A. Experiments

Experiment 1: Mini-Preps

Week: 25/08/2025

Purpose:

Extraction of plasmid DNA from selected clones to perform Sanger sequencing. For DH5α clones, the goal was to prepare DNA for transformations into K16MG55 and Sanger clones.

Methods and Procedures:

Culture Preparation ("Culture" protocol)
Prepared overnight cultures using selected clones:
- Large culture: 9 flasks × 125 mL of overnight culture + 2 mL of colony.
- Small culture: 9 flasks × 25 mL of overnight culture + 0.4 mL of colony.
Clones included:
- Non-Sanger: B1, J1, I1, K1 (DH5α)
- Others: A1, B1, C1, D1, E1, F2, H1, I1, J1, K1, L1, M1, P1, R1, S1, X1, V1, W1 (DH5α)
Mini-Prep Procedure ("Miniprep for PCR and Sanger" protocol)

Results:

Plasmid DNA was successfully extracted from all selected clones.
DNA is ready for PCR amplification and Sanger sequencing.
Yield and purity were assessed (if applicable, record concentrations or A260/A280 values here).

Notes:

Ensure all ethanol is removed during washing to avoid interference with downstream applications.
For clones with low growth, increasing the initial colony volume can improve yield.
Store DNA at –20°C to maintain stability until use.

Week: 01/09/2025

Purpose:

Extract plasmid DNA from selected clones to perform PCR and Sanger sequencing.

Methods and Procedures:

Mini-Prep Procedure ("Miniprep for PCR and Sanger" protocol)
Clones processed: A1, F1, U1, O1 (K16MG55).
DNA from U1 was quantified using Nanodrop: 122.6 ng/µL.
Prepared DNA for Sanger sequencing ("Preparation for Sanger Sequencing" protocol).

Results:

Plasmid DNA successfully extracted from all clones.
DNA is ready for PCR amplification and Sanger sequencing.

Notes:

U1 showed good concentration on Nanodrop, suitable for downstream applications.
DNA storage: –20°C until use.

Week: 08/09/2025

Purpose:

Extract plasmid DNA from selected clones for PCR and Sanger sequencing.

Methods and Procedures:

Mini-Prep Procedure ("Miniprep for PCR and Sanger" protocol)
Clones processed: S1, J1, K1, U1, R1, O1, E1, W2 (K16MG55)
DNA quantified using Nanodrop.

Results (Nanodrop concentrations):

S1: 55.7 µg/mL
J1: 114.8 µg/mL
K1: 64.8 µg/mL
U1: 65.8 µg/mL
R1: 169.8 µg/mL
O1: 41.4 µg/mL
E1: 36.2 µg/mL
W2: 138.5 µg/mL
Mini-prep also performed for M1 for PCR application.

Notes:

All DNA samples were suitable for downstream PCR and Sanger sequencing.
Store DNA at –20°C until use

Experiment 2: Sanger Sequencing Preparation

Week: 25/08/2025

Purpose:

Prepare plasmid DNA and primers for Sanger sequencing to verify the presence of expected inserts and detect possible mutations. Some clones required re-sequencing due to mutations or poor coverage at sequence ends.

Methods and Procedures:

Clones and primer combinations:

B1, J1, I1, K1 – initial Sanger

Observations:

B1: one band of correct size, but mutation Leu → Met detected → change colonies.
I1 & J1: mutation in RBS → not usable.
K1: primer modified to sequence from start → resubmit.

A1, B1, C1, D1, E1, F2, H1, I1 – primer L33.
J1, K1, L1, M1, P1, R1, S1, X1, V1, W1 – primer L33.
E1, F2, I1 – primer L1326.

Objective: improve coverage at the beginning of the insert due to poor accuracy at sequence ends in previous Sanger results.

L1, J2, M1, P1, R2, S1, X1, V2, W1 – primer L1326.

Results:

All samples were prepared and sent to sequencing companies.
Some clones required resubmission due to detected mutations or incomplete coverage at sequence ends for DH5α colonies.

Notes:

Sequencing errors often occur near the ends of sequences; using alternative primers (like L1326) helps cover these regions.
B1, I1, J1, and K1 required adjustments due to detected mutations.
Ensure proper labeling to avoid mix-ups during sequencing submission.

