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

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 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

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 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.
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 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.
RFP: 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.
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.

WEEK 4

We had the same problems making competent cells (contaminations...). We had the help of our instructor where he point 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: 'DH5alpha 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 comtamination 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.

Day 3

Chitinase: We digest the megapriming product by DpnI and transformed it into DH5alpha competent cells.
MdlB biobrick: Interlab: We launched starters for a second CFUs test.

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 DH5alpha cells: turns out they were Bacillus Subtilis strains (Smell check).
We had to run another megapriming.
MdlB biobrick: Interlab: We ran the CFUs test.

Day 5

Chitinase:We digest the megapriming product by DpnI and transformed it into new DH5alpha competent cells.
MdlB biobrick:

MdlB :

25/06/18 Digestion-ligation : [MdlB dig] = 60 ng.µL-1 / 1.89 / 2.04 [RFP dig] = 74 