicates that our hydrogel matrix supports bacterial viability. However, we did not observe a significant difference in growth between the motile and non-motile strains, suggesting that motility may not play a crucial role in bacterial escape from the hydrogel under these conditions. Further experiments could involve testing different hydrogel compositions or crosslinking densities to assess their impact on bacterial containment.

13 August 2025

Hydrogel bacterial survival test results

Conclusion: Our bacteries are still alive inside our hydrogel after 24h. We shall repeat our test during a longer period of time

28 August 2025

Hydrogel bacterial escape test

Follow the same protocol as Wednesday 30/07/2025. Consider preparing a stock solution of approximately 50 mL of LB supplemented with antibiotic in an Erlenmeyer flask, to facilitate LB handling during the protocol.

References: 1, 2, 3 corresponds to the triplicate of each cell sample, xM corresponds to W3110 non motile + pBAD33, Δ corresponds to W3110 ΔompT + pBAD33, * corresponds to the tubes in which the marbles have been crushed, t = time

Results interpretation: We compared our current results with those obtained from the same test conducted on Thursday 31/07/2025 to ensure that our measurements were both representative and accurate. After refining our technique for producing alginate marbles, we achieved improved results. To minimize operator error, we combined data from both the July 31 test and the current experiments for our final results graph.

Résultats: Results interpretation: We observed growth for all samples except for the t=0h samples, which is expected since they were processed immediately after encapsulation. The growth of both motile and non-motile strains over time indicates that our hydrogel matrix supports bacterial viability. However, we did not observe a significant difference in growth between the motile and non-motile strains, suggesting that motility may not play a crucial role in bacterial escape from the hydrogel under these conditions. Further experiments could involve testing different hydrogel compositions or crosslinking densities to assess their impact on bacterial containment.

27 June 2025

pAIDA-I from AddGene

Pick cells forms the AddGene tube with a toothpick than transfer into a Cm + LB plate and incubate overnight at ambient temperature

30 June 2025

pAIDA-I stock

Preculture:Inoculate 1 colony of E.coli pAIDA-I into a 5 mL Erlenmeyer flask of LB. Shake overnight at 37°C

01 July 2025

Plasmid Miniprep Kit for pAIDA-I

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18 June 2025

Nanodrop results: pAIDA-I : 18 ng/µL

Preculture:Inoculate 1 colony of E.coli pAIDA-I into a 5 mL Erlenmeyer flask of LB. Shake overnight at 37°C

02 July 2025

pAIDA-I stock

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18 June 2025

Nanodrop results: pAIDA-I : 24,9 ng/µL

Preculture: Inoculate 1 colony of E.coli pAIDA-I into a 5 mL Erlenmeyer flask of LB. Shake overnight at 37°C

03 July 2025

pAIDA-I stock

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18 June 2025

Nanodrop results: pAIDA-I : 28,8 ng/µL

08 July 2025

Clonage PCR of Tyr1 in pAIDA

Here we use :

• 1 µL of Template DNA (pAIDA)
• 5 µL of Tyr1
• PFU in separate composants that contains : 5 µL of 10X PFU Buffer, 4 µL of dNTPs, 2 µL of DMSO, 0,8µL of PFU enzyme

Thermocycling Conditions for a Routine PCR:

• Initial Denaturation: 95°C for 5 minutes
• Denaturation: 95°C for 1 min
• Annealing: 56°C for 1 min
• Extension: 68°C for 13 minute
• 29 cycles of Denaturation - Annealing - Extension
• Final Extension: 68°C for 20 minutes
• Hold: 4°C indefinitely

09 July 2025

Clonage PCR of Tyr1 in pAIDA : DpnI treatment

Add 1 µL of DpnI in the PCR cloning products. Incubate at 37°C for 3h.

Clonage PCR of Tyr1 in pAIDA : transformation into DH5 alpha super-competent

Preparation of LB + Cm plates.

Under sterile conditions ( Bunsen burner flame ) :

• Add 5 µL of the Dpn1-digested product in 50 µL of DH5 alpha
• Incubate in ice 30 min
• Apply heat shock : 1 min at 42 °C
• Incubate in ice 1 min
• Add 1 mL of LB
• Incubate at 37 °C with shaking for 45 min and 1 hour max
• Centrifuge at 1100 rpm for 1 min
• Remove 900 µL of supernatant and resuspend the remaining 100 µL by pipeting
• Spread 100 µL of bacteria on LB + cm plates

10 July 2025

Result of Clonage PCR of Tyr1 in pAIDA :

Colony PCR Protocol with oLS 4.1, oLS 4.2 and pAIDA Tyr 1 cells

We do this for 6 colonies of pAIDA Tyr1 and 1 colony of pAIDA (negative control) Follow the same protocol as Thursday, 26 June. This time we do 60°C and 1 minute.

Results: Colonies 1, 5 and 6 have a 1kb, colonies 2 and 4 have the same result as negative control. So Colonies 1, 5 and 6 have tyr1 in pAIDA.

Pre-culture preparation of pAIDA Tyr1 1, 5 and 6 to do a Miniprep Inoculate colony 1 of pAIDA Tyr1 into a 5 mL Erlenmeyer flask of LB with Chloramphenicol. Same for pAIDA Tyr1 colonies 5 and 6. Shake overnight at 37°C.

11 July 2025

Miniprep of pAIDA Tyr1 1,5 and 6

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.

Nanodrop results: pAIDA Tyr1 1 : 24,75 ng/µL; OD= 1,60 pAIDA Tyr1 5 : 18,75 ng/µL; OD= 1,50 pAIDA Tyr1 6 : 37,45 ng/µL; OD= 1,78

Samples 1 and 5 do not meet the required purity and concentration criteria for sequencing. A new culture and miniprep will be necessary to obtain higher quality DNA.

15 July 2025

Pre-culture preparation of pAIDA Tyr1 6

Inoculate colony 6 of pAIDA Tyr1 into a 5 mL Erlenmeyer flask of LB with Chloramphenicol. Shake overnight at 37°C.

Miniprep of pAIDA Tyr1 1,5 and 6

Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.

Nanodrop results: pAIDA Tyr1 1 : 33,64 ng/µL; OD= 2,00 pAIDA Tyr1 5 : 34,6 ng/µL; OD= 1,44 pAIDA Tyr1 6 : 36,047 ng/µL; OD= 1,65

We placed the tubes from colony 1 (today) and colony 6 (last week) in the speedvac because their OD values are good, and we are going to send them for sequencing tomorrow.

Motility Test of W3110 Δ amp T compared to W3110 motile

Dessert-like LB agar petri dish preparation (final LB agar concentration should be 0.3%)

LB agar final volume = 20mL

LB agar initial concentration = 1.5%

Calculation:

Vi = (0.3x20mL)/1.5 = 4mL of LB agar

Vf - VLB agar = VLB liquid = 20mL - 4mL = 16mL LB liquid

Once the LB agar kind of solidifies (the idea is for it not to be completely solid), we pick one W3110 colony to transfer as a single point on the petri dish and we do the same for a W3110 Δ amp T colony.

Incubate at 37°C (carefully, since de LB agar is not completely solid for this test)

17 July 2025

Result of motility test :

Test for verification of melanin expression : transformation of pAIDA-Tyr1 into w3110ΔompT and w3110

Preparation of supercompetent cells W3110ΔompT and W3110 Follow the same procedure as described on Tuesday, 03/06.

Transformation of pAIDA-Tyr1 and pAIDA (for the control) into w3110ΔompT and w3110 Follow the same procedure as described on Tuesday, 03/06. And use chloramphenicol petri dishes (CM) + Tyrosine for the transformation with pAIDA-tyr1 and chloramphenicol petri dishes for the transformation with pAIDA.

21 July 2025

Results of the transformation

We have colonies on all the plates, so the transformation worked.

Sequencing of pAIDA Tyr1 with oLS4.2 With a reverse primer, we do a sequencing of pAIDA-tyr1.

The results show us that we find Tyr1 in pAIDA with the colonie 1 and we have not a signal in colonie 6.

Sequencing of pAIDA Tyr1 with oLS4.1 With a forward primer, we do a sequencing of pAIDA-tyr1.

The results with the two primers show us that Tyr1 is in pAIDA.

22 July 2025

Preculture DilutionCollect the precultures of pAIDA-tyr1 into W3110 ΔompT, W3110 and and the contrôle tag His of the Laetitia team

• 1. Add 1 mL of preculture to a 500 mL flask containing100 mL of LB for each samples
• 2. Incubate for 3h to observe the stationary phase and perform the western Blot
• 3.At OD = 0,5, induction with 200 uL of IPTG

OD results for e.coli W3110 delta ompT and W3110

	t=1h	t=1h30	t=2h	t=2h30	t=2h40	t=3h
W3110 delta ompT	0,244	0,502	0,933	1,17	1,2	1,3
W3110	0,202	0,533	0,905	1,16	1,17	1,3
	t=1h	t=1h30	t=1h40	t=1h50	t=2h20	t=2h30	t=2h40	t=3h
Control	0,109	0,293	0,429	0,577	0,908	0,98	1,01	1,086

western Blot :Follow the same procedure as described on Friday 04/07

The total length of the protein is actually 1008pb , which corresponds to 37KDa.

The results show us we have expression only of the contrôle and no in w3110 ΔompT and w3110. So we will make preculture of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team

23 July 2025

Test for verification of melanin expression : western Blot

Preparation of LB+tyr+IPTG+Cm plates :

• 200ml of LB
• 260uL of Cm
• 100uL of IPTG
• 100mg of Tyr for 1g/L de concentration
• Streaking on plates of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT and w3110 .

Preculture of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team. Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C. For the control tag His of the Laetitia team, inoculated 1 colony into a 5 mL LB + Km Erlenmeyer flask. Shake overnight at 37°C.

24 July 2025

Preculture Dilution of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team.

Under sterile conditions (Bunsen Burner flame)

• 1. Collect the precultures of pAIDA-tyr1 into pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110, pAIDAw3110ΔompT, pAIDAw3110, w3110 ΔompT,w3110 and contrôle tag His of the Laetitia team.
• 2. Add 500uL of preculture to a 250 mL flask containing 50 mL of LB for each samples .
• 3. incubate for 3h to observe the stationary phase and perform the western Blot .
• We perform 2 dilutions in two different Erlenmeyer flasks: one that will be induced with 100uL of IPTG, and the other without IPTG .

OD Mesurements 600nm

	t=1h30	t=3h
pAIDA-Tyr1 W3110 delta ompT	0,7	1,24
pAIDA-Tyr1 W3110	0,75	1,2
pAIDA w3110	0,7	1,3
pAIDA w3110 delta ompT	0,76	1,2
W3110 delta ompT	0,7	1,4
W3110	0,7	1,2
Control tag His of the Laetitia team	0,5	1,05

Western BlotFollow the same procedure as described on Friday 04/07

western Blot Results without IPTG : The total length of the protein is actually 1008pb , which corresponds to 37KDa.

We found the same result like the first western blot. Conclusion: our colony 1 doesn’t exprime the tyrosinase. western Blot Results with IPTG

Here we have expression only of pAIDA in w3110 ΔompT and w3110 no in pAIDA-tyr1w3110 ΔompT and pAIDA-tyr1w3110. Conclusion: our colony 1 doesn’t exprime the tyrosinase. So we must make another PCR Colony with the colony 5,6,7,8,9,10,13 and 14. The samples that successfully contain tyr1 in pAIDA will be used to perform another western blot, and our control will be pAIDA in w3110 ΔompT or pAIDA in w3110.

25 July 2025

Colony PCR Protocol with oLS 4.1, oLS 4.2 and pAIDA Tyr 1 cells

We do this for 8 colonies of pAIDA Tyr1 and 1 colony of pAIDA (negative control) Streaking on LB+Cm plates over the weekend for colonies 5,6,7,8,9,10,13,14.