Week: 1/09/2025

Purpose:

Prepare plasmid DNA from selected clones for PCR and Sanger sequencing, ensuring coverage of the insert using different primers for K16MG55 colonies.

Methods and Procedures:

For Sanger sequencing, prepared two tubes per clone one with primer L33 and one with primer L1326

Results:

All mini-preps were successfully performed.
DNA prepared with two primers ensures complete coverage of the insert for accurate Sanger sequencing.

Notes:

Using two primers helps sequence regions near the beginning and end of the insert, which are prone to sequencing errors.

Experiment 3: PCR Verification

Week: 25/08/2025

Purpose:

Verify the presence of the expected 1.5 kb insert in 17 mini-prep plasmids extracted from DH5α clones into A1 and F1.

Methods and Procedures:

("PCR" protocol)

Results:

PCR verified the presence of the 1.5 kb insert in all tested mini-preps.
Starter cultures for mini-preps and PCR for all transformants were successfully established.

Week: 01/09/2025

Purpose:

Verify that K16MG55 transformants A1 and F1 contain the expected 1.5 kb insert.

Methods and Procedures:

("PCR" protocol)

Results:

Bands at 1.5 kb observed for both clones.
DNA verified for downstream Sanger sequencing.

Week: 08/09/2025

Purpose:

Check for correct insert in clones prepared for mini-prep and future transformations.

Methods and Procedures:

("PCR" protocol)

PCR performed for clones S1, J1, K1, U1, R1, O1, E1, W2, M1 (K16MG55)

Results:

Clear bands at 1.5 kb for all transformations.
Night cultures can be used for further applications.

Notes:

DreamTaq master mix contains loading dye; no additional dye needed for gel.
Gel visualization in BET is low quality; confirm bands with ladder.
PCR confirms successful transformation and presence of insert before scaling cultures.

Experiment 4: Bacterial Transformation

Week: 25/08/2025

Purpose:

Transform K16MG55 competent E. coli with plasmids prepared from DH5α clones to obtain colonies for PCR and Sanger sequencing. DH5α transformants had low efficiency, so K16MG55 was preferred.

Methods and Procedures:

("Transformation of E. coli K16MG55" protocol)

Results:

Control PIBA37STAR: no growth, transformation sterile.
Colonies obtained for most transformants; however, M1, L1, I1, K1 did not grow.
Some cultures were contaminated; corresponding cell pellets were not usable.

Notes:

Transformation efficiency higher in K16MG55than DH5α.
Proper handling of SOC and pre-warmed plates is crucial for yield.
Contaminated cultures must be discarded and repeated (e.g., M clone).

Week: 1/09/2025

Purpose:

Transform K16MG55 competent cells with selected mini-preps to obtain new colonies for PCR and Sanger sequencing.

Methods and Procedures:

("Transformation of E. coli K16MG55" protocol)

Transformation performed with mini-preps: M1, O1, U1, E1, I1, J1, K1, L1, R1, S1, W2, plus control PIBA73STAR.

Results:

Control PIBA73STAR: no growth.
Colonies obtained for most transformants.
M1, L1, I1, K1 did not grow or were contaminated; M1 will be repeated.
Remaining transformants were suitable for downstream PCR and Sanger.

Notes:

Ensure sterile conditions throughout to avoid contamination.
Pre-warmed plates and careful spreading with beads improve transformation efficiency.
Negative controls are essential to verify sterility of SOC and procedures.

Experiment 5: Purification from Filtrates – Method A

Week: 25/08/2025

Purpose:

To test Protocol A for extracting and purifying proteins from extracellular vesicles of bacteria expressing Vnp-mNeongreen-6xHis. The goal is to recover and purify the protein of interest from bacterial supernatant, then validate its presence and purity by SDS-PAGE, and fluorescence measurement.

