Experiment 1: Cloning and Transformation

Week: 1: 04/08/2025

Purpose:

Perform test cloning of two different DNA fragments (VNP green and VNP cis his) into linearized plasmids to evaluate whether functional vesicles can be obtained.

Methods and Procedures:

  • Prepared recombination mixes using EnzymeX to calculate plasmid/insert ratios.
  • Constructed 3 tubes: Tube1 with Vnp-mNeonGreen-6xHis, Tube2 control (empty plasmid), Tube3 Vnp-6xHis.
  • Circularization achieved by insertion of VNP fragments into plasmid PRL81.
  • Transformed E. coli DH5α with TLTC method and heat shock.
  • Plated transformants (100 µL and pellet) on Cm selection plates.
  • Poured new agar plates with selective antibiotics (Cm).

Results:

  • Successful preparation of transformation mixes and plating.
  • Plates incubated overnight at 37°C.

Notes:

  • Plasmid circularization necessary for survival under Cm selection.
  • Fluorescence of mNeonGreen will be used as a reporter for successful cloning.
  • Fgt = fragment
  • Cm = chloremphenicol

Spreadsheet for the volumes used in the clonage

Tube 1(Fgt Vnp m neongreen- 6xhis)

 Tube 2 control (empty plasmide)

Water

5,1ul

6,8

Fgt Vnp m neongreen- 6xhis

1,7ul

X

Cutsmart Buffer 10X

1ul

1ul

T5 exo (1/10)

1ul

1ul

Fgt E

1,2ul

1,2ul

Spreadsheet for the volume used in the clonage

Eau

2,8ul

Fgt Vnp- 6xhis (1/10) (attention 1ul dans 9ul)

4ul

Tampon cutsmart 10X

1ul

T5 exo (1/10)

1ul

Fgt E

1,2ul

Experiment 2: Colony Observation and PCR Setup

Week: 1: 05/08/2025

Purpose:

Check colonies obtained from June 2 transformation and set up PCR for EGE37 (DH5α) and EGE1345 (DH5α) as controls.

Methods and Procedures:

  • Observed colonies on plates: control (no insert) showed none or very few colonies, while VNP and VNP-Green showed tens to hundreds of colonies.
  • Performed streaking of EGE37 and EGE1345 strains.
  • Prepared PCR reactions with DreamTaq polymerase using diluted primers.
  • Prepared mega master mix for 15+ reactions.
  • Collected colonies and prepared suspensions for PCR templates.
  • Ran PCR cycles and prepared agarose gels (1% and 1.5%).
  • Performed agarose gel electrophoresis of transformants 1 and 3.

Results:

  • Colonies with inserts successfully grew, indicating plasmid recircularization.
  • PCR reactions prepared and run with DreamTaq.
  • Gel electrophoresis performed for verification.

Petri dish

100 uL of bacteria

Pellet

Sans insert

No colonies

A few colonies

With VPN

10 colonies

50 colonies

With VPN Green

10 colonies

100 colonies

Notes:

  • Resistance marker used: Cm (not Ampicillin, to avoid interference with vesicle formation).
  • PCR program: ~1.5 min elongation for ~1.3 kb fragment.

Experiment 3: TB Medium, Gel Interpretation, Cultures

Week: 1: 06/08/2025

Purpose:

Prepare TB medium, interpret PCR gel results, and set up cultures for vesicle purification.

Methods and Procedures:

  • Prepared ~4.5 L TB medium and 500 mL phosphate buffer (autoclaved separately).
  • Observed Cm + ATC plates for fluorescence induction: fluorescence seen at ATC20 but absent at ATC100.
  • Poured new agar plates with Cm20, Cm+ATC100, and Amp+ATC100.
  • Interpreted gels: 1% agarose gel confirmed presence of inserts; 1.5% gel showed some colonies lacking insert.
  • Selected clones 1.5 and 1.6 (transformant 1) and clones 3.3 and 3.4 (transformant 3).
  • Set up overnight cultures of EGE37 and EGE1345 in TB medium for vesicle extraction.
  • Performed duplication of plates with sterile velvet and OD600 measurements.

Results:

  • TB medium successfully prepared.
  • PCR confirmed insert presence in multiple colonies.
  • Clones selected for further experiments.
  • Cultures of EGE37 and EGE1345 growing in TB medium.
Screenshot Screenshot

Photos of the PCR migration of transformants 1 (right) et 3 (left) on agarose gel 1,5% and 1% made the 05/08/2025

Notes:

  • High ATC concentration may reduce fluorescence due to toxicity or mislabeling of plates.