28 July 2025

pAIDA-I stockColony PCR Protocol with oLS 4.1, oLS 4.2 and pAIDA Tyr 1 cells

Follow the same protocol as Thursday, 26/06. This time we do 60°C and 1 minute.

Colonies 5, 6 and 7 have a 1kb like tyr1, colonies 8, 9, 10, 13 and 14 have the same result as negative control. So Colonies 5, 6 and 7 have tyr1 in pAIDA. So we will make pre-culture of colony Colonies 5, 6, 7 and pAIDAw3110ΔompT.

Test for verification of melanin expression : Preculture of colonies 5,6,7 and pAIDA w3110

Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C

29 July 2025

Test for verification of melanin expression Preculture Dilution of colonies 5,6,7 and pAIDA w3110

Under sterile conditions (Bunsen Burner flame)

• Collect the precultures of colonies 5,6,7 and pAIDAw3110 (the control).
• Add 500uL of preculture to a 250 mL flask containing 50 mL of LB for each samples .
• Incubate for 3h to observe the stationary phase and perform the western Blot .
• At OD=0,5 induction with 100uL of IPTG.

	t=1h	t=1h30	t=2h	t=2h20	t=3h
pAIDA W3110 control	0,228	0,534	-	-	1,3
pAIDA-Tyr1 W3110 delta ompT colonie 5	0,102	0,185	0,382	0,527	1,3
pAIDA-Tyr1 W3110 delta ompT colonie 6	0,104	0,199	0,386	0,527	1,25
pAIDA-Tyr1 W3110 delta ompT colonie 7	0,109	0,203	0,393	0,538	1,3

Western BlotFollow the same procedure as described on Friday 04/07

We do not have expression of tyrosinase in colonies 5,6 and 7 with the anti-His tag antibody. The anti-His tag antibody did not detect any protein bands in these samples.

30 July 2025

Test for verification of melanin expression : Preculture of pAIDA-tyr1w3110 ΔompT, pAIDA-tyr1w3110 and pAIDAw3110 (the control)

Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C.

31 July 2025

Test for verification of melanin expression : Preculture Dilution of colonies pAIDA W3110, pAIDA-tyr1 w3110 and pAIDA-tyr1 3110ΔompT

Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C.

Under sterile conditions (Bunsen Burner flame)

• 1. Collect the precultures of pAIDA-tyr1 inw3110 and pAIDA-tyr1 inw3110ΔompT and pAIDAw3110 (the control).
• 2. Add 500uL of preculture to a 250 mL flask containing 50 mL of LB for each samples .
• 3. Incubate for 3h to observe the stationary phase and perform the western Blot .
• 4. At OD=0,5 induction with 100uL of IPTG 200uM.

OD Mesurements 600nm

	t=1h	t=1h30	t=2h45	t=3h	t=4h
pAIDA W3110 Δ ompT	0,035	0,094	0,3	0,604	1,33
pAIDA W3110	0,2	0,625	-	1,25	-
pAIDA Tyr1 w3110	0,193	0,624	-	1,4	-

Western BlotFollow the same procedure as described on Friday 04/07

In this western Blot, we used : pAIDA W3110 as control, which has His tag at the N-terminal and a Myc tag at the C-terminal. And the mouse anti Myc antibody. We can see the expression of la tyrosinase in w3110 Δ ompT even if it is weak . This means that the sequencing results are correct : the tyrosinase gene is present in pAIDA W3110Δ ompT. So, we will do another western Blot with colony PCR samples 5,6 and 7 transformed in W3110Δ ompt ;to check if these colonies also express tyrosinase

Test for verification of melanin expressionPreculture of colonies PCR 5,6 and 7 transformed in W3110Δ ompt Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C

1 August 2025

Test for verification of melanin expression : Preculture dilution of colonies PCR 5,6 and 7 transformed in W3110Δ ompt

OD Mesurements :

	t=1h	t=3h
pAIDA W3110 delta ompT	0,4	1,3
pAIDA-Tyr1 W3110 delta ompT colonie 5	0,02	0,04
pAIDA-Tyr1 W3110 delta ompT colonie 6	0,4	1,3
pAIDA-Tyr1 W3110 delta ompT colonie 7	0,4	1,3

Western BlotFollow the same procedure as described on Friday 04/07

We did the western Blot only with the control and colonies 5 and 7 because we got the sequencing results of colony 6 and the signal was not good . In colonies 1 et 7 we see the tyrosinase experession. But we schooled have one band, not two.Maybe the tyrosinase was cut. So, we will do another western Blot with and without IPTG, both in dans pAIDA-Tyr1 w3110ΔompT and pAIDA-Tyr1 w3110, for the colonies 5 and 7 to show clearly that tryrosinase expression is in strain w3110ΔompT.

Preculture of colonies PCR 1and 5 transformed in W3110Δ ompt , colonies PCR 1 and 5 transformed in W3110, pAIDAW3110Δ ompt ( control) and pAIDAW3110 ( control)

04 August 2025

Test for verification of melanin expression

Preculture dilution of colonies PCR 1 and 5 transformed in W3110Δ ompt colonies PCR 1 and 5 transformed in W3110, pAIDAW3110Δ ompt ( control) and pAIDAW3110 ( control)

OD Mesurements :

	t=1h	t=1h10	t=1h20	t=1h30	t=3h	t=3h20
pAIDA W3110 delta ompT	0,5	-	-	-	1	1,2
pAIDA-Tyr1 W3110 delta ompT colonie 1	0,47	0,38	0,5	-	1	1,2
pAIDA-Tyr1 W3110 delta ompT colonie 5	0,41	0,44	0,5	-	1	1,2
pAIDA W3110	0,34	0,32	-	0,5	1	1,3
pAIDA-tyr1 W3110 colonie 1	0,26	0,33	-	0,5	1	1,3
pAIDA-tyr1 W3110 colonie 5	0,3	0,36	-	0,5	1,2	1,4

Western BlotFollow the same procedure as described on Friday 04/07

In this western blot, we see expression of colonies 1 and 5 only in the Δ+ strain, which is w3110Δompt induced with 1 mM IPTG. In w311, we only have the control expressed. This strain does not have the same physiological properties as w3110Δompt, so tyrosinase is not expressed. This confirms that tyrosinase is well expressed in w3110Δompt when induced. Therefore, we will do more western blots with colonies 1 and 5 at different IPTG concentrations to know at which concentration tyrosinase is expressed in a good amount.

Preculture of colonies 1 and 5 transformed in W3110Δompt.

5 August 2025

Preculture dilution of colonies PCR 1 and 5 transformed in W3110Δ ompt

OD measurement

For colony 1

	t=1h30	t=2h5	t=3h
0uM IPTG	0,2	0,56~0,6	1,2
50uM IPTG	0,2	0,6	-
200uM IPTG	0,2	0,6	-
500 uM IPTG	0,2	0,6	-
1 mM	0,2	0,6	1,4

For colony 5

	t=1h30	t=2h5	t=3h
0uM IPTG	0,2	0,6	1,4
50uM IPTG	0,2	0,6	-
200uM IPTG	0,2	0,6	-
500 uM IPTG	0,2	0,6	-
1 mM	0,2	0,6	1,2

Western BlotFollow the same procedure as described on Friday 04/07

From these results, we see that tyrosinase is well expressed in colony 1 at 200 um and 500 um [IPTG], and in colony 5 at 1 mM. Therefore, we will do other western blots of these colonies at three different temperatures and times: 30°C, 37°C and 16°C overnight, to see if the amount of tyrosinase expression changes or stays the same.

06 August 2025

Preculture dilution of colonies PCR 1 and 5 transformed in W3110Δ ompt.

OD Mesurements

For colony 1 at 37°C

	t=1h	t=1h30	t=2h	t=2h30	t=3h	t=16°C over night
200uM IPTG	0,1	0,2	0,4	0,7	1,7	1,9
500uM IPTG	0,09	0,2	0,4	0,7	1,7	1,9

For colony 5 at 30°C

	t=1h	t=2h	t=3h	t=16°C over night
200uM IPTG	0,1	0,2	0,4	0,7	1,7	1,9
500uM IPTG	0,09	0,2	0,4	0,7	1,7	1,9

For colony 5 at 30°C

	t=1h	t=2h	t=3h	t=16°C over night
1mM IPTG	0,1	1,1	0,3	0,2	0,6	1,4

Western Blot

With the different concentrations and temperatures, we can see that tyrosinase is expressed, even if it is cut at different sizes.

18 August 2025

The PCR Colony doesn’t necessary because the researchers from Sweden optimized them plasmid pAIDA-tyr1 and we see in them article the expression of tyrosinase and melanin expression . So we will make western blot to verify if we have the same thing like our plasmid pAIDA-Tyr from addgene.

Preculture of pAID-Tyr1 w3110, pAIDA w3110ΔompT, colony 1´, 3´ and 4´ pAID-Tyr1 w3110ΔompT

Inoculate 1 colony in to 5 ml LB + cm Erlenmeyer flask. Shake overnight at 37C.

These preculture will be used both to perform western blot and to test the enzymatic activity of tyrosinase.

19 August 2025

Results of transformation of the pAIDA-Tyr1 With Supercompetent Cells W3110 and W3110 Δ ompt

Colony PCR Protocol with oLS 4.1 and 4.2 and pAIDA Tyr1 cells Follow the same protocol as Thursday, 26/06. This time we do 60°C and 1 minute.

The PCR Colony doesn’t necessary because the researchers from Sweden optimized thème plasmid pAIDA-tyr1 and we see in them article the expression of tyrosinase and melanin expression . So we will make western blot to verify if we have the same thing like our plasmid pAIDA-Tyr from addgene.

20 August 2025

Test for verification of melanin expression : visual test

Preparation plates :

For our plasmide pAIDA FROM addgene( induced by IPTG) :

• 10 mL LB
• 20uL of 10mM Cu2+
• 20uL IPTG (200uM)
• 13uL Cm

For the plasmide of David Hornstrom team( induced by rhamnose) :

• 10 mL LB
• 20uL Cu2+ to weigh 10 mg for 4 mL H2O
• 100 uL Rhamnos ( 1g/L)
• 13uL Cm

Take 1 colony for each plasmid and steak on each plate, then incubate at 37C over night.

We have the same results with the plasmide of Sweden team or ours, whatever antibody used.

21 August 2025

Test for verification of melanin expression : visual test results

We see the melanin production in the plamsmid induced by rhamnose and no in the plasmid induced by IPTG.

So we will make another test of plasmid induced by IPTG.

Take 1 colony and steak on the plate, then incubate at 37C over night.

Preparation plates :

plasmide induced by IPTG :

• 10 LB
• 20uL of 10mM Cu2+
• 20uL IPTG (200uM)
• 13uL Cm

Enzymatic activity assay

22 August 2025

Test for verification of melanin expression : visual test results

We can clearly see the appearance of some black spots, which indicate that melanin is being produced.

26 August 2025

Activity test for pAIDA-Tyr1 Sweden

Preculture

27 August 2025

Preparation of M9 media

For 1L :

• 100 mL M9 10x
• 887 mL water sterile
• 10 mL Glucose 40%
• 2 mL MgSO4 1M
• 1 mL CaCl2 0,1M

Preparation of M9 media agarose 1,5 %

• 150 mL M9 media
• Add 1,5 g of agarose
• Boil to dissolve the agarose and sterilize in the microwave

Activity test with pAIDA Tyr-1 Sweden

We adjust the DO at 0,05 for a final volume of 25ml.