Methods and Procedures:

("Protocol A: Extraction of Vesicles from the Supernatant of K16MG55 Vnp-mNeongreen and Vnp-6xHis Bacteria" protocol)

Results:

Multiple samples collected at key stages (supernatant, post-Triton-X, flow-through, washes, elution, dialysis)
SDS-PAGE deposits:

Vnp.mNeongreen: 17.5 µL (from 35 µL, 100 mL culture)
Vnp-6xHis: 10 µL (from 20 mL culture)

Resin pellet loss observed after Step 4 centrifugation
Final protein yield limited

Notes:

Loss of resin during centrifugation likely reduced purification efficiency
Initial resin volume (100 µL) may be insufficient → increase resin volume
Critical loss steps: decanting and washes
Retest with optimized resin volume and careful handling during centrifugation

Experiment 5 – Vesicle Purification – Vnp-Mneongreen-6xHis K16MG55 - Method C

Week: 25/08/2025

Purpose:

The goal of Protocol C is to isolate, purify, and analyze vesicles containing Vnp-Mneongreen-6xHis from the filtered supernatant of bacterial cultures. This allows:

Concentration of intact vesicles for analysis and purification.
Controlled lysis of vesicles to release the target protein.
Purification of mNeongreen-6xHis protein using Ni-NTA affinity chromatography.
Assessment of protein purity and quantity at various stages (SDS-PAGE, Western blot, fluorescence).

Methods and Procedures:

("Protocol C (also from vesicles). K16MG55 Vnp-green and Vnp-6xhis" protocol)

Notes:

Protocol C performed on 250 mL of filtered supernatant from the previous day.
Volume recovered after scraping the filter: ~1 mL.

Experiment 5 – Extraction and Purification of Proteins from Bacterial Pellets - Method B

Week: 25/08/2025 — Vnp-mNeongreen-6xHis K16MG55

Purpose:

The purpose of this experiment was to test Protocol B for protein extraction from bacterial pellets using E. coli strains EGE314 Vnp-mNeongreen and Vnp-6xHis. The aim was to extract and purify the target protein Vnp-mNeongreen-6xHis and evaluate the efficiency and purity at each step.

Methods and Procedures:

("Protocol B: Protein Purification from Lysed Bacterial Pellet (Batch Method)" protocol)

Adjustments:

During dialysis/concentration with Vivaspin 500, centrifugation was done at 7,800×g instead of 12,000×g (maximum allowed by equipment).

Results:

Cell pellets and lysates were successfully obtained for both strains.
Soluble fraction contained detectable Vnp-mNeongreen-6xHis.
Ni-NTA purification successfully captured His-tagged protein; flow-through and wash fractions showed minimal protein.
Elution fractions contained concentrated Vnp-mNeongreen-6xHis, confirmed by SDS-PAGE and Western Blot.
Dialysis and glycerol addition provided stable protein samples for storage at −80°C.
Fluorescence measurements confirmed retention of mNeongreen activity after purification.

Notes:

Low-speed centrifugation during batch Ni-NTA was critical to prevent resin loss.
Collecting SDS-PAGE samples at each step allowed monitoring of protein yield and purity throughout the protocol.

Week: 01/09/2025 — Vnp-mNeongreen-6xHis K16MG55

Purpose:

The purpose of this experiment was to refine Protocol B to improve protein purity by reducing nonspecific bands (contaminants) in the purified fractions. This was achieved by testing different numbers of wash steps during Ni-NTA purification of Vnp-mNeongreen and Vnp-6xHis proteins.

Methods and Procedures:

("Protocol B: Protein Purification from Lysed Bacterial Pellet (Batch Method)" protocol)

Used cell pellets from 50 mL overnight cultures (supernatants removed during previous centrifugation on 25/08/2025).
Test of different wash steps during Ni-NTA purification:

1 pellet Vnp-mNeongreen + 1 pellet Vnp-6xHis: 3 washes
1 pellet Vnp-mNeongreen: 4 washes
1 pellet Vnp-mNeongreen: 5 washes

SDS-PAGE and Western Blot were performed for all wash and eluate fractions to evaluate protein purity.
Fluorescence measurements at 506 nm were used to monitor mNeongreen activity.