Experiment 4: Minipreps, Nanodrop, Digestion, Vesicles

Week: 1: 07/08/2025

Purpose:

Perform minipreps of EGE37 and EGE1345, check plasmid DNA quality, and test fluorescence at different ATC concentrations.

Methods and Procedures:

  • Observed fluorescence: ATC20 (ATC with a 20ug/ml concentration) showed strong signal; ATC100 (ATC with a 100ug/ml concentration) plates showed none.
  • Performed minipreps of EGE37 and EGE1345 to extract plasmid DNA.
  • Measured DNA concentration and purity using Nanodrop.
  • Prepared enzymatic digestions of plasmid DNA with restriction enzymes.
  • Ran agarose gels of digested and undigested samples.
  • Filtered vesicles from cultures of EGE37 and EGE1345 using vacuum pump.
  • Prepared overnight cultures of clones 1.5, 1.6, 3.1, and 3.3 with Cm.

Results:

  • Minipreps yielded plasmid DNA (confirmed by Nanodrop).
  • Digestions prepared for gel analysis.
  • Vesicle filtration performed with a lot of fluorescence in the pellets and not much in the filtration lysate (noted as very slow).
  • The filter seems to be containing the vesicles.
Screenshot

Photo of the petri dish Cm20 + ATC20 (left) and Cm20 + ATC100 (right)

Screenshot

Photo of the pellet containing the bacteria with Vnp mneon green vesicles (right) and Vnp 6xhis vesicles (left)

Screenshot Screenshot

Photo of the filtration lysate from the filter containing vesicules for Vnp mneon green (right) and the filter before scraping containing Vnp mneongreen vesicules (right)

Notes:

  • Nanodrop cannot distinguish plasmid vs genomic DNA → digestion required for confirmation.
  • Possible cytotoxicity of ATC100 reducing fluorescence.
  • The vesicules might mostly be in the bacterias.

Experiment 5: Colony Observation, Miniprep Attempt, Night Cultures

Week: 1: 08/08/2025

Purpose:

Repeat minipreps for selected clones and prepare night cultures for next week.

Methods and Procedures:

  • Observed repicked colonies (1.1-1.6, 3.1-3.6). Fluorescence is better on ATC100 plates from tuesday than the monday ones.
  • Attempted minipreps for clones 1.5, 1.6, 3.3, 3.4 with new kit, but forgot ethanol addition → failed prep.
  • Waliya prepared cryotubes for the same clones and agarose gels for digestion from June 5.
  • Checked migration results against restriction maps.
  • Prepared overnight cultures of clones 1.5, 1.6, 3.3, 3.4 (stored at 4°C).
  • Cultures will be incubated on Monday for repeat minipreps.

Results:

  • Minipreps failed due to missing ethanol step in the kit.
  • Cryotubes and gels prepared successfully.
  • Night cultures ready for next week.

Notes:

  • Reminder: always check new kit reagents for preparation requirements.

Experiment 6: Mini-Preps, SDS-PAGE, Digestion

Week 2 : 12/08/2025

Purpose:

To extract plasmid DNA (clones 1.5, 1.6, 3.3, 3.4), verify by restriction digestion, and assess visualisation of vesicle production by SDS-PAGE.

Methods and Procedures:

  • Performed mini-preps for clones 1.5, 1.6, 3.3, and 3.4.
  • Prepared SDS-PAGE gels (12%) for vesicle protein analysis (ENG1-4, E1-6xHis, E3-6xHis).
  • Nanodrop quantification of plasmid DNA.
  • Digestion with HindIII and SpeI.
  • Ran agarose gel for digested plasmids.
  • Prepared overnight cultures for PRL81 plasmids (empty, Vnp-mNeonGreen, Vnp-6xHis).

Results:

  • Nanodrop results: 1.5 = 73.5 ng/µL, 1.6 = 86.2 ng/µL, 3.3 = 70.9 ng/µL, 3.4 = 94.3 ng/µL.
  • SDS-PAGE revealed weak bands near 30 kDa for mNeonGreen.
  • Agarose gel confirmed expected digestion patterns for clones 1.5, 1.6, and 3.4.
Screenshot

Photo of the agarose gel of the enzymatic digestions of clones 1.5, 1.6, 3.3 and 3.4.

Well order:

  • Size marker
  • 1.5 digested → nNeonGreen
  • 1.6 digested → nNeonGreen
  • Plasmid without insert, undigested (error: it should have been digested)
  • 3.3 → 6xHis (cloning without insert)
  • 3.4 → 6xHis (correct cloning)
The plasmids were digested with SpeI and HindIII:
  • For nNeonGreen (1.5 and 1.6) we expect two fragments at 3015 bp and 1711 bp.
  • For 6xHis (3.3 and 3.4) we expect two fragments at 3015 bp and 1133 bp.
These expected results were observed for clones 1.5, 1.6, and 3.4.
  • Clone 3.3 showed an abnormal fragment migration pattern.