Delta ompt pAIDATry-1: 4,6mL of preculture (DO: 0,27)W3110 pAIDATry1: 4,3mL of preculture (DO: 0,29)

Add in each erlenmeyer 25mL of Lb and 200µL of Rhamnose with the right volume of periculture

Incubate at 37°C and 180rpm during 3hours

Centrifugate at 3270g during 10minutes

Resuspend the pellet in 10mL of tampon

Tampon préparation :

• 40µL of cooper
• 5mL of Tris-HCL pH 7.2
• 100mg of tyrosine
• Add water of a finale volume of 100mL
• Put the solution in the microwave until it’s clear

Incubate at 30°C, 220rpm during 15hours

28 August 2025

Activity test with pAIDA Tyr-1 Sweden

Measurement of Melanin in the supernatantCentrifugate at 13 000 rpm during 5min, Measure the supernatant at 400nm, the blank is the tampon made

17 June 2025

Shikimate pathway amplification: Mutation AroF

Preparation of E. coli CC118 and E. coli CC118 pKNG101 Pre-cultures for Supercompetent Cell Preparation

Inoculate 1 colony of E. coli CC118 into a 5 mL LB flask.

Inoculate 1 colony of E. coli CC118 pKNG101 into a 5 mL LB flask.

Incubate overnight at 37°C with shaking.

18 June 2025

Preculture Dilution

Follow the same procedure as described on Tuesday, 03/06 but this time with E.coli CC118

OD Measurement by Spectrophotometer

Measure the OD of the cells in 1 mL cuvettes

CC118 : 0,77

CC118 pKNG 101 : 0,32

Centrifugation of CC118 cells

Follow the same procedure as described on Friday 13/06 (with DH5 alpha) this time with 50 mL of CC118, so only one falcon.

Preparation of Supercompetent Cells CC118

Follow the same procedure as described on Tuesday, 03/06.

Plasmid Miniprep Kit for E. coli CC118 pKNG 101

Follow the NEB protocol below for T1110S (50-prep) :

Buffer Preparation Guidance (NEB #T1110)

• Add supplied Monarch RNase A to Monarch Buffer B1 for a final concentration of 100 μg/ml. Mix and store at 4°C.
• For T1110S (50-prep) kit, add 60 μl of Monarch RNase A to Monarch Buffer B1.
• Add isopropanol to the Monarch Buffer BZ prior to use (0.43 volumes of isopropanol per volume of Monarch Buffer BZ).
• For T1110S (50-prep) kit, add 3.6 ml of isopropanol to Monarch Buffer BZ.
• Add ethanol to the Monarch Buffer WZ prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Buffer WZ).
• For T1110S (50-prep) kit, add 20 ml of ethanol to Monarch Buffer WZ.
• Always keep all buffer bottles tightly closed when not in use.

Plasmid Miniprep Protocol using Centrifugation (NEB #T1110)

Plasmid Miniprep Protocol

• 1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard the supernatant. For a standard miniprep to prepare plasmid for restriction digestion or PCR, we recommend 1.5 ml of culture, which is sufficient for most applications. Ensure cultures are not overgrown; 12–16 hours is usually ideal for optimal growth.
• 2. Resuspend the pellet in 200 μl of Monarch Buffer B1 (pink •). Vortex or pipet mix to ensure cells are completely resuspended. There should be no visible clumps.
• 3. Add 200 μl of Monarch Buffer B2 (blue •), gently invert the tube 5-6 times, and incubate at room temperature for 1 minute. Do not vortex. The color should change to dark pink, and the solution should be transparent and viscous. Handle the sample gently to reduce the risk of shearing chromosomal DNA, which can be co-purified as a contaminant. Avoid incubating longer than one minute to prevent irreversible plasmid denaturation.
• 4. Add 400 μl of Monarch Buffer B3 (yellow •), and gently invert the tube until neutralized. Do not vortex. The color should be uniformly yellow and a precipitate will form. Incubate for 2 minutes. Gentle but uniform mixing will ensure complete neutralization without shearing chromosomal DNA.
• 5. Centrifuge the lysate for 2–5 minutes. The pellet should be compact; spin longer if needed. Spin time should not be less than 2 minutes. For culture volumes > 1 ml, we recommend a longer spin (~5 minutes) to ensure efficient RNA removal by RNase A and a more compact pellet, which will lower the risk of clogging the column.
• 6. Carefully transfer the supernatant to the Monarch Spin Column S2D and centrifuge for 1 minute. Discard the flow-through.
• 7. Re-insert the Monarch Spin Column S2D in the Monarch Spin Collection Tube and add 200 μl of Monarch Buffer BZ (wash 1). Centrifuge for 1 minute and discard the flow-through. This is a high-salt wash step that helps remove any residual RNA, protein, and other contaminants. Incubate for 5 minutes after adding Monarch Buffer BZ and before centrifugation if the plasmid will be used for transfection.
• 8. Wash by adding 400 μl of Monarch Buffer WZ (wash 2) and centrifuge for 1 minute and discard the flow-through. Centrifuge again for 1 minute and discard the flow-through.
• 9. Transfer the column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not touch the flow-through. If in doubt, re-spin for 1 minute.
• 10. Add ≥ 30 μl of Monarch Buffer EY (preference if it’s with H2O but this time I did it with the Buffer EY) to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA. Nuclease-free water (pH 7– 8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of Monarch Buffer EY is used, but the DNA will be less concentrated. For larger size plasmids (≥ 15 kb), incubate the column with an elution buffer at room temperature for 5 minutes to maximize the yield. Alternatively, the elution buffer can be heated to 50°C before use.
• 11. Use a Nanodrop Spectrophotometer to measure pKNG DNA.
• 12. Store the DNA at -20°C.
• 13. Nanodrop result : pKNG 101 : 52 ng/µL

20 June 2025

Test : Transformation with Supercompetent Cells with CC118 and pKNG 101

Follow the same procedure as described on Tuesday, 03/06.

For the last step, spread 6µl of bacteria onto a LB + Streptomycine plate for each plasmid (CC118 (control) and CC118 pKNG 101) (using a spreader/pasteur pipette)

Contamination Test

Spread 6 µL of E. coli CC118 on half of 3 plates and 6 µL of E. coli CC118 pKNG 101 on the other half of each plate (on LB agar + ampicillin, LB agar + kanamycin and LB agar + chloramphenicol)

24 June 2025

PCR and Electrophoresis of genomic DNA of W3110 with oLS 2.1 + 2.2 and oLS 2.3 + 2.4 using Q5® High-Fidelity 2X Master Mix following NEB Protocol

Follow the same procedure as described on Thursday 19/06. This time we do 2 PCR tubes.

For W3110, we pick an isolated colony with a toothpick and put it in 30 µL of H2O. Heat at 92°C for 10 min to lyse cells.

F.1 (aroF mut) : primers = oLS 2.1, oLS 2.2 ; Template DNA = W3110

F.2 : primers = oLS 2.3, oLS 2.4 ; Template DNA = W3110

For the 25-35 cycles, we put the 2nd step at 72°C.

F.1 and F.2 = 0.5 kB

Wednesday 25/06

Digestion of pKNG101 with BAMH1:

In a 1.5 mL Eppendorf tubes add (for a final volume of 30µL):

• 20µL pkng101
• 5 µL H20
• 3µL Buffer R CutSmart
• 2 µL BAMHI

Incubate for 3 hours at 37°C

PCR and DNA Cleanup of F.1 and F.2, following NEB Protocol (#T1130)

Buffer Preparation

• Add isopropanol to the Monarch Buffer BZ prior to use (0.43 volume of isopropanol per volume of Buffer BZ).
• For T1130S (50-prep) kit, add 18 ml of isopropanol to Monarch Buffer BZ.
• Add ethanol to the Monarch Buffer WZ prior to use (4 volumes of ≥ 95% ethanol per volume of Buffer WZ).
• For T1130S (50-prep) kit, add 20 ml of ethanol to Buffer WZ.
• Always keep all buffer bottles tightly closed when not actively in use.

Standard Cleanup Protocol using Centrifugation

1. Add 5 volumes (e.g., 250 μl) of Monarch Buffer BZ to 1 volume (e.g., 50 μl) of sample. Mix well by pipetting up and down or flicking the tube. Do not vortex. Using a sample volume of 20-100 μl is recommended. For samples less than 20 μl, adjust the volume with TE or nuclease-free water to 20-100 μl. For diluted samples larger than 800 μl, load 800 μl first, proceed with step 2, and repeat as needed.

1. 2. Insert the Monarch Spin Column S1A into the Monarch Spin Collection Tube and load the sample onto the column. Spin for 1 minute, then discard the flow-through.
2. Re-insert the column into the collection tube. Wash by adding 200 μl of Monarch Buffer WZ and spin for 1 minute. Discarding flow-through is optional.
3. Repeat wash (step 3).
4. Centrifuge again for 1 minute and discard the flow-through.
5. Transfer the column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column does not touch the flow-through. If in doubt, re-spin for 1 minute.
6. Add 5-20 μl of H2O to the center of the matrix to elute DNA. Wait for 1 minute, and spin for 1 minute.

SLIC

Two different T4 polymerases are used (from two different kits, including one from the former iGEM 2024 team). Test in two different ratios: 1-1(1µL vector and 1µL of each primer) or 1-2 for each T4 polymerase. To be performed on ice

Preparation of tubes:

In tube ratio 1-1:

• 5,6 µL H20
• 1µL Buffer
• 1 µL PKNG vector
• 1 µL PCR fragment F1
• 1 µL PCR fragment F2
• 0,4 µL T4 polymerase

In tube ratio 1-2:

• 3,6 µL H20
• 1 µL Buffer
• 1 µL PKNG vector
• 2 µL PCR fragment F1
• 2 µL PCR fragment F2
• 0,4 µL T4 polymerase

Lastly, T4 polymerase is added to the tubes on ice.

Incubate for 2min45 at room temperature

Return to ice

CC118 cell transformation: Follow the same procedure as described on Tuesday, 03/06. For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube (CC118 ratio 1-1 T4 pol 2024 and CC118 ratio 1-2 T4 pol 2024, and CC118 ratio 1-1 T4 pol 2025 and CC118 ratio 1-2 T4 pol 2025) (using glass beads)

Incubate at 37°C overnight

26 juin

Colony PCR Protocol with oLS 3.2, oLS 3.3 and CC118 pKNG 101 Aro F 2024 ratio 1-2 cells

We do this for 24 colony.

1. Pick a well-isolated colony with a toothpick of CC118 pKNG 101 AroF
2. Transfer the colony into a Sm + LB plate, than into a PCR tube
3. Prepare a PCR mix in an Eppendorf tube:
   • 5 µL of EconoTaq (2X)
   • 0.5 µL of Forward Primer (10 µM)
   • 0.5 µL of Reverse Primer (10µM)
   • H2O to complete until final volume
4. For 24 PCR tubes, we prepare 260 µL of PCR mix so everything x26 + 104 µL of H2O
5. To finish, vortex the PCR mix
6. Add 10 µL of PCR Mix into every PCR tube and vortex a little.
7. Transfer PCR tubes to a PCR machine and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

STEP	TEMP	TIME
Initial Denaturation	94°C	5 minutes
30 Cycles	94°C	30 seconds
*50–72°C	we did 60°C	30 seconds
72°C	1 min/kb	we did 1 min 30
Final Extension	72°C	5 minutes
Hold	16°C

Electrophoresis:

• We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel.
• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB. (I forgot)

Every vector is empty. We have to try again.

Colony PCR Protocol with oLS 3.2, oLS 3.3 and CC118 pKNG 101 Aro F 2025 ratio 1-2 cells

We do this for 8 colony. Follow the same protocol as today. The electrophoresis will be done tomorrow.

27 June

Colony PCR Protocol with oLS 3.2, oLS 3.3 and CC118 pKNG 101 Aro F 2025 ratio 1-2 cells

We finished yesterday's protocol with the electrophoresis.

Every vector is empty. We have to try again.

01 July

PCR and Electrophoresis of genomic DNA of W3110 with oLS 2.1 + 2.2 and oLS 2.3 + 2.4 using Q5® High-Fidelity 2X Master Mix following NEB Protocol

• Follow the same procedure as described on Thursday 19/06. This time we do 2 PCR tubes.
• For W3110, we pick an isolated colony with a toothpick and put it in 30 µL of H2O. Heat at 92°C for 10 min to lyse cells.
• F.1 (aroF mut) : primers = oLS 2.1, oLS 2.2 ; Template DNA = W3110
• F.2 : primers = oLS 2.3, oLS 2.4 ; Template DNA = W3110
• For the 25-35 cycles, we put the 2nd step at 72°C.