Results:

Increasing the number of washes reduced nonspecific bands, indicating fewer contaminants.
3 washes removed some nonspecific bands but left minor contamination.
4 washes further improved purity.
5 washes achieved the highest purity, though yield slightly decreased.
Dialyzed proteins remained stable after concentration at 7,800×g.
Fluorescence measurements confirmed that mNeongreen retained its activity after purification.

Notes:

Adjusting the number of washes is effective to optimize purity at the expense of minor reduction in yield.
Centrifugation speed limitation (7,800×g) during Vivaspin concentration did not significantly impact protein recovery.
Collecting samples from each wash fraction is crucial to monitor which step removes the most contaminants.

Week: 07/09/2025 — Vnp-mNeongreen-6xHis K16MG55

Purpose:

The purpose of this experiment was to test protein purification strategies for Vnp-mNeongreen-6xHis variants, including:

Elution with increasing imidazole concentrations for VG (Vnp-mNeongreen) to evaluate contamination and protein yield.
TEV protease cleavage for A1 and U1 constructs to remove tags and test protein cleavage efficiency.
Optimizing purification for maximum purity and retention of protein activity.

Methods and Procedures:

("Protocol B: Protein Purification from Lysed Bacterial Pellet (Batch Method)" protocol)

1. Protein production and induction

Tested different induction conditions: 2 h induction in 5 mL cultures for most constructs; overnight cultures used for vesicle-associated proteins.
Prepared induced and non-induced controls, including empty plasmid or uninduced cultures, to verify the induction system.
Selected clones: out of 25 clones, 22 transformed, 15 produced protein, 11 functional, 5 used for time constraints.

2. Purification with increasing imidazole concentrations (VG)

VG100: eluted sequentially with NPI-100 then NPI-500.
VG250: eluted sequentially with NPI-100, NPI-250, then NPI-500.
Observed contamination and protein loss; concluded 3 standard washes without changing imidazole concentration are more efficient.

3. TEV protease cleavage (A1 and U1)

Incubated 2 µL TEV in 500 µL NPI-20 buffer overnight at 4°C for A1 and U1.
A1CT and U1CT incubated overnight at 4°C without TEV.
Following day: continued dialysis and added glycerol (50% final) to concentrate proteins (~200 µL) for −80°C storage.

4. SDS-PAGE analysis preparation

Diluted protein samples to optimize band resolution.
Planned to run separate gels for each sample to avoid overlapping bands.
Included TEV-only control to identify TEV migration.

5. Additional purification strategies

Tested elution with imidazole after column was emptied.
Planned re-purification with Ni-NTA resin magnetic to remove excess TEV protease.

Results:

A1(K16MG55) purification: three bands observed.

Two bands likely correspond to RPA WT (potential miscleaved forms).
One lower band likely corresponds to TEV protease.
Conclusion: successful purification of RPA WT

U1(K16MG55) purification: no detectable band; protein may be absent or degraded.
VG constructs (VG100 and VG250): significant contamination and protein loss observed with stepwise imidazole elution. Using 3 washes without changing imidazole concentration appears more efficient.

Notes:

TEV incubation amount may need optimization; 2 µL might be excessive — testing 0.5 µL is suggested.
Aliquoting protein (~10 µL) is recommended to adapt to experimental needs and probe usage.
Running SDS-PAGE with diluted samples improves band resolution.
For VG constructs, stepwise imidazole elution caused more contamination than standard washing; protocol should favor simpler washes.
Successful purification of A1(K16MG55) demonstrates that TEV cleavage and Ni-NTA purification can yield functional RPA WT protein.

Week: 15/09/2025 — A1 Vnp-mNeongreen-6xHis

Purpose:

The purpose of this experiment was to optimize TEV protease cleavage during A1(K16MG55) protein purification by testing different TEV volumes. The goal was to find the optimal TEV amount that effectively cleaves the tag while minimizing TEV contamination in the final protein sample.

Methods and Procedures:

("Protocol B: Protein Purification from Lysed Bacterial Pellet (Batch Method)" protocol)

1. Testing A1(K16MG55) purification with different TEV volumes

Tested TEV volumes: 2 µL, 1 µL, 0.5 µL, 0.2 µL.
Lysed one cell pellet and divided the total lysate into 4 Falcon tubes (15 mL each), 1.25 mL per tube.
Adjusted purification volumes proportionally:

Ni-NTA resin: 100 µL → 25 µL per tube.
Other buffer and reagent volumes divided accordingly.

