WEEK 1

Training

During the first week, we had several training sessions, under the supervision of Gauthier DANGLA-PELISSIER (Instructor), to get acquainted with the lab work, master everyday protocols (PCR/Colony-PCR, competent cells preparation, transformation, cloning, minipreps, PCR clean-ups...), and establish a classic workflow.

Day 1: Lab setup

We recovered lab equipment from our home university, cleaned-up the lab, and had a first meeting to establish the project's workflow.

Day 2: Preparing competent cells stock and transformation

We prepared a stock of competent cells using the following protocol. Afterward, we prepared Petri dishes and we transformed the cells to test their efficiency using a plasmid provided by our instructor.

Day 3: Colony-PCR and agarose gel electrophoresis

The next day we had colonies on our Petri dishes and wanted to check if they acquired the transformed plasmid with the right inserted gene. We picked out some positive colonies and ran a colony-PCR. We then migrated the PCR products on an agarose gel.

Day 4: DNA purification miniprep

We purified the plasmid with the right inserted gene from the positive colonies using a DNA purification kit. In a further step, we digested the purified plasmid with respective restriction enzymes to double-check the presence of the inserted gene.

Day 5: Western-blot

The last day, we checked the induced production rate of the relative protein to the inserted gene (Produced in a preliminary step by our instructor) using the following western-blot protocol.

WEEK 3

Day 1

pLacI promoter biobrick

We recovered the transformation plates and ran a colony PCR on the obtained positive colonies.

RFP

We recovered the transformation plates and launched overnight starters to purify the plasmid later on. (no colony-PCR because the colonies are red: RFP expression).

Chitinase

- Results of the chitinase transformation: →2 colonies for the insert ratio →5 colonies for ratio 4 →1 colony for ratio 5 - Negative Control Transformation Results: No Bacteria - PCR colony of chitinase: - Results of the PCR colony: no band at 1000 bp, but a band at 250 bp therefore bad insert in the plasmid - PCR of chitinase (-0.2 µL of chitinase IDT) - Hybridization temperature gradient to find the optimal ™ (-1 µL of chitinase IDT) - Results of the gel

Methionine-gamma-lyase

We recovered the methionine-gamma-lyase biobrick (from the iGEM 2018 kit) and transformed it in DH5alpha competent strains.

DH5alpha competent cells

- Starter of competent cells - Preparation of buffers 1 and 2 for competent cells

InterLab

We recovered the 8 transformations and launch of 8 starters in duplicates.

Day 2

pLacI promoter biobrick

Miniprep of pLacI: concentration 13 ng / µL of DNA → Remake because concentration too low

RFP

- RFP miniprep: concentration 148 ng / µL

Chitinase

- Gel extraction from the chitinase gel - Digestion - PCR- clean up - overnight chitinase ligation 16 ° C

DH5alpha competent cells

- Culture DH5a - Aliquots of competent - Competent verification by transformation with RFP

Methionine-gamma-lyase

- MGL PCR colony - Check of the cPCR by gel agarose - Starter Positive Colonies on Agarose Gel

InterLab

- First measurement tests on TECAN. - 3 calibration measurements (Water and LUDOX, silica beads and Fluorescein). - Overnight crop recovery, 1/10 dilution and DO600 measurement at TECAN (all OD600 are good except for one with OD600 above 4). - Additional dilution (for an OD600 of 0.02) and taking aliquots at t = 0, t = 3 and t = 6 growth at 37 C. - Measure at TECAN of the different aliquots.

Thanks

Day 3

Mégaprimer - we received the 9 megaprimer to add the 6-His tag - impoundment for final concentration of 100μMolar

pLacI promoter biobrick

- miniprep with the butterfly kit. Nanodrop results: 109ng / μl 260/280: 1.92; 260/230: 2.10

P fu DNA Polymerase - Received for megapriming

Chitinase

- Ligation on bench (same quantity as the day before) + CTRL with RFP alone league - Processing in Gautier's DH5alpha of bench-top and overnight ligation (from 19/06)

MDLB - Ligation on bench (same quantity as the day before) + CTRL with RFP alone league - Processing in Gautier's DH5alpha of bench-top and overnight ligation (from 19/06)

Skill - Starter DH5-apha to remake skills the next day

Methionine-gamma-lyase

- miniprep with the promega kit. Nanodrop results: 233 ng / μL. - Mégaprimer overnight →tag his (PCR megapriming)

InterLab

- Data analysis and launch of new starters to redo the measures of 19/06.

Day 4

InterLab

- Resumption of the 19/06 measurements and data analysis.

Methionine-gamma-lyase

- Digestion by DpnI (will cut the methylated GATC sites) of the PCR product magapriming - Transformation of DpnI digestion product in DH5a using two techniques: Rolland's and Gauthier's.

Skill : - 200ml culture from starter DH5a - 100 μL aliquot of DH5a - Verification of the competence by transformation with the RFP.

MdlB : - Observation of colonies of transformations →few white colonies (bench-top ligation + effective overnight ligation) - CPCR on these colonies + transplanting + gel - launch of STARTER on the well 20. - Realization of the Breaking Bugs logo

Day 5

MdlB : - STARTER of the day before →red so corresponds to the RFP →Miniprep above + clean up + nanodrope + digestion test

skills : - Re-transformation of versions 3 and 4 of DH5alpha (supposedly competent)