X = PCR 1 (we did it a second time)

Plasmid Miniprep Kit for E. coli CC118 pKNG 101, pAIDA-I

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.
• Result Nanodrop :
  • pKNG 101 : 19,78 ng/µL
  • pAIDA-I : 18 ng/µL

03 July

Digestion of pKNG101 with BAMH1:

• Follow the same protocol as described on Wednesday 25/06

DNA Cleanup of pKNG 101, following NEB Protocol (#T1130)

• Follow the same protocol as Wednesday, 25/06.
• Result Nanodrop : pKNG 101 : 52.23 ng/µL

SLIC of pKNG 101 with fragments 1 and 2.

• We followed a new SLIC protocol.
• We use CC118 cells for the transformation

Preparation of Cloning reaction:

Component	Volume	Final Concentration
Linearized vector (pKNG)	0.5 µL (10ng/µL)	100ng
Insert (fragment 1 and 2)	1.5 µL (a 1/10 dilution)
10X BSA	1 µL
10X NEB Buffer 2	1 µL
H2O	Up to 10 µL

Calculate the amount of the insert needed as follows:

ng of insert needed = [100 ng (ng of vector used) × size of the insert (kb)] / size of vector (kb) × molar ratio of insert:vector

Here we have:

• Vector : 6.982 kb, 100 ng
• Insert : 0.43 kb
• Molar ratio of the vector:insert = 1:3

Insert needed = 100 ng vector × 0.43 kb insert / 6.982 kb vector × 3/1 = 18.5 ng

We calculate how much of F.1 and F.2 we need to have 18.5 ng.

• F.1 : Insert needed/Concentration of the fragment = 18.5/37.76 = 0.4 µL
• F.2 : 18.5/101.91 = 0.2 µL

[Since we put H2O to have our fragment, we add 0.2 µL of Tris pH 8 for our 10mM fragment.]

Attention: As you need to treat the reaction mixture with T4 DNA polymerase for a short duration you should keep the vial of reaction mixture at room temperature rather than on ice. Be sure to mix the vector and insert completely before adding the T4 DNA polymerase

1. Add 0.5 µL of T4 DNA Polymerase (3U/µL, NEB) to the mixture, mix immediately, and incubate at room temperature (~25°C) for 1 min or 50°C for 30 sec.
2. To stop the reaction, place the reaction mixture on ice immediately and incubate on ice for 10 min.
3. (You may use 10 µL (final volume) of the reaction mixture in 100µL of competent cells.)
4. Keep chemically competent E.coli cells on ice for 10 minutes to thaw.

CC118 cell transformation: Follow the same procedure as described on Tuesday, 03/06. For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube (CC118 ratio 1-1 T4 pol 2024 and CC118 ratio 1-2 T4 pol 2024, and CC118 ratio 1-1 T4 pol 2025 and CC118 ratio 1-2 T4 pol 2025) (using glass beads)

Incubate at 37°C overnight

Friday 04/07

Result of the SLIC:

It didn't work, we have to do it again and change the ratio.

SLIC of pKNG 101 with fragments 1 and 2.

• Follow the same procedure as described on Thursday 03/07
• This time we do it with a ratio 1:5 and we do 4 tubes (2 tubes with a ratio 1:3 like yesterday and 2 tubes with ratio 1:5)

CC118 cell transformation: Follow the same procedure as described on Tuesday, 03/06. with 1 tube 1:5 and 1 tube 1:3

For the 2 other tubes:

1. Incubate the cells on ice for 20 min
2. Heat shock the cells at 42°C for 45 sec
3. Incubate the cells on ice for 2 min
4. Add 200 µL LB broth to the cells
5. Incubate the cells at 37°C for 1 hr
6. Spread the cells onto agar plates containing appropriate antibiotics
7. Incubate the plates at 37°C overnight

For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube

07 July

Respread of contaminated SLIC colonies

Results of Transformation with SLIC from last week : Our petri dishes were contaminated, we repoured the colonies we thought could correspond to our on new petri dishes and incubated them at 37°C

08 July

Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F 2024 ratio 1:3 et 1:5 cells

• Follow the same protocol as Thursday, 26/06

Results:

we did not find our SLIC desired product

09 July

Electrophoresis of Digested Pkng101 overnight

• Follow the same protocol as Thursday 19/06 for the electrophoresis

The plasmid wasn't digested

Digestion of pKNG101 with BAMH1, following Promega Protocol (Usage information)

In a 1.5 mL Eppendorf tubes add (to get 1ng for digested plasmid):

• 16,6µL pkng101
• 2µL E Buffer
• 0,2 µL BSA
• 0,5 µL BAMHI

Overnight at 37°C no shaking

10 July

PKNG

Electrophoresis of digested Pkng101 overnight

• Follow the same protocol as Thursday 19/06

The plasmid was digested, it was at 6KB

SLIC aroF

Test in two different ratios: 1-2(1µL vector and 2µL of each primer) or 1-3 with the T4 polymerase of our kit. To be performed on ice

Preparation of tubes:

In tube ratio 1-3:

• 1,6 µL H20
• 1µL Buffer (R.1)
• 1 µL PKNG vector
• 3 µL PCR fragment F1
• 3 µL PCR fragment F2
• 0,4 µL T4 polymerase

In tube ratio 1-2:

• 3,6 µL H20
• 1 µL Buffer (R 2.1)
• 1 µL PKNG vector
• 2 µL PCR fragment F1
• 2 µL PCR fragment F2
• 0,4 µL T4 polymerase

Lastly, T4 polymerase is added to the tubes on ice.

Incubate for 2min45 at room temperature

Return to ice

CC118 cell transformation: We used 3µL of our mix and 50µL CC118. Follow the same procedure as described on Tuesday, 03/06. For the last step, spread 100µl of bacteria onto a LB + Streptomycine plate for each tube (using glass beads)

Incubate at 37°C overnight

11 July

Colony PCR Protocol with oLS 2.5, oLS 3.6 and CC118 pKNG 101 Aro F 2024 ratio 1-2 cells

• Follow the same protocol as Thursday, 26/06. We do this for 5 colony

The plasmid is empty we have to try again

result

Slic

• Follow the same protocol as Wednesday, 25/06. This time we did the ratio 2/2 (2µL of pkng for 2µL fragment)

15 July

Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F 2024 ratio 2/2

• Follow the same protocol as Thursday, 26/06.

17 July

Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F

Follow the same procedure as described on Thursday 26/06

18 July

result slic

21 July

Colony PCR Protocol with oLS 2.5, oLS 2.6 and CC118 pKNG 101 Aro F

• Follow the same procedure as described on Thursday 26/06

The vector contains AroF !!!!

Preculture DH5alpha pKng101-AroF* colony 6, 8, 11 for Miniprep

Test for verification of melanin expression : western Blot

Pre-culture of pAIDA-tyr1 into W3110 ΔompT, W3110 and the contrôle tag His of the Laetitia team (CHI)

Control tag His of the Laetitia team

• Inoculated 1 colony into a 5 mL LB + Cm Erlenmeyer flask. Shake overnight at 37°C.

For the contrôle tag His of the Laetitia team, inoculated 1 colony into a 5 mL LB + Km Erlenmeyer flask. Shake overnight at 37°C.

22 July

Miniprep pkng 101 AroF*, colony 6, 8, 11

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.

23 July

Result of transformation pkng101-AroF* in W3110 ΔompT:

W3110 is resistant to streptomycin so we need to change pkng101, and do the Slic again.

24 July

Transformation of pKng101 AroF* colony 6 in W3110

• Follow the same procedure as described on Tuesday, 17/06.

Preculture of CC118 pkng AroF* colony 11, 8, and 4

• Inoculate colony 3 into a 5 mL Erlenmeyer flask of LB with Streptomycin

25 July

Result of the transformation of pKng101 AroF* colony 6 in W3110

28 July

Slic of AroF

• Follow the same protocol as Wednesday 25/06. This time we did the ratio 2/2 (2µL of Plasmid for 2 µL of DNA fragments)

Result of Slic AroF

Preculture of DH5 alpha pko3

• Add 5mL of LB and 6,5µL of Cm incubate overnight at 30°C

PCR for linearized PKO3 using Q5® High-Fidelity 2X Master Mix following NEB Protocol with oLS 4.3 and oLS 4.4

• Follow the same procedure as described on Thursday 19/06. For the 72°C step we did 2:50

29 July

Result of PCR for linearized PKO3 using Q5® High-Fidelity 2X Master Mix following NEB Protocol with oLS 4.3 and oLS 4.4

• Follow the same procedure as described on Thursday 19/06.

PCR and Electrophoresis of genomic DNA of W3110 with oLS 4.5 + 2.2 and oLS 2.3 + 4.6 using Q5® High-Fidelity 2X Master Mix following NEB Protocol

• Follow the same procedure as described on Monday 23/06.

Slic of AroF

• Follow the same protocol as Wednesday 25/06. This time we did the ratio 2/2 (2µL of Plasmid for 2 µL of DNA fragments)

31 July

slic

We did it again

• Follow the same protocol as Wednesday 30/07/2025

PCR for linearized PKO3 using Pfu Turbo with oLS 4.3 and oLS 4.4

Add for 50 µL final:

• 2,5 µL oLS 4.3
• 2,5 µL oLS 4.4
• 2µL pKO3
• 5µL dNTP
• 5µL Buffer
• 1 µL Pfu Turbo

For the second step we did 56°C and the elongation time was 6min.

For the Slic Follow the same protocol as Wednesday 30/07/2025

For the Transformation incubate at 30°C with shaking during 1H20

Monday 04/08/2025

Results of Slic PCR for linearized PKO3 using Pfu Turbo and PKO3 linearized with Q5 master Mix

05 August

PCR for linearized PKO3 using Q5® High-Fidelity 2X Master Mix following NEB Protocol with oLS 4.3 and oLS 4.4

• Follow the same procedure as described on Thursday 19/06. For the 72 step we did 3min30 and put the Tm at 56°C. We did that with 4 tubes using our primer and PI's primers. He did the manipulation in parallel

Slic

• Follow the same protocol as Wednesday 25/06. We did the transformation in DH5alpha

06 August

Slic

• Follow the same protocol as Wednesday 25/06

PCR and Electrophoresis of genomic DNA of W3110 with oLS 4.5 + 2.2 and oLS 2.3 + oLS 4.6 using Q5® High-Fidelity 2X Master Mix following NEB Protocol

• Follow the same protocol as Tuesday 24/06

07 August

Colony PCR with olS 4.7 and 4.8

There was an issue with olS 4.8. It didn't hybridize completely on the plasmid.

So we did 3 difference Colony PCR with 3 couple of oligos différents to try to amplify the fragment of AroF

• Tube a: olS 4.5 and olS 2246 (it hybridizes on PKO3, gave by our PI)
• Tube b: olS 4.8 and olS 4.5
• Tube c: olS 4.7 and olS 4.6

Follow the same protocol as Thursday 26/06

Results

08 August

Result of electrophoresis of the 3 Colony PCR:

• Tube a: olS 4.5 and olS 2246 (it hybridizes on PKO3, gave by our PI)
• Tube b: olS 4.8 and olS 4.5
• Tube c: olS 4.7 and olS 4.6

The oligos of the tubes A are going to be used for the next Colony PCR.

Only the colony 3, 4, 6 on the Pfu petri dish contain the fragment AroF.

Colony PCR Protocol with oLS 4.7/4.8 and oLS 4.5 and 2246(iGEM Slic)

• Tube a: olS 4.5 and olS 2246 (gave by our PI it hybridizes on PKO3)
• Tube d: olS 4.7 and olS 4.8

For the PI's Slic i lost the products. So I only did 2 Colony PCR. None of this colony have the fragment.

