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 DH5α 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
For the whole week, we ran into a lot of problems making competent cells, so we spent a lot of time optimizing the protocol.
Day 1
Biobricks design: We designed Hmas, MdlB, MdlC and Hyd1 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
pLacI promoter biobrick: We recovered the biobrick from iGEM 2018 kit to assemble with other biobricks.
Chitinase: We took the PCR product and verified the amplification on an agarose gel. We ran into hybridization 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 DH5α strains .
Day 4
Chitinase: We ran another cloning process on the previous PCR product and transformed it into DH5α 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.
Interlab: We received a training on the measurement devices for the device and created the respective programs.
Biobrick design: we designed a new biobrick (OmpT-AIDAC) a cleavage sequence.
RFP biobrick: We recovered the biobrick (From the iGEM 2018 kit) that will be used for white-red screenings.
Day 5
pLacI promoter biobrick: We transformed the plasmid in DH5alpha competent cells.
Chitinase: We transformed DH5α strains with the plasmid containing the chitinase sequence.
Interlab: We recovered the 8 biobricks (From the iGEM 2018 kit) and transformed them into DH5α 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.
RFP biobrick: We transformed the biobrick into DH5alpha competent cells.
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.
DH5α competent cells: We prepared a new DH5α 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.
DH5α competent cells: We launched DH5α 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 DH5α competent strains.
DH5α 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
DH5α 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.
WEEK 4
We had the same problems making competent cells (contaminations...). We had the help of our instructor who pointed out a lot of flaws with what we were doing in the process.
Day 1
Chitinase: We took stocked strains and launched starters to purify the DNA the next day.
MdlB biobrick: We tried cloning the gene sequence into PSB1C3 and transformed the prouct into DH5α competent cells. Furthermore, we ran a PCR on the IDT sequence to renew our stock.
DH5α competent cells:
Interlab: We ran measurments for the third time and launched the CFUs protocol.
Methionine-gamma-lyase: We sent the magrapriming product to sequencing to check if we managed to add the Tag-his.
Day 2
Chitinase: We purified the DNA using a miniprep kit and had 225,87 ng/uL in concentration. Afterward, we sent the product for sequencing, and simultaneously, launched a megapriming to add the Tag-his for purification later-on.
RFP biobrick: We amplified it to renew our stock for contamination purposes.
Interlab: We had a training on how to do dilution series.
Methionine-gamma-lyase: The sequencing results were positve: we managed to add the Tag-his to our sequence.
MdlB biobrick: We recovered the transformation plates and ran a colony-PCR on the supposedly positive colonies.
Hyd1 biobrick: We received the gene sequence from IDT, ran a PCR on it and stocked it.
Day 3
Chitinase: We digest the megapriming product by DpnI and transformed it into DH5α competent cells.
MdlB biobrick: We tried cloning the gene sequence, a second time, into PSB1C3 and transformed the product into DH5α competent cells. Furthermore, we ran a PCR on the IDT sequence to renew our stock.
Interlab: We launched starters for a second CFUs test.
Hyd1 biobrick: We migrated the PCR product on an agarose gel, and did a second PCR after a first failure. After a second failure, we ran a gradient-PCR to determine the correct hybridization temperature.
Day 4
Chitinase: We recovered the transformation plates. No colonies were observable whereas the sequencing results showed positive results (The chitinase sequence was inserted into the PSB1C3 plasmid. We suspected the DH5α cells: turns out they were Bacillus Subtilis strains (Smell check). We had to run another megapriming.
MdlB biobrick: We used the same strains we used for the chitinase.
Interlab: We ran the CFUs test.
Hyd1 biobrick: We performed a PCR clean-up and migrated the PCR product.
Day 5
Chitinase: We digested the megapriming product by DpnI and transformed it into new DH5α competent cells.
Hyd1 biobrick: We cloned the gene sequence in PSB1C3 and trasnformed into DH5α competent cells.
WEEK 5
Day 1
Chitinase: Interlab: We counted the boxes and calculated the CFU/mL. Afterward, we prepared new Petri dishes to run another CFU test. Hyd1 biobrick: MdlB biobrick: MdlC biobrick: We received the gene sequence from IDT and ran a PCR and gradient-PCR on it, but we failed. Methionine-gamma-lyase: Hmas biobrick: We received the gene sequence from IDT and ran a PCR on it and failed.
Day 2
Interlab: We made the dilution series and incubated the plates overnight. Hmas biobrick:
Day 3
Interlab: We counted the boxes and calculated the CFU/mL. We then elaborated a wrokflow for the flow cytometry measurements.
Day 4
Day 5
Chitinase: 02/07/18: results of the transformation of the megaprime chit1: 97,3 colonies obtained/petri box Starter thrown for a miniprep Miniprep
03/07/18:
send to the sequencing 04/07/18 - Slide of the Parisian meet-up 06/07/18 Results of the sequencing: negative → not of tag his Colony PCR to dominate crossing on the tag HIS
MdlB: 03/07/18 Slic MdlB (PCR slic 1 and 4)
04/07/18
slic MDLB with pSB1C3 digested (pRFP digested) and transformer
05/06/18
Obtaining of 9 colonies potentially good, test CPCR and frost? 1 well with a band approximately of the good size? Starter
06/07/18
Miniprep and test of digestion on the starter (postponed to Monday)
HYD1:
02/07/18 Test of the colonies of the transformation 29/06
Colony PCR on 23 colonies : there are 8 good colonies Starter of 2 of them for the next day
03/07/18
Miniprep 2 starter Digestion by EcoRI and SpeI (500ng not decisive Results, we begin again by putting starter of the other good colonies on 3 colonies tested in test 04/07/18
digestion of 5 other colonies parmies 8 good ones miniprep digestion ecoRI SpeI 1 clones which seem in good
05/07/18
Sending to the sequencing
Lipase A: in 02/07/18: Reception by IDT (500ng of the part A) put back in water in 10µl. One wait for the reception of the part B to be able to make a slic.
PLacI 03/07/18 Launch of the megapriming of pLacI to add the RBS by Quentin 04/07/18
Digestion Dpn1 Transformation
05/07/18
starter
06/07/18 miniprep sending to the sequencing
Mgl :
02/07/18
Digestion of PsB1C3-RFP by XbaI and SpeI
Digestion of PsB1C3-RFP by EcoRI and PstI
Reception of primers for SLIC-1, MGL-2, MGL-3 and SLIC-4 PCR Q5 of fragments PlacI and MGL-tagHis for the SLIC PCR squeaky clean up? PLacI (47ng/µ L) and MGL-His (61ng/µ L) Frost extract: Psb1C3 E/P (19ng/µ L) and Psb1C3 X/S (29ng/µ L) 03/07/18 SLIC of pLac I + MGL-His in PsB1C3 transformation of the product of SLIC at DH5a PCR of pLacI-Slic and MGLHis-Slic with development by baits Slic-1 and Slic-4 Clean up of the product PCR of PlacI-MGLHis-Slic 04/07/18 cPCR of the transformation of pLacI-MGL-His: 36 colonies? 30 positive colonies
Starters of colonies: 7/18/29
05/07/18 Minipreps of starters: 7? 251 ng/µ L/18? 150 ng/µ L/29? 189 ng/µ L Séquençage of minipreps Transformation