Team:Aix-Marseille/Notebook
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 biobrick: 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: We recovered the transformation plates and ran a colony PCR on the obtained positive colonies.
We amplified the chitinase IDT gene sequence to recover the stock.
Methionine-gamma-lyase: We recovered the methionine-gamma-lyase biobrick (from the iGEM 2018 kit) and transformed it in DH5alpha competent strains.
InterLab: We recovered the 8 transformations and launched 8 starters in duplicates.
Day 2
pLacI promoter biobrick: We purified the inserted RFP gene using DNA purification kits and restart because the final DNA concentration was too low (13 ng/µL).
RFP biobrick: We purified the inserted RFP gene using DNA purification kits.
Chitinase: We ran a gradient PCR to find the optimal primers hybridization temperature. We then extracted the DNA using a gel extraction kit, digested the gene with respective enzymes, and ran ana overnight ligation in a PSB1C3 plasmid at 16°C.
DH5alpha competent cells: We prepared a new DH5aplha competent cells stock and verified their efficiency using an RFP plasmid.
Methionine-gamma-lyase: We ran a colony-PCR on the positive colonies and verified it on an agarose gel.
InterLab: We launched the 3 calibration measurements and ran the first cell measurements.
Day 3
pLacI promoter biobrick: We purified the DNA using a different miniprep kit.
Chitinase: We ran a ligation in PSB1C3 at room temperature.
DH5alpha competent cells: We launched DH5-alpha starter to renew the competent cells stock.
Methionine-gamma-lyase: We purified the DNA using the Promega miniprep kit. Afterward, we launched an overnight megapriming using specific primers to add a Tag-his to our protein so we can purify it.
InterLab: Data analysis and launch of new starters to run a second measurement test.
MdlB biobrick: We ran a ligation in PSB1C3 at room temperature.
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.
DH5alpha competent cells - 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: We recovered the overnight starters, and purified the DNA using minpreps kits, measured the DNA concentration on the NanoDrop and ran a digestion test.
DH5alpha competent cells: We double-checked the competent cells efficiency by transforming the RFP plasmid.