Performed Ni-NTA batch purification following standard Protocol B steps.

2. Dialysis of TEV-treated samples

Eluted fractions centrifuged sequentially: 500 g → 1000 g → 2000 g → 5000 g (2 min each).
Transferred samples to Vivaspin 500 columns for dialysis/concentration.
Centrifugation: 12,000 g, 4°C, 45 min.
Buffer exchange: 3× with PBS to remove imidazole.
Samples taken for SDS-PAGE: "Dialyzed Eluate (Protocol B)".

3. Removal of TEV using MagneHis™ Ni beads

Concentrated protein (~200–300 µL) mixed with 30 µL MagneHis™ Ni beads.
Gentle mixing: tube inverted 10 times, incubated 2 min at room temperature.
Magnetic separation: 30 sec to remove beads bound to TEV.
Supernatant recovered: contains purified A1 protein without TEV.
Samples prepared for SDS-PAGE.

4. Addition of glycerol for long-term storage

Protein concentrated after dialysis, added PBS + 50% glycerol (~200 µL).
Aliquoted according to downstream needs.
Stored at −80°C.

Results:

Observed that higher TEV volumes (2 µL) caused excess TEV contamination ("big blob" on gel).
Lower TEV volumes (0.5–1 µL) maintained effective cleavage while reducing TEV presence.
SDS-PAGE after MagneHis™ Ni treatment effectively removed TEV from the protein sample.
Final protein remained concentrated and stable after dialysis and glycerol addition.

Notes:

Aliquoting protein allows flexibility for downstream assays.
TEV volume optimization is crucial to balance tag cleavage and TEV contamination.
Magnetic Ni beads are effective for removing excess TEV after cleavage.
Dialysis and glycerol addition provide stable long-term storage conditions.

There was contamination when I had to load the wells but we notice that the TEV is not the second band.

For 0.2 µL, we notice that we keep the two bands but as we saw previously it is not TEV but therefore we do not know what it is.

For 0.5; 1; 2 µL, we notice that we keep the two bands also perhaps from the TEV?

Here we compare with the reference TEV, the sample was poorly deposited but we see that the TEV is much lower.

Experiment 6: SDS-PAGE Overproduction Tests

Week: 08/09/2025 — First SDS-PAGE screening

Objective:

Verify whether our K16MG55 transformants overproduce the protein of interest (RPAwt and Lipase) through SDS-PAGE.

Methods and Procedures:

Measured OD of overnight cultures: ~1.9.
For each transformant (A1, F1, O1, U1, M1, J1, S1, E1, W2; negative control = A1 non-induced (K16MG55)):

Collected 1 mL culture → centrifuged 10 min at 3000 g.
Discarded supernatant, resuspended pellet in 200 µL cracking buffer (10% of OD).
Heated 10 min at 95°C to denature bacteria and proteins.

SDS-PAGE: tested 5 µL and 10 µL sample loading (to avoid protein smearing).

Mini-cultures for verification:

5 mL LB + 5 µL Amp (100 mg/mL) + colony.
Tested: E1, W2, M1, S1, A1, F1, A1 non-induced (K16MG55).
At OD > 0.5 → induced with 1 µL aTc (1 mg/mL, final conc. = 200 ng/mL), 2 h incubation.
Volumes normalized to equivalent OD = 2 (e.g., OD 8 → 0.5 mL, OD 1 → 2 mL).

Expected sizes:

A1(K16MG55) RPAwt + VNP + TEV site + 6xHis = 38.7 kDa
RPAwt without VNP = 36.6 kDa
VNP alone = 2.1 kDa
U1(K16MG55) Lipase + VNP + TEV site + 6xHis = 38.8 kDa
Lipase without VNP = 36.7 kDa

Observations:

SDS-PAGE showed 2 slightly more intense bands around ~40 kDa and just below.
Likely corresponding to protein of interest with and without VNP.
Same observation for U1 lipase.
Loading 5 µL → clearer bands, less smearing.
Loading 10 µL → stronger signal but dirty gels.