SDS PAGE:

Gel 1 : Sample ENG1 à ENG4, two concentrations tested:
  • 15 µL sample + 5 µL with CB load buffer
  • 10 µL Sample + 10 µL with CB load buffer
Gel 2 : Sample E1-6xHis / E3-6xHis : Same variation in terms of protein and load buffer concentration for the Vnp 6xhis vesicules.
Screenshot

Photo of the SDS PAGE gel with different protein and buffer concentrations of VNP 6xhis vesicules (left) and VNP mneongreen vesicules (right)

Notes:

  • Culture volume (50 mL) may be insufficient; 500 mL recommended for vesicle production.
  • Clones 1.5 and 3.4 selected for further work.
  • ENG1 and ENG4: The first round of scraped vesicles from the filter and 4th round for VNP mneongreen vesicles.
  • E1 and E3: The 1 and third round of scraping from the filter of Vnp 6xhis vesicles.

Experiment 7: iGEM Inserts, Cultures, Transformations

Week 2 : 13/08/2025

Purpose:

To prepare iGEM inserts, start large cultures of selected clones for vesicle filtration, and perform transformations with Fragment A, mNeonGreen, and empty plasmid.

Methods and Procedures:

  • Prepared lyophilized iGEM inserts by adding 50 µL sterile water and vortexing.
  • Set up 250 mL TB cultures with chloramphenicol for clones 1.5 and 3.4 in 1 L Erlenmeyers.
  • Measured OD600 values multiple times during incubation.
  • Transformation and cloning performed for Fragment A, Vnp-mNeonGreen (positive control), and empty plasmid.

Spreadsheet of volumes used for this cloning

Linear Plasmid + A (RPA1163_WT)

Linear plasmid

Plasmid linear

Water

4,8 µL

6,8

RPA

1,7 µL

X

Cutsmart buffer 10X

1 µL

1 µL

T5 exo (1/10)

1 µL

1 µL

Fgt A (RPA1163_WT)

1,2 µL

1,2 µL

Table of volumes used for this cloning

m Neon Green

Water

5,1 uL

RPA

1,7 µL

Cutsmart Buffer 10X

1 µL

T5 exo (1/10)

1 µL

Fgt A (RPA1163_WT)

1,2 µL

Results:

  • OD600 results (dilution 1/5): Clone 1.5 = 0.14, Clone 3.4 = 0.15.
  • ATC added to reach 200 ng/mL concentration.
  • Cultures incubated overnight at 37°C with shaking.

Notes:

  • 24 inserts renamed for easier labeling and management.
  • Gel SDS-PAGE washed with acetic acid and reused.

Experiment 8: Transformation Analysis, PCR, Filtration

Week 2 : 14/08/2025

Purpose:

To analyze transformations, confirm inserts by PCR, test vesicle filtration, and set up overnight cultures.

Methods and Procedures:

  • Observed colonies from previous transformations (Fragment A, mNeonGreen, empty plasmid).
  • Replica plating by velvet on Cm20 + ATC20 and Cm20 + ATC100 plates.
  • Prepared PCR reactions with insert-specific primers; ran agarose gel.
  • Started vesicle filtration for clone 1.5 (mNeonGreen).
  • Performed new transformations with controls and mNeonGreen.
  • Prepared overnight cultures for EGE314 (MG1655) and EGE1236 (1236 BL21) (wild-type strains).

Results:

  • Unexpected colonies observed in negative control plates, suggesting possible plate mix-up.
  • PCR strategy: expect 1.5 kb band if insert present, 5 kb if absent.
  • Filtration was very slow; pump efficiency suspected as the cause of the experimental failure.

100 µL of bacteria

Pellet

Control

~ 50 colonies

~ hundreds of colonies

mNéonGreen

1 colonies

~50 colonies

Fragment A

~ 50 colonies

~ ten colonies

Spreedsheet of colonies obtained from the transformations and clonings of Fragment A, mNeonGreen, and no-insert (Wednesday, June 11)

Notes:

  • Plan to test stronger vacuum pump at Mendel laboratory.
  • Wild-type strains (EGE314, EGE1236) expected to yield more vesicles.

Experiment 9: Troubleshooting PRL81, Linearization

Week 2 : 15/08/2025

Purpose:

To analyze transformations from June 12, troubleshoot PRL81 plasmid issues, and linearize PRL81 by PCR.

