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 2
Day 1
Biobricks design: We designed Hmas, MdlB and MdlC biobricks.
Megaprimers design: We designed megaprimers to add the Tag-his to our biobriks sequences.
Chitinase: We received the chitinase gene sequence from IDT and ran a PCR to amplify the gene.
Methionine-gamma-lyase': We recovered the biobrick from iGEM 2018 kit.
Day 2
Chitinase: We took the PCR product and verified the amplification on an agarose gel. We ran into into hybridyzation problems so we had to determine it using a gradient-PCR.
Day 3
Chitinase: We cloned the PCR product in a PSB1C3 plasmid.
Methionine-gamma-lyase: We transformed the biobrick (From the iGEM 2018 kit) into BL21 competent strains beacuse we ran into problems making competent DH5alpha strains .
Day 4
Chitinase: We ran another cloning process on the previous PCR product and transformed it into DH5alpha competent cells.
Methionine-gamma-lyase: We recovered the transformation plates and ran a colony-PCR on selected colonies. Afterward, we launched starters on the positive colonies.
Day 5
Chitinase: We transformed DH5alpha strains with the plasmid containing the chitinase sequence.
Interlab: We recovered the 8 biobricks (From the iGEM 2018 kit) and transformed them into DH5alpha competent cells.
Methionine-gamma-lyase: We purified the DNA using a miniprep kit and stoked it.
MdlB biobrick: We received the biobrick and ran a PCR on it.
Competent cells : 12/06/18 Competent cells made by the origin DH5-alpha (less recombination for the copy of DNA) for the next cloning of the chitinase But: error: centrifugation of the starter instead of putting increasing by the starter and then centrifugation in 4°C Transformation of the competent cells to test the skill of bacteria with an unknown plasmid of the iGEM on 2016 of a miniprep (normally) Expected results: 1 band for the not digested plasmid, and 2 bands for the digested plasmid. Obtained results: 2 bands for the non-digested plasmid, and 2 bands for the digested plasmid. The plasmid non-digested was already digested, so the digestion was completely unnecessary. And cell competence can’t be quantified by transformation because we won’t obtain colonies. Digestion of the plasmid outcome of miniprep for the negative control of the transformation and PCR clean - up for the negative control of the previous transformation 13/06/18 Transformation results: no colonies obtained for the undigested plasmid, nor for the digested plasmid, this is normal because they were found both digested. Therefore, we can’t deduce the skill rate of the bacteria prepared in comparison to BL21 from “training week 1” and uncontaminated 14/06/18 Results of the transformation: for the competent DH5-alpha: → without plasmid: 4 colonies → with plasmid: 75 colonies per box against BL21 ratio of 506 colonies per box of BL21 with plasmid and no colonies without plasmid
OmpT-AIDAC : Biobrick of the biobrick of: OmpT-AIDAC cleavage sequence made and ordered Interlab 14/06/18 Yorgo’s TECAN training for interlab: 8 biobricks to transform at DH5-alpha 6 with GFP and 2 systems Tetr repression tecan use 15/06/18 Transformation of 8 biobricks (Yorgo) for the Interlab RFP : 14/06/18 Output of RFP (plate 1 position 23O) → RFP will serve as plasmid support for biobricks: white-red screen 15/06/18 DH5-alpha transformation by the Plasmid Containing LacI and RFP
LacI: 14/06/18 LacI BBa8 R0011 without RBS so we will try with promoter-RBS because difficult assembly (plate 4 position 5H) of the kit iGEM → re-hydration and storage at -20 ° C → LacI will serve as an inducible promoter for the production of biobricks 15/06/18 DH5-alpha transformation by the Plasmid Containing LacI
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
Methionine-gamma-lyase: We digestion magapriming product by DpnI (To cut the methylated GATC sites), and transformed it into DH5alpha competent strains.
DH5alpha competent cells: we renewed our stock and verified their efficiency using the RFP plasmid.
InterLab: We ran a second measurement test.
MdlB biobrick: We recovered the transformation plates and ran colony-PCR on the positive colonies.
Extracurricular: We made our breaking bugs logo using E. coli strains that express the GFP.
Day 5
DH5alpha competent cells: We double-checked the competent cells efficiency by transforming the RFP plasmid.
MdlB biobrick: We recovered the overnight starters, and purified the DNA using minpreps kits, measured the DNA concentration on the NanoDrop and ran a digestion test.