Conclusion:

Preliminary evidence of overproduction, but gels not convincing.

Week: 08/09/2025 — Repeat with TB medium

Objective:

Repeat SDS-PAGE with improved culture conditions (TB medium).

Methods and Procedures:

Mini-cultures: 5 mL TB + 5 µL Amp + colony.
Tested clones: A1, A1 NI, U1, U1 NI, F1, M1, S1, J1, R1, W2, E1, O1 (K16MG55).
Incubation → induced at OD ~2 with 1 µL aTc, 2 h.
SDS-PAGE prepared as above.

Observations:

No clear overproduction visible.

Hypotheses:

aTc induction too low.
Protocol not properly optimized.
Plasmid backbone issue.
Enzyme is toxic → bacteria release proteins into vesicles (in supernatant, not pellet).

Week: 15/09/2025 — Larger screening (Test 2 & 3)

Test 2: K16MG55 transformants:

Colonies tested: A1, A1 NI, U1, U1 NI, F1, W2, E1, J1, S1, R1, O1, M1 (K16MG55).
Overnight cultures: 5 mL LB + 5 µL Amp.
Next day: transferred 100 µL preculture into 5 mL TB + 5 µL Amp (in plastic tubes, OD directly measurable).
Induction at OD ~0.5 → +1 µL aTc, 2 h.
Samples: 1 mL culture → centrifuged 10 min at 3000 g, pellet resuspended in 200 µL cracking buffer.

Observation:

S1(K16MG55) did not grow (OD = 0.004, culture remained clear).

Test 3: DH5α transformants (wild-type from Waliya & Matthieu):

Colonies tested: A3–A12 (DH5α).
Same culture and induction protocol as above.
Control: A3 NI.

Observation:

Cultures grew well, induction performed.
Pellets prepared for SDS-PAGE.

General outcome of SDS-PAGE experiments:

Direct TB culture (no LB preculture):

Clearer overproduction in A1, U1, O1, W2, F1, R1, E1 (K16MG55).
No overproduction in M1, J1 (K16MG55).
U1 (K16MG55) shows basal expression even without induction (leaky promoter).

LB → TB preculture transfer:

No visible overproduction.
Likely medium switch reduces induction efficiency.

Gueguen's feedback:

Current results not convincing enough.
Recommendation: test ~20 DH5α colonies to find potential overproducers.

Direct culture in TB (no LB preculture):

Clear overproduction observed in A1, U1, O1, W2, F1, R1, E1 (K16MG55).
No detectable overproduction in M1 and J1.
Notably, U1 appeared expressed even without aTc induction.

LB preculture (5 mL LB → 100 µL into 5 mL TB):

No visible overproduction detected under these conditions.

Experiment 8 – Protein Quantification

Week: 01/09/2025

Objective:

Quantify protein concentration using a BSA calibration curve and Nanodrop measurements (Thermo Scientific protein assay kit).

Methods and Procedures:

("Protein Quantification (ThermoScientific)" protocol)

Results – Sample "VG Protocol B":

Initial OD = 1.4 (too high, required dilution).

Dilution scheme tested:

Dilution	Sample volume	Preparation
×10	5 µL sample	+ 1 mL working reagent
×50	2.5 µL sample	+ 1 mL working reagent
×100	0.5 µL sample	+ 1 mL working reagent

Week: 15/09/2025 — Quantification of Purified Proteins

Goal: Measure the concentration of purified proteins from the recent purification batches
Methods and Procedures: (“Protein Quantification (ThermoScientific)” protocol)

Enzyme (K16MG55	DO	Concentration (µg/mL)	Quantity in the Falcon (µg)
A1 (18/07)	0,117	152,6	22,89
A1 (22/07)	0,104	131	19,65
A1 (22/07)	0,084	96	14,4
O1	0,017	12,87	1,93
U1	0	0	0
U1	0,011	8,33	1,25
M1	0,006	4,54	0,681

B. Cultures

Culture 1: Day Cultures for Mini-Preps

Week: 25/08/2025

Purpose:

Prepare daytime cultures from overnight colonies to perform mini-preps the following day.