Methods and Procedures:

  • Checked transformations (controls and mNeonGreen).
  • Tested stronger vacuum pump for vesicle filtration efficiency.
  • Observed PCR results from filtrations under UV (no fluorescence).
  • Identified issue: PRL81 plasmid was circular, not linearized.
  • Prepared PCR to linearize PRL81 using primers L2172 and L2713.
  • Prepared overnight cultures of EGE314 and EGE1236.

Results:

  • Control plate unexpectedly had colonies: The plasmid was not properly linearized.
  • PCR designed to produce 4 kb fragment for PRL81, 3.1 kb for PIB37.
  • No fluorescence observed in filtrations.

100 µL

Pellet

Control

1 colonie

10 colonies

m Neon Green

10 colonies

100 colonies

Notes:

  • Lost linearized PRL81 plasmid : It must re-linearize by PCR.
  • Plan established for next week: Monday (day culture), Tuesday (competent cells), Wednesday (transformation).

Week 3 : 18/08/2025

Purpose:

Verification of plasmid linearization (agarose gel), preparation of competent cells (EGE12), sequencing (Sanger) for inserts, first cloning attempts with PIBA37, and setup of overnight cultures for plasmid mini-preps.

Methods and Procedures:

  • PCR and agarose gel migration (1%) with safe dye.
  • Competent cell preparation protocol (storage at -80°C).
  • Sequencing sample prepared (12 µL plasmid + 3 µL primer L33, 20 µM).
  • Cloning of 9 fragments + control into PIBA37, followed by transformation.
  • Plate preparation with ampicillin.
  • Overnight cultures (EGE37, EGE1345, and strain with Vnp-6xHis).

Results:

  • Agarose gel: PRL81 plasmid linearization successful, PIB37 unsuccessful (redone).
  • Sanger sequencing sample sent.
  • Competent cells aliquoted and stored.
  • Transformation with PIBA37 carried out.

Notes:

  • Re-test purification with Vnp-mNeonGreen.
  • For competent cells, centrifugation must be done at 4 °C.
  • PIBA37 plasmids stored, overnight cultures incubated.

Week 3 : 19/08/2025

Purpose:

Purification of linearized plasmids, verification by Nanodrop, mini-preps (EGE37, EGE1345, HB42), and bacterial transformations with PIB37 constructs.

Methods and Procedures:

  • Agarose gel purification of plasmids → Nanodrop quantification.
  • Mini-preps performed for three strains (EGE37, EGE1345, HB42).
  • Transformations carried out using PIB37 + Vnp-mNeonGreen + Vnp-6xHis + control.
  • Competent cells used: PBL21 and MG55.
  • Plate preparation with Ampicillin.
  • PCR setups for remaining fragments.
  • Culture OD monitoring: Vnp-mNeonGreen (OD600 = 0.5), Vnp-6xHis (OD600 = 0.75).
  • ATC induction: 20 µL added.

Results:

  • Nanodrop confirmed DNA presence.
  • Successful mini-preps.
  • Transformation completed by 19h15.

Notes:

  • Approximately 80 Amp plates were prepared.

Experiment 10 (Aout 18-20, 2025)

Week 3 : 20/08/2025 - 22/08/2025

Purpose:

To transform and clone bacterial strains with the competition genes in order to obtain colonies carrying the desired plasmids. Test the filtration protocol with a type of filter that has a hydrophobic MCE 0.1um membrane to avoid clotting.

Methods and Procedures:

  • Performed bacterial transformation with the constructs containing the competition genes.
  • Plated the transformed cells on a selective medium containing the appropriate antibiotic.
  • Incubated overnight at 37 °C.
  • Repeated the entire transformation and cloning procedure a second time due to the absence of colonies after the first attempt.
  • Retake of the filtration protocol with a vacuum pump.

Results:

  • First attempt (June 18-19) : No colonies observed on selective plates.
  • Fluorescence where noted among the filters for Vnpmneon green with the 0.1um MCE filter.
  • Second attempt (June 20): Again, no colonies appeared.
Screenshot

Photo of the test of MCE filter 0.1 for Vnp mneon green (middle), 0,1 PVDF filter for mneongreen (left) and MCE filter for Vnp 6xhis (right)

Notes:

  • The failure was traced back to the use of non-viable or low-quality competent cells, which prevented successful transformation.
  • DNA constructs and cloning strategies were not the cause of the problem.
  • The next step is to prepare fresh competent bacteria and repeat the transformations with the same plasmids.
  • The new filter is the one that is going to be used for further filtration.