12 August

Shikimate pathway amplification: Mutation AroF

Plasmid Miniprep Kit for PKO3 AroF of colony 3,6,4

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.
• 3, 4,6: 15 ng/µL

Transformation of PKO3 AroF colony 3,6,4 into W3110 Δ OmpT

• Follow the same procedure as described on Tuesday, 03/06.
• Transform 3µL plasmid into 50µL of Cell
• Incubate with shaking at 30°C during 1H20
• Spread 50µL on CM and incubate overnight at 42°C

13 August

Shikimate pathway amplification: Mutation AroF

Result Transformation pko3 AroF* into W3110 ΔompT (first recombination event)

Preparation of LB + Sucrose petri dish for the second recombination event

For 5 petri dishes, one for each colony add:

• 12 mL of Sucrose 50% (it must be filtered)
• 88 mL of LB

We want a concentration final of 6% Sucrose.

14 August

Transformation pko3 AroF* into W3110 ΔompT

Streak into plate:

• Streak positives clones () into LB agar + Sucrose 6% (1 plate for 1 colony)
• Incubated for 72 hours at 12°C

18 August

Results of spreading W3110 Δ OmpT pko3 AroF

We put this plates at 37°C

19 August

Shikimate pathway amplification: Mutation AroF

Results of spreading W3110 Δ OmpT pko3 AroF after we put the plate at 37°C

20 August

Shikimate pathway amplification: Mutation AroF

The sequencing results are horrible. I may have extracted another plasmid.

21 August

Shikimate pathway amplification: Mutation AroF

Colony PCR DH5 alpha PKO3 Arof with oLS 2246 and oLS 4.5

• Follow the same protocol as Thursday, 26/06.

I have one colony right (the 9)

Friday 22/08/2025

Shikimate pathway amplification: Mutation AroF

Miniprep of the colony 9

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.

Slic

• Follow the same protocol as Wednesday, 25/06. We did the ratio 2/2.

Monday 25/08/2025

Shikimate pathway amplification: Mutation AroF

Colony PCR W3110 Delta OmpT PKO3 Arof with oLS 2246 and oLS 4.5

• Follow the same protocol as Thursday, 26/06.

27 August

Shikimate pathway amplification: Mutation AroF

Colony PCR DH5 alpha PKO3 Arof with oLS 2246 and oLS 4.5

• Follow the same protocol as Thursday, 26/06.

17 June 2025

Preparation of 10% glycerol solution for W3110 :

• Vi = 10% (Cf) * 200 mL (Vf) / 80% (Ci) = 25 mL glycérol 80%
• Added H2O to final volume of 200 mL

Preparation of Electrocompetent Cells W3110 cells

• 1. Pre-chilled on ice for 15min
• 2. Two washes with 35mL of 10% glycerol (keep on ice)
• 3. Transferred 35 mL from the Erlenmeyer into a 40mL Falcon tube
• 4. Centrifuged at 5000 rpm 10 min 4°C
• 5. Discarded supernatant and resuspended in 35mL 10% glycerol (using two sticks)
• 6. Repeated centrifugation 5000 rpm 10 min 4°C
• 7. Discarded supernatant
• 8. Resuspended in 35mL 10% glycerol (using two sticks)
• 9. Final Centrifugation 5000 rpm 10 min 4°C
• 10. Discarded supernatant
• 11. Resuspended in 250 uL 10% glycerol (using two sticks)

Electroporation of pKOBEG Vector into W3110

• Prepared two 1,5mL Eppendorf tubes:
  • CONTROL : 60 µL W3110 in 10% glycerol
  • pKOBEG : 60 uL W3110 in 10% glycérol + 1 uL vecteur pKOBEG
• 1. Incubated on ice for 15min
• 2. Transferred les 60 µL into electroporation cuvettes
• 3. Performed electroporation using the appropriate machine
• 4. Add 1mL of LB to each cuvette (control and vector-containing sample) and resuspend.
• 5. Transfer into two 1.5 mL Eppendorf tubes
• 6. Incubate in a shaker at 37°C, 750 rpm for 1 hour
• 7. Centrifuge at 5000 rpm for 5 minutes
• 8. Remove 900 µL of supernatant
• 9. Resuspend the pellet in the remaining 100 µL
• 10. Plate serial dilutions on LB agar + Chloramphenicol for selection
• 11. Store the remaining WT and pKOBEG bacteria at 4°C

01 July

Pre-culture Dilution

Under sterile conditions (Bunsen Burner flame)

1. Collect the precultures of E. coli W3110 ΔompT
2. Add 250 µL of preculture to a 125 mL erlenmeyer flask containing 25 mL of LB for W3110 ΔompT
3. Incubate with with chalking for 2 hours at 37°C

OD Measurement via spectrophotomètre

• Measure the OD of cells in 1 mL cuvettes, targeting OD600 = 0.5.
• W3110 ΔompT:

Preparation of Electrocompetent Cells W3110 ΔompT Cells:

• Follow the same protocol as Friday 13/06.

09 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG :

Preculture Dilution of W3110 Δ ompT pKOBEG :

Under sterile conditions (Bunsen burner flame)

1. Retrieve the W3110 Δ ompT pKOBEG preculture
2. Add 1 mL of preculture to a 500 mL Erlenmeyer flask containing 100 mL of LB
3. Incubating at 30°C with shaking at 180 rpm until A600 reaches 0,2 - 0,4

DO measure of W3110 Δ ompT pKOBEG :

• After 3 hours : DO 0,35

Induction of W3110 Δ ompT pKOBEG :

• When the DO reaches 0,2-0,4 : Add 0,05% of L-arabinose in the W3110 Δ ompT pKOBEG culture.
• Incubate at 30°C until an A600 of 0,6-0,8.

DO measure of W3110 Δ ompT pKOBEG :

• Result DO 0,8

Optional : (we did it)

• Incubate 15 min at 42°C

Prepare electrocompetent cells :

• Follow the same protocol as Friday 13/06

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol :

• Here we use 1 µL of Template DNA (pKD4)
• Reaction Setup: Follow the same protocol as Thursday 19/06 for 25µL

Transfer PCR tubes to a PCR machine and begin thermocycling.

Thermocycling Conditions for a Routine PCR :

STEP	TEMP	TIME
Initial Denaturation	98°C	3 minutes
25–35 Cycles	98°C	40 seconds
*57°C	57°C	40 seconds
72°C	72°C	40 seconds
Final Extension	72°C	2 minutes

Electrophoresis :

• We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel for a PCR with pKD4.
• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

2 : PCR pKD4 (oLS 1.3 and oLS 1.4)

• The band of the kanamycin is very light, so we did it again with a different temperature of hybridization.

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol :

• Reaction Setup: Follow the same protocol as Thursday 19/06 for 25µL
• Thermocycling Conditions for a Routine PCR : Follow the same protocol as Wednesday 09/07 but for the hybridization : 62 °C

Electrophoresis :

• We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel for a PCR with pKD4.
• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

Prepare electrocompetent cells :

• Follow the same protocol as Friday 13/06.
• And freeze aliquots (100 µL) in liquid nitrogen, then store at -80°C.

10 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG :

PCR clean up of pKD4 :

• Follow the same protocol as Wednesday, 25/06
• We mix the 2 tubes of PCR that we did Wednesday 09/07
• Nanodrop result : pKD4 : 64,85 ng/µL

Electroporation 600ng of PCR pKD4 :

• Follow the same protocol as Friday 13/06
• We had 10 µL of pKD4 into 50 µL of cells competent two times to test on different concentrations of DL glutamic acid on plate LB.
• Select on Kan LB 10 mM DLGlu plates and Kan LB 2 mM DLGlu.
• Incubate on at 37°C

Preparation of DL-glutamic acid monohydrate (DL-glu) and LB agar, Kan plates :

Preparation of DL glu stock :

• Molecular Weight of DL-glu : 165,145 g/mol
• Preparation of the stock solution : DL-Glu is miscible in water : solubility 8,83 mg/ml
• Weigh 441,5 mg of DL-glu
• Add 50 mL of distilled water to dissolve
• Filter to sterilize
• Store at 4°C
• The concentration : C = m / (V × M) = 0,4415 g / (0,05 L × 165,145) = 0,053 mol/L so it's 53 mM

Plate of 20 µL LB, Kanamycin and 10 mM DL Glu :

• Volume of DL glu stock to preleve : 20 mL × (10 mM / 53 mM) = 3,8 mL

Plate of 20 µL LB, Kanamycin and 2 mM DL Glu :

• Volume of DL glu stock to preleve : 20 mL × (2 mM / 53 mM) = 0,8 mL

Pre-culture preparation of pAIDA Tyr1 1, 5 and 6 to do a Miniprep

• Inoculate colony 1 of pAIDA Tyr1 into a 5 mL Erlenmeyer flask of LB with Chloramphenicol.
• Same for pAIDA Tyr1 colonies 5 and 6.
• Shake overnight at 37°C.

11 July

Result of Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 :

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 :

Colony PCR Protocol with oLS 1.1 and 5'KAN, oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4 :

• We do this for 8 colony and in two tubes of different primers :
• oLS 1.1 and 5'KAN
• oLS 1.2 and 3'KAN

1. Pick a well-isolated colony with a toothpick
2. Transfer the colony into a Kan + DL Glu LB agar plate (and incubate 37°C), than into a PCR tube
3. Prepare a PCR mix in an Eppendorf tube with oLS 1.1 and 5'KAN
   • 90 µL of EconoTaq (2X)
   • 3,6 µL of Forward Primer
   • 3,6 µL of Reverse Primer
   • 83 µL H2O to complete until final volume
4. Prepare a PCR mix in an Eppendorf tube with oLS 1.2 and 3'KAN
   • 90 µL of EconoTaq (2X)
   • 3,6 µL of Forward Primer
   • 3,6 µL of Reverse Primer
   • 83 µL H2O to complete until final volume
5. To finish, vortex the PCR mix
6. Add 20 µL of PCR Mix into every PCR tube and vortex a little.
7. Transfer PCR tubes to a PCR machine and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

STEP	TEMP	TIME
Initial Denaturation	94°C	5 minutes
30 Cycles	94°C	30 seconds
56°C	56°C	30 seconds
72°C	72°C	1 min 30
Final Extension	72°C	5 minutes

Electrophoresis :

• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

Colonies 6 and 7 have positive results!

The band is about 2kb == strange. Normally it gives 1.2kb.

15 July

DO preculture of E. coli W3110 Δ ompT pKOBEG pKD4

• It's the same as the zero LB.

The result of the save of the PCR colony:

We think that maybe the cells die on the plate because there is so little colony :/

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Colony PCR Protocol with oLS 1.1 and 5'KAN, oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:

• We do this for 8 colony and in two tubes of different primers (clone 9 to 16):
• oLS 1.1 and 5'KAN
• oLS 1.2 and 3'KAN

1. Pick a well-isolated colony with a toothpick
2. Transfer the colony into a Kan + DL Glu LB agar plate (and incubate 37°C), than into a PCR tube
3. Prepare a PCR mix in an Eppendorf tube with oLS 1.1 and 5'KAN:
   • 90 µL of EconoTaq (2X)
   • 3,6 µL of Forward Primer
   • 3,6 µL of Reverse Primer
   • 83 µL H2O to complete until final volume
4. Prepare a PCR mix in an Eppendorf tube with oLS 1.2 and 3'KAN:
   • 90 µL of EconoTaq (2X)
   • 3,6 µL of Forward Primer
   • 3,6 µL of Reverse Primer
   • 83 µL H2O to complete until final volume
5. To finish, vortex the PCR mix
6. Add 20 µL of PCR Mix into every PCR tube and vortex a little.
7. Transfer PCR tubes to a PCR machine and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

STEP	TEMP	TIME
Initial Denaturation	94°C	5 minutes
30 Cycles	94°C	30 seconds
56°C	56°C	30 seconds
72°C	72°C	1 min 30
Final Extension	72°C	5 minutes

Electrophoresis:

• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

17 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:

• Here we use 2 µL of Template DNA (pKD4)
• Reaction Setup: Follow the same protocol as Thursday 19/06 for 50µL
• Transfer PCR tubes to a PCR machine and begin thermocycling.
• Thermocycling Conditions for a Routine PCR as Wednesday 09/07

Electrophoresis:

• We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel for a PCR with pKD4.
• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

PCR clean up of pKD4:

• Follow the same protocol as Wednesday, 25/06
• Nanodrop result: pKD4 : 31 ng/µL

Electroporation 600ng of PCR pKD4:

• Follow the same protocol as Friday 13/06
• We had 15 µL of pKD4 into 50 µL of cells competent
• Select on Kan LB 20 mM DLGlu plates
• Incubate overnight at 37°C

18 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Colony PCR Protocol with oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:

• Follow the same procedure as described on Tuesday 15/07

The result of W3110 Δ ompT pKOBEG pKD4

The colonies 4, 5 and 10 are positive (~1200 pb) !