Methods and Procedures: (“culture” protocol)

17 DH5α clones (labeled A–R, as per mini-preps)
Two culture volumes:

9 Erlenmeyer flasks × 125 mL overnight culture
9 Erlenmeyer flasks × 25 mL overnight culture

Results:

Cultures grew well and were ready for mini-prep the next day.

Week: 01/09/2025

Purpose:

Prepare starter cultures for mini-preps and PCR from selected clones.

Methods and Procedures:

Clones A1, F1, U1, K16MG55: same culture volumes as above.
Starter cultures for mini-preps from all transformants OK: S1, J1, K1, U1, R1, O1, E1, W2 (K16MG55).

Results:

All cultures reached appropriate density for downstream mini-preps and PCR.

Week: 09/09/2025

Purpose:

Prepare cultures for mini-prep from M1 (K16MG55).

Methods and Procedures: (“culture” protocol)

Results:

Culture of M1 grew successfully, ready for mini-prep.

Notes:

Ensure proper shaking and incubation for optimal growth.
Maintain sterility to avoid contamination for downstream mini-preps and PCR.

Culture 2: Overnight and Large-Scale Cultures

Week: 25/09/2025

Purpose:

Prepare starter cultures for large-scale overnight cultures to test purification protocols with high biomass.

Methods and Procedures: (“Large-Scale Protein Culture” protocol)

Clones: K16MG55 Vnp.mNeongreen and K16MG55 6xHis.
In 15 mL flasks: 3 mL LB + 3 µL Amp (100 mg/mL) + colony of interest.
Incubate over the weekend at 37°C, 200 rpm.

Results:

Starter cultures successfully grew and were ready for subsequent large-scale overnight cultures.

Week: 01/09/2025

Purpose:

Produce large-scale overnight cultures for F1, A1, U1 (K16MG55) and test inserts for PCR/Sanger sequencing. Also, produce bacteria and vesicles for extraction protocols B and C.

Methods and Procedures: (“Large-Scale Protein Culture” & “Miniprep for PCR and Sanger” protocol)

Small-scale overnight culture (for mini-prep/PCR/Sanger):

Clones: F1, A1, U1 (K16MG55)

Large-scale culture (for vesicle/bacterial production):

Clones: A1 (K16MG55)
Starter cultures for U1, O1, R1, S1 (K16MG55)

Results:

Cultures grew to expected density.
Small-scale cultures ready for mini-prep and PCR.
Large-scale cultures ready for vesicle/bacterial extraction.

Week: 08/09/2025

Purpose:

Produce overnight cultures for O1, U1, R1, S1(K16MG55) and backup starters for A1, U1(K16MG55).

Methods and Procedures: (“Large-Scale Protein Culture” protocol)

Backup cultures:

A1 and U1 (K16MG55)starters prepared for potential repeat of large-scale cultures
M1 (K16MG55) pellet stored at –80°C

Large-scale overnight culture from starters:

500 mL TB + 500 µL Amp + starter culture
Incubate 37°C, 200 rpm until DO > 0.8
Induce with 100 µL aTc (final 200 ng/mL) overnight

Issues observed:

U1(K16MG55): adhesion problem on cheese plate → culture failed (corrected afterwards)

Notes:

Monitor DO carefully before induction with aTc.
Filtration and vesicle recovery must be done gently to avoid loss.
Store supernatants and pellets appropriately for downstream SDS-PAGE and purification.

Culture 3 – Glycerol Stocks & Fresh Plates

Week: 15/09/2025

Objective:
Freeze validated transformants as glycerol stocks at –80 °C and prepare fresh plates. Colonies on agar plates are only stable for ~2 weeks, so repicking is necessary to maintain usable stocks.

Methods and Procedures: (“Large-Scale Protein Culture” & “Storage of Transformed Bacteria “ protocol)

For all K16MG55 transformants: 4xA1, 3xO1, 3xM1, 2xU1.
For A1, F1, O1, M1, U1, R1, J1, E1, W2

Repicked onto fresh LB + Amp plates.
Also prepared glycerol stocks.

Storage of transformed bacteria For A1, F1, O1, M1, U1, R1, J1, E1, W2 (K16MG55).