21 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Preparation of pre-cultures for E. coli W3110 Δ ompT pKOBEG pkd4

• Inoculated 1 colony into a 5 mL LB KAN 20 mM DL Glu Erlenmeyer flask for the colonies 4, 5 and 10.
• Incubated overnight at 37°C with shaking

Streak into plate:

• Streak positives clones (4, 5 and 10) into LB agar + chloramphenicol plates to verify the sensitivity of cells
• Streak positives clones (4, 5 and 10) into LB agar + KAN + 20 mM DL Glu plate
• Streak positives clones (4, 5 and 10) into LB agar + KAN plate
• Incubated overnight at 37°C

22 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Result of positive clone on Kan DL Glu or only KAN:

The colony 10 didnt grow without the DL Glu !!!!

Result of the test for the cm sensitivity

The plasmide pKOBEG is cmR so it's still in the cells…

Verification of W3110 Δ ompT if they are AMP, CM, SM sensible:

• Streak a colony of W3110 Δ ompT into LB agar plate and different antibiotic
• Streak a colony of BL21 (our control) into LB agar plate and different antibiotic

Preparation of Electrocompetent Cells W3110 Δ ompT pKOBEG pkd4:

• Follow the same protocol as Friday 13/06, the centrifugation is made with the program: 5000 rpm for 8 min.
• And at the end of the preparation of electrocompetent cells, resuspend in 100 µL of glycerol 10%.
• Stockage at -80°C

23 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Results of W3110 Δ ompT on LB and antibiotic plates:

W3110 Δ ompT grow on Streptomycin plate :/

24 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Streak the colony 10 of W3110 Δ ompT pKOBEG pKD4 Δ murI into LB agar KAN 20 mM GLU plate and incubate at 42°C (to eliminate pKOBEG)

25 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Streak multiple colonies of W3110 Δ ompT pKOBEG pKD4 Δ murI of the plate LB agar KAN 20 mMGLU did the 24 july into a LB agar Kan plate and into a LB agar Cm plate to compare which colonies grow on plate CM.

Incubate on paillasse over weekend

28 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

results: W3110 Δ ompT pKOBEG pKD4 Δ murI

After the incubation on the Δ murI at 42°C they are no longer Cm resistant!

Preparation of pre-cultures for E. coli W3110 Δ ompT pKOBEG pkd4 Δ murI

• Inoculated 1 colony into a 5 mL LB KAN 20 mM DL Glu Erlenmeyer flask.
• Incubated overnight at 37°C with shaking

29 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Preparation of Electrocompetent Cells W3110 Δ ompT pKOBEG pKD4 Δ murI:

• The dilution of the preculture is made with 2µL of the preculture (DO: A600 nm: 1) in 20 mL LB KAN 20mM DL GLU.
• Follow the same protocol as Friday 13/06, the centrifugation is made with the program: 5000 rpm for 8 min.
• And at the end of the preparation of electrocompetent cells, resuspend in 100 µL of glycerol 10%.

Electroporation of pCP20:

• Electroporation with 1 µL of pCP20 and follow the same protocol as Friday 13/06 but for the incubation it's 1h20 at 30°C
• Streak into LB agar CM, AMP and 20mM DL Glu plate
• Incubate two night at 30°C

31 July

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

result of the electroporation of pCP20 into W3110 Δ ompT Δ murI pKD4:

Left: control W3110 Δ ompT Δ murI pKD4

And right: W3110 Δ ompT Δ murI pKD4 pCP20

W3110 Δ ompT pkd4 pCP20 Δ murI:

• Grow W3110 Δ ompT, pKD4, pCP20 Δ murI colonies in 1 mL of LB 20 mM DL Glutamate (without antibiotic) at 30°C to an A600 of 0,5.
• DO at 11 am: 0,2
• DO at 4 pm: 0,09
• DO at 6 pm: 0,1
• Incubation overnight at 30°C with shaking

01 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

W3110 Δ ompT pkd4 pCP20 Δ murI:

• Grow W3110 Δ ompT, pKD4, pCP20 Δ murI colonies in 1 mL of LB DL Glutamate (without antibiotic) at 30°C to an A600 of 0,5:
• DO after 14h incubation: 5
• Dilution of the culture: 10 µL culture in 1 mL LB 20 mM DL Glu
• Incubation at 30°C with shaking
• DO after 2h30: 0,3
• DO after 3h10: 0,3

Streak on LB 20 mM DL Glu plate and LB plate and incubate over weekend on bench

04 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

It is not supposed to grow without D-L-glutamate, which means that our strain is no longer auxotrophic to D-L-glutamate. We have to find a strain which is auxotrophic.

• Streak multiple colonies of W3110 Δ ompT pKOBEG pKD4 Δ murI of the plate LB agar Kan did Friday 25/07/2025 into a LB agar Kan plate and into a LB agar Kan plate +20 mM D-L glutamate to see if they are still auxotrophic to D-L glutamate.
• Incubate 30°C overnight.
• Streak again the 22 colonies of the plate from Thursday 17/07 into a LB agar Kan plate and into a LB agar Kan plate + 20 mM D-L glutamate to see if they are still auxotrophic to D-L glutamate.
• Incubate 30°C overnight.

05 August

Result of clone on Kan DL Glu or only Kan:

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

Verification of colony 10 W3110 Δ ompT pKD4 to see if we can use it:

• Streak colony 10 of W3110 Δ ompT pKOBEG pKD4 Δ murI of the plate LB Kan 20mM D-L glutamate did Monday 21/07 into a LB agar Kan plate to see if they are still auxotrophic to D-L glutamate.
• Incubate 30°C overnight.

Electroporation 600ng of PCR pKD4:

• Follow the same protocol as Friday 13/06
• We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
• With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 558 µl of 2YT)
• Select on Kan LB 20 mM DLGlu plates
• Incubate overnight at 37°C

06 August

Result of clone on Kan DL Glu and colony 10 on Kan:

It did not work, we need to do the electroporation again

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

Electroporation 600ng of PCR pKD4:

• Follow the same protocol as Friday 13/06
• We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
• With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 900 µl of 2YT)
• Select on Kan LB 20 mM DLGlu plates
• Incubate overnight at 37°C

07 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:

• We don't have PKD4 anymore. Here we use 2 µL of Template DNA (pKD4)
• Follow the same protocol as Thursday 17/07

Thermocycling Conditions:

STEP	TEMP	TIME
Initial Denaturation	98°C	3 minutes
25 Cycles	98°C	10 seconds
57°C	57°C	30 seconds
72°C	72°C	45 seconds
Final Extension	72°C	2 minutes
Hold	16°C

Result of clone on Kan DL Glu and PCR

Nothing has grow

PCR clean up of pKD4:

• Follow the same protocol as Wednesday, 25/06
• Nanodrop result: pKD4: 15,7 ng/µL

Electroporation 600ng of PCR pKD4:

• Follow the same protocol as Friday 13/06
• We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
• With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 558 µl of 2YT)
• Select on Kan LB 20 mM DLGlu plates
• Incubate overnight at 37°C

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

Pre-culture of W3110 ΔompT pKOBEG

• Inoculate 1 colony of W3110 ΔompT pKOBEG into 5 mL LB + Cm in a sterile 25 mL flask.
• Shake overnight at 30°C

08 August

Result of electroporation of pKD4

It didn't work, we need to retry by creating new W3110 ΔompT pKOBEG electrocompetent

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

Preculture Dilution of W3110 Δ ompT pKOBEG

• Follow the same protocol as Wednesday 09/07

Prepare electrocompetent cells:

• Follow the same protocol as Friday 13/06

Electroporation 600ng of PCR pKD4:

• Follow the same protocol as Friday 13/06
• We had 15 µL of pKD4 into 50 µL of cells competent W3110 Δ ompT pKOBEG
• With 2YT add 20mM of D-L glutamate. (342 µL of D-L-glutamate + 558 µl of 2YT)
• Select on Kan LB 20 mM DLGlu plates
• Incubate overnight at 37°C

11 August

Result of electroporation of pKD4 in W3110 Δ ΔompT pKOBEG

No results, we need to try again and create new electrocompetent cells

Pre-culture of W3110 ΔompT pKOBEG and W3110 ΔompT

• Inoculate 1 colony of W3110 ΔompT pKOBEG into 5 mL LB + Cm in a sterile 25 mL flask. Shake overnight at 30°C
• Inoculate 1 colony of W3110 ΔompT into 5 mL LB in a sterile 25 mL flask. Shake overnight at 30°C

12 August

Construction of isogenic mutant murI of W3110 Δ ompT:

Preculture Dilution of W3110 Δ ompT until a DO 0,6:

• in 20 mL LB, dilution 1/100

Preparation of Electrocompetent Cells W3110 ΔompT Cells:

• Follow the same protocol as Friday 13/06.

Electroporation of PCR pKOBEG in W3110 ΔompT Cells:

• Follow the same protocol as Friday 13/06
• Streak on LB Cm plate
• Incubate at 30°C

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

Preculture Dilution of W3110 Δ ompT pKOBEG:

• Same protocol as Wednesday 09/07 but in 20 mL LB, dilution 1/100

Induction of W3110 Δ ompT pKOBEG with arabinose when DO 0,2 - 0,4:

• Same protocol as Wednesday 09/07

Prepare electrocompetent W3110 Δ ompT pKOBEG cells:

• Follow the same protocol as Friday 13/06

Electroporation 600ng of PCR pKD4 in W3110 Δ ompT pKOBEG:

• Follow the same protocol as Friday 13/06
• We had 10 µL of pKD4 into 50 µL of cells competent
• Select on Kan LB 20 mM DLGlu plates
• Incubate on at 37°C

13 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Colony PCR Protocol with oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:

• Follow the same procedure as described on Tuesday 15/07

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Colony PCR oLS 1.2 and 3'KAN of W3110 Δ ompT pKOBEG pKD4:

• Follow the same procedure as described on Tuesday 15/07

The result of W3110 Δ ompT pKOBEG pKD4

The colony 1 is positive (~1200 pb)!

14 August

Construction of isogenic mutant murI of W3110 Δ ompT:

Preculture Dilution of W3110 Δ ompT until a DO 0,6:

• in 20 mL LB, dilution 1/100

Preparation of Electrocompetent Cells W3110 ΔompT Cells:

• Follow the same protocol as Friday 13/06.

Electroporation of PCR pKOBEG in W3110 ΔompT Cells:

• Follow the same protocol as Friday 13/06
• Streak on LB Cm plate
• Incubate at 30°C

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

Preculture Dilution of W3110 Δ ompT pKOBEG: (30°C)

• Same protocol as Wednesday 09/07 but in 20 mL LB, dilution 1/100

Induction of W3110 Δ ompT pKOBEG with arabinose when DO 0,2 - 0,4:

• Same protocol as Wednesday 09/07

When DO 0,8:

• Incubate 15 min at 42°C

Prepare electrocompetent W3110 Δ ompT pKOBEG cells:

• Follow the same protocol as Friday 13/06

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:

• Follow the same protocol as Wednesday 09/07

The electrophoresis reveal no band …

18 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:

• Follow the same protocol as Wednesday 09/07

Result: no band

There is a problem with our plasmide, maybe there is a DNAase in it … So we tried another plasmid pKD4 from someone else.

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:

• Follow the same protocol as Wednesday 09/07

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 colony 10:

• The isolated colonies from colony 10 are analyzed by PCR on colony (primer oLS 1,2 and 3'KAN).
• Incubate the backup to 37°C.

Result: There was a problem with the tube caps, which were not suitable, and the water evaporated in the PCR machine. So there is no band …

19 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4 colony 10:

• The isolated colonies from colony 10 are analyzed by PCR on colony (primer oLS 1,2 and 3'KAN).
• Incubate the backup to 37°C

Result: no band at 1200 pb

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG:

PCR and Electrophoresis of pKD4 with Del-murI-W3110-6-DW (oLS 1.3) and Del-murI-W3110-3-DW (oLS 1.4) using Q5® High-Fidelity 2X Master Mix following NEB Protocol:

• Follow the same protocol as Wednesday 09/07
• We did it with 5 differents Q5 master mix to verify if it's the problem

Result:

PCR clean up of pKD4:

• We did the clean up on the mix 1, 2 and 4 of pKD4
• Follow the same protocol as Wednesday, 25/06
• Nanodrop result:
  • pKD4 tube 1: 22 ng/µL
  • pKD4 tube 2: 45 ng/µL
  • pKD4 tube 4: 66 ng/µL

Electroporation 600ng of PCR pKD4:

• Follow the same protocol as Friday 13/06
• We had 15 µL of pKD4 into 50 µL of cells competent two times to test the addition of DL GLU during the 1 hour incubation at 37°C with shacking.
• Select on Kan LB 20 mM DLGlu plates.
• Incubate on at 37°C

21 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

PCR Colony with W3110 Delta ompT pKOBEG pKD4 (primer oLS 1,2 and 3'KAN):

• Streak the backup on Kan 20 mM DL GLU and on Kan to verify if the clones grow without the Glutamate and if the auxotrophy are working.
• Incubate the backup to 37°C

Control: W3110 Delta ampT pKOBEG

Red dot: clones of the plates when we add DL GLU in the 1 hour incubation after the electroporation

1 à 6: clones of the plates when we do not add DL GLU in the 1 hour incubation after the electroporation

All clones are positive (1200 pb)

But there are also capable to grow without DL GLU …

It's possible that there is a mutation or maybe a duplication of the gene murI …

Results PCR colony and growth

25 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Pre-culture preparation

• Inoculate 1 colony of W3110 delta ampT delta murI pKOBEG pKD4 (the colony red dot) into a 5 mL Erlenmeyer flask of LB Kan 20 mM DL GLU and same with the W3110 delta ampT.
• Shake overnight at 37°C.

26 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

Preculture Dilution

• Follow the same procedure as described on Tuesday, 03/06

Growth curve:

• Incubate the culture at 37°C with shaking and every 15 min take the DO 600 nm and report it
• For the blank: use LB for the WT and for W3110 delta ampT murI pKOBEG pKD4 whitout Glu
• and use LB 20mM DL GLU for W3110 delta ampT murI pKOBEG pKD4 which grow on LB 20mM GLU

Time (min)	20 mM DL GLU add	W3110 delta ampT murI pKOBEG pKD4	W3110 delta ampT
t=0	0,061	0,046	0,052
15	0,047	0,059	0,09
25	0,038	0,064	0,125
40	0,053	0,126	0,243
55	0,054	0,214	0,37
70	0,065	0,355	0,575
85	0,072	0,555	0,755
95	0,083	0,67	0,95
105	0,105	0,84	1,124

The problem is that we dilute the LB when we add the 20mM GLU so there is no more the same concentration of composites of LB. This is not possible to compare the result.

But we can say that the bacteria are not auxotrophic for glutamate since they grow as the WT

27 August

Construction of isogenic mutant murI of W3110 Δ ompT pKOBEG pKD4:

• Streak the colony red dot on M9 media agarose 1,5% KAN 20mM GLU
• and on M9 media agarose 1,5% KAN
• to see if they can grow without glutamate
• But nothing has grow on it

17 June

Transformation of iGEM Distribution Kit Parts into DH5 alpha to Construct the pmurI Complementation Plasmid

1. Puncture the desired DNA brick tube with a clean pipette tip (specify which DNA blocks being used)
2. Add 10 µL of sterile H2O and mix by pipetting up and down, then transfer to a 1.5mL Eppendorf tube
3. Aliquot 1 µL into a new Eppendorf tube (placed horizontally to visualize droplet) keep this aliquot on ice for transformation. Store remaining 9 µL at -20°C
4. Add 50 µL of DH5 alpha competent cells to the 1 µL DNA aliquot
5. Incubate on ice for 30 minutes
6. Perform heat shock at 42°C for 45 seconds
7. Immediately return to ice for 2 minutes
8. Add 700 µL of sterile LB broth
9. Incubate with shaking at 37°C for 1 hour
10. Centrifuge at 5000 rpm for 5 minutes
11. Plate cells on appropriate selective agar plates
    • A1, I19, C1, O10: Cm
    • O9, G7: Amp

18 June

Transformation of iGEM Distribution Kit Parts into DH5 alpha to Construct the pmurI Complementation Plasmid

• Follow the same procedure as described on Tuesday, 17/06.

23 June

Transformation of iGEM Distribution Kit Parts into DH5 alpha to Construct the pmurI Complementation Plasmid

• Follow the same procedure as described on Tuesday, 17/06, this time with A12 and C1.
• For the last step, plate cells on appropriate selective agar plates (C1 on Chloramphenicol and A12 on Kanamycin)

27 June

Golden gate assembly pMurI

In a 0.5 µL PCR tube add:

• 75 ng plasmide A12: volume to pipette from undiluted tube: 2,36 µL
• 2 ng promoteur A1: volume to pipette from tube 1/20: 1,89 µL
• 1,2 ng RBS I19: volume to pipette from tube 1/100: 1,7 µL
• 6,8 ng terminator C1: volume to pipette from tube 1/20: 1,9 µL
• 45 ng PCR murI purified (clean up): volume to pipette from tube 1/2: 1,75 µL
• 2 µL T4 DNA ligase buffer (10x)
• 1 µL NEB Golden Gate mix
• 7.4 µL H2O (for a final volume of 20 µL)

Thermocycler program:

STEP	TEMP	TIME	CYCLES
Cycle	37°C	1 minute	30 cycles
Cycle	16°C	1 minute
Final Extension	65°C	5 minutes

Transformation with Supercompetent Cells

• Follow the same procedure as described on Tuesday, 03/06.
• For step 1 and 2 we used 2 µL of PCR product (GG assembly = pMurI-HA) and 50 µL of DH5alpha supercompetent cells.
• Before step 8, we spread 100 µL of each tube on the LB + Kanamycine + IPTG 0.1M + xGal plate.
• Incubate overnight at an ambient temperature protected from light.

Transformation with NEB 5-alpha competent Cells (C29871) following High Efficiency Transformation Protocol of NEB

1. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into a transformation tube on ice.
2. Add 1-5 µl containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature SOC into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50-100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

• At step 2, we add 2 µL of GG assembly (pMurI-HA)
• At the end, we spread 100 µL of each tube on the LB + Kanamycine + IPTG 0.1M + xGal plate. Incubate overnight at an ambient temperature protected from light.

30 June

Result

Colony PCR Protocol with OG35, OG36 and pMurI-HA cells

• We do this for 23 colony, 22 white colonies and 1 blue colony (negative control).
• Follow the same protocol as Thursday, 26/06.
• This time we do 56°C and 45 seconds.

It didn't work, we have to try again.

Western Blot of pMurI-HA

The total length of the protein is 258pb, which corresponds to 28,6KDa.

We will prepare 4 gels at 15%.

Preparation of the lower gel (separating gel):

Add in the following order:

• 7 mL of H2O
• 10 mL of 30% Acrylamide
• 2,6 mL of 3M Tris, pH 8,8
• 200 uL of 10% SDS
• Add 20 uL of TEMED under a fume hood
• Add 150 uL of 10% APS, (weigh 10g for 10 mL)

Preparation of the upper gel (stacking gel)

Add in the following order:

• 5,6 mL of H2O
• 1,32 mL of 30% Acrylamide
• 1 mL of 1M Tris, pH 6,8
• 200 uL of 10% SDS
• Add 12 uL of TEMED under a fume hood
• Add 80 uL of 10% APS, (weigh 10g for 10 mL)

Pouring the gels:

• Pour the separating gel into the casting frame, add water on top to prevent polymerization at the surface, blot with absorbent paper once set, add the stacking gel.

01 July

Plasmid Miniprep Kit for pMurI 8, 12, 17 and 19

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.
• Result Nanodrop:
  • pMurI 8: 37 ng/µL
  • pMurI 12: 40 ng/µL
  • pMurI 17: 36 ng/µL
  • pMurI 19: 64 ng/µL

Colony PCR Protocol with OG35, OG36 and pMurI

• We do this for 4 different pMurI (8, 12, 17, 19) and A12 (negative control, empty vector).
• Follow the same protocol as Thursday, 26/06.
• For 5 PCR tubes, we prepare 60 µL of PCR mix so everything x6 + 18 µL of H2O
• Instead of picking a colony, we need 10ng of DNA (the µL depends on the DNA concentration) so we dilute DNA in H2O to have 3 µL final volume and take 1µL of it.

DNA	DNA (µL)	µL of H2O
pMurI 8	0.8	2.2
pMurI 12	0.75	2.25
pMurI 17	0.8	2.2
pMurI 19	0.5	2.5

This time we do 56°C and 1 minute 30 seconds for the PCR.

Abnormal results

Colony PCR Protocol with oLS 3.2 oLS 3.3 and pMurI

• We do the same with primers oLS 3.2 and oLS 3.3 for 4 different pMurI (8, 12, 17, 19) and A12 (negative control, empty vector)

Abnormal results

Digestion and Electrophoresis of pMurI with Ecor1/Xma1 and Ecor1/Spe1

Reaction Mix Setup:

• 2 µL of rCutSmart Tampon
• 0.2 µL of Enzyme 1
• 0.2 µL of Enzyme 2
• H2O QSP 10 µL
• The DNA must be 300ng

DNA	DNA (µL)	µL of H2O
pMurI 8	8.1	1.9
pMurI 12	7.5	2.5
pMurI 17	8.3	1.7
pMurI 19	4.7	5.3
A12	9.4	0.6

• Add 10 µL of DNA in a 1.5 Eppendorf Tube, than add 10 µL of Mix.
• Vortex and heat at 37°C for 30 minutes.
• This time, we do this for pMurI 8, 12, 17, 19 and A12 (empty vector) with Ecor1/Xma1 and Ecor1/Spe1 enzymes. So we need 60 µL of Reaction Mix:
  • 12 µL of rCutSmart Tampon
  • 1 µL of Enzyme 1
  • 1 µL of Enzyme 2
  • H2O 46 µL

Electrophoresis:

• We add 100 mL TAE 0.5X to 1g of agarose to create agarose gel.
• We used a Quick-Load® Purple 1 kb Plus DNA Ladder from NEB.

We added too much Ladder.

For Ecor1/Xma1 we wanted 2931pb and 220pb for A12, and a linearized band for the rest.

For Ecor1/Spe1 we wanted ~2980pb and ~460pb and for A12, and 2931pb and 1134pb for the rest

Starter

• Follow the same protocol as described on Monday, 30/06 for the clone pMurI 17, 8, 12, 19.

02 July

Sequencing

• We send to sequencing with LightRun our plasmid pMurI 19 and pMurI 12 by adding in a tube: 5 µL of plasmid, 2.5 µL of primer (OG35) and 2.5 µL of H2O
• JFB470 = pMurI 19
• JFB469 = pMurI 12

Pre-culture preparation of pAIDA-I and iGEM Brick A12 to make a stock

• Inoculate 1 colony of A12 into a 5 mL Erlenmeyer flask of LB and with Kanamycin. Same for pAIDA-I with Chloramphenicol.
• Shake overnight at 37°C.

03 July

Pre-culture preparation of pMurI 12, pMurI 19 and pok-HA for Western Blot

• Inoculate 1 colony of pok-HA into a 5 mL Erlenmeyer flask of LB and with Kanamycin. Same for pMurI 12 and 19.
• Shake overnight at 37°C.

04 July

OD measurement using a spectrophotometer for pMurI (colonie 12 and 19) and pok-HA

• Measured cell OD in 1 mL cuvettes to target OD₆₀₀ ≈ 0.8.
• pMurI 12: 2.9
• pMurI 19: 3.6
• pok-HA: 2.9

Western Blot of pMurI-HA

The total length of the protein is actually 858pb, which corresponds to 32KDa.*

The pok-HA is our positive control to ensure that our anti-HA antibodies are working. We are testing two different anti-HA antibodies (the iGEM anti-HA antibodies and one from the team of Laeticia).

Gel preparation (Monday 30/06)

• Centrifuge 1 mL of pre-culture at 9000 rpm for 4 min.
• Discard the supernatant and resuspend the pellet in loading buffer as follows:
  • 72.5 µL of TPC 2X Buffer for pok-HA,
  • 62.5 µL for pMurI 12,
  • 90 µL for pMurI 19 to have a 0.4 OD/10µL

The calculation used helps determine the volume of loading buffer to add based on the culture's OD. We aim for an OD of 0,4 in 10 µL for each electrophoresis well. Use the formula Vf × Cf = Ci × Vi, therefore Vf = (Ci × Vi) / Cf

1. Incubate at 95°C for 10 min.
2. Assemble the gel system and fill the space between the plates with a 10X ZY running buffer to the top.
3. Load the gel: 5 µL of protein ladder and 10 µL of each sample (2 times to test the two different antibodies)
4. Run the gel 200 V, 50 min for a 10% gel and 55 min for a 15% gel
5. Transfer to nitrocellulose: Prepare the transfer sandwich as follows, soaking all components in transfer buffer beforehand:
   • 3 whatman papers
   • The gel
   • 1 nitrocellose membrane
   • 3 whatman papers
6. Note: Proteins migrate downward, so the gel must be placed directly on top of the nitrocellulose membrane.
7. Transfer using a semi-dry transfer system: 7 min at 25 V, 1,3 A
8. Blocking buffer preparation:
   • For a 3% BSA: weigh 300 mg BSA and add 10 mL of 1X PBS
   • For a 5% milk: weigh 500 mg powdered milk and add 10 mL of 1X PBS
9. Place the nitrocellulose membrane into trays on a shaker and discard the other papers and gel
10. Add 10 mL of 3% BSA for the membrane which would be incubated with iGEM anti-HA antibody and add 10 mL of 5% milk for the membrane which would be incubated with anti-HA antibody from the Laeticia teams.
11. Incubate 1 h
12. Add 10µL of the corresponding anti HA antibodies in each tray to obtain a 1:1000 dilution.
13. Incubate 1h
14. Wash 3 times with 1X PBS 5 min each
15. Add 10 mL of dilution buffer containing the alkaline phosphatase conjugated secondary antibody.
16. Wash 2 times with 1X PBS 5 min each
17. Wash once with 1X PA buffer 5 min
18. Revelation buffer preparation:
    • 10 mL of 1X PA buffer
    • 10 µL MgCl2
    • 70 µL NBT
    • 30 µL BCIP
19. Add the revelation buffer and monitor the color development

07 July

Result of Western Blot:

We don't have positive result: lack of tag HA of our pMurI

Golden gate assembly pMurI

• Follow the same procedure as described on Friday 27/06
• In a 0.5 µL PCR tube add:
  • 75 ng plasmide A12: volume to pipette from undiluted tube: 1 µL
  • 2 ng promoteur A1: volume to pipette from tube 1/20: 1 µL
  • 1,2 ng RBS I19: volume to pipette from tube 1/100: 1 µL
  • 6,8 ng terminator C1: volume to pipette from tube 1/20: 1 µL
  • 45 ng PCR murI purified (clean up): volume to pipette from tube 1/2: 0.7 µL
  • 2 µL T4 DNA ligase buffer (10x)
  • 1 µL NEB Golden Gate mix
  • 7.4 µL H2O (for a final volume of 20 µL)

Transformation with NEB 5-alpha competent Cells (C29871) following High Efficiency Transformation Protocol of NEB

• Follow the same procedure as described on Friday 27/06

08 July

Rehydration of murI oligos from IDT

Primer Name	µL H2O for Rehydration
oLS 4.1 tyr1UP	265
oLS 4.2 tyr1DO	252

→ Stock concentration: 100µM

Dilution to 10µM (Final volume: 200µL):

1. Vortex the stock oligo solution
2. Incubate on ice for 1 hour
3. Vortex again
4. In a 1.5 mL Eppendorf tube, add 20µL of oLS and 180µL of H2O

Colony PCR Protocol with OG35, OG36 and pMurI-HA cells

• We do this for 7 colonies, 6 white colonies and 1 blue colony (negative control).
• Follow the same protocol as Thursday, 26/06.
• This time we do 56°C and 1 minute 30 seconds.

Colony PCR Protocol with OG35, oLS 3.3 and pMurI-HA cells

• We do the same with primers OG35 and oLS 3.3 for the same 7 colonies, 6 white colonies and 1 blue colony (negative control).

Result of Transformation with pMurI and PCR colony

Pre-culture preparation of pMurI 4, 5 and 6

• Inoculate 1 colony of pMurI 4, 5 and 6 into a 5 mL Erlenmeyer flask of LB with Kanamycin.
• Shake overnight at 37°C

09 July

Plasmid Miniprep Kit for pMurI 4, 5 and 6.

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday, 18/06.
• pMurI 4 = 29.5 ng/µL
• pMurI 5 = 64.7 ng/µL
• pMurI 6 = 76.05 ng/µL

11 July

Golden gate assembly pMurI

• Follow the same procedure as described on Friday 27/06
• In a 0.5 µL PCR tube add:
  • 75 ng plasmide A12: volume to pipette from undiluted tube: 1 µL
  • 2 ng promoteur A1: volume to pipette from tube 1/20: 1 µL
  • 1,2 ng RBS I19: volume to pipette from tube 1/100: 1 µL
  • 6,8 ng terminator C1: volume to pipette from tube 1/20: 1 µL
  • 45 ng PCR murI purified (clean up): volume to pipette from tube 1/2: 0.7 µL
  • 2 µL T4 DNA ligase buffer (10x)
  • 1 µL NEB Golden Gate mix
  • 7.4 µL H2O (for a final volume of 20 µL)
• In another 0.5 µL PCR tube add:
  • 75 ng plasmide A12: volume to pipette from undiluted tube: 1 µL
  • 75 ng promoteur A1: volume to pipette from tube 1/20: 1.1 µL
  • 75 ng RBS I19: volume to pipette from tube 1/100: 0.8 µL
  • 75 ng terminator C1: volume to pipette from tube 1/20: 0.6 µL
  • 45 ng PCR murI purified (clean up): volume to pipette from tube 1/10: 1.5 µL
  • 2 µL T4 DNA ligase buffer (10x)
  • 1 µL NEB Golden Gate mix
  • 12 µL H2O (for a final volume of 20 µL)
• For the program, we do 60°C during 5 minutes instead of 65°C.

Transformation with NEB 5-alpha competent Cells (C29871) following High Efficiency Transformation Protocol of NEB

• Follow the same procedure as described on Friday 27/06

15 July

Colony PCR Protocol with OG35, OG36 and pMurI-HA cells

• We do this for 8 colonies, 7 white colonies and 1 blue colony (negative control).
• Follow the same protocol as Thursday, 26/06.
• This time we do 56°C and 1 minute 30 seconds.

Colony PCR Protocol with OG35, oLS 3.3 and pMurI-HA cells

• We do the same with primers OG35 and oLS 3.3 for the same 7 white colonies.

Result of Transformation with Golden Gate and PCR colony:

16 July

Pre-culture preparation of pMurI 3, 4 and 5 for a Miniprep and POK-HA

• Inoculate colony 4 of pMurI into a 10 mL Erlenmeyer flask of LB with Kanamycin.
• Same for pMurI 3, 5 and pok-HA with Kanamycin,
• Shake overnight at 37°C

17 July

Miniprep of pMurI 3, 4 and 5

• Follow the NEB protocol for T1110S (50-prep) as described on Wednesday 18/06.
• Result Nanodrop:
  • pMurI 3: 51 ng/µL
  • pMurI 4: 41 ng/µL
  • pMurI 5: 39 ng/µL
• We sent pMurI 3, 4 and 5 for sequencing.

Pre-culture preparation of pMurI 3, 4, 5 and Pok-HA for WesternBlot

• Inoculate colony 3 of pMurI into a 5 mL Erlenmeyer flask of LB with Kanamycin.
• Same for pMurI 4, 5 and pok-HA with Kanamycin,
• Shake overnight at 37°C

18 July

Western Blot of pMurI-HA

The total length of the protein is actually 858pb, which corresponds to 32KDa.*

The pok-HA is our positive control. We use our antibodies.

• Follow the same procedure as described on Friday 04/07

It is difficult to read, but we think 3 and 5 have HA and not 4.

21 July

Sequencing of pMurI 3, 4 and 5 with oLS 3.3

• With a Reverse primer (murDO) we do a sequencing of pMurI 3, 4 and 5
• KCH602 = Colonie 3
• KCH605 = Colonie 4
• KCH608 = Colonie 5

3 has a mutation

4 is unreadable

5 is good but has some empty space, we need to do another sequencing to be sure everything is alright and redo a Western blot only with 5.

Pre-culture preparation of pMurI 5 and Pok-HA for WesternBlot

• Inoculate colony 3 of pMurI into a 5 mL Erlenmeyer flask of LB with Kanamycin.
• Same for pok-HA with Kanamycin,
• Shake overnight at 37°C

22 July

Western Blot of pMurI-HA

The total length of the protein is actually 858pb, which corresponds to 32KDa.*

The pok-HA is our positive control. We use our antibodies.

• For step 3, in 450 µL of loading buffer, add 50µL of β-mercaptoéthanol 25% to obtain a loading buffer + β-mercaptoéthanol 2.5%.
• Discard the supernatant and resuspend the pellet in loading buffer, to aim for an OD of 0,2 in 10 µL, as follows:
  • 220 µL of TPC 2X Buffer + β-mercaptoéthanol 2.5% for pok-HA,
  • 110 µL for pMurI 12.
• Follow the same procedure as described on Friday 04/07

We have murI with HA at the top and maybe some remains at the bottom of it.

28 July

Sequencing of pMurI colony 5

• We send to sequencing with LightRun our plasmid pMurI 19 and pMurI 12 by adding in a tube: 5 µL of plasmid, 2.5 µL of primer and 2.5 µL of H2O
• KCH594: pMurI 5 with primer oLS 3.5
• KCH595: pMurI 5 with primer oLS 3.4

07 August

Pre-culture of pMurI 5 for Miniprep and W3110 ΔompT pKOBEG

• Inoculate 1 colony of pMurI 5 into 5 mL LB + Kan in a sterile 25 mL flask.
• Shake overnight at 37°C

08 August

Sequencing of pMurI colony 5

• The sequencing tubes have been lost, we have to send it again.
• We send to sequencing with LightRun our plasmid pMurI 19 and pMurI 12 by adding in a tube: 5 µL of plasmid, 2.5 µL of primer and 2.5 µL of H2O
• KCH603: pMurI 5 with primer oLS 3.5
• KCH604: pMurI 5 with primer oLS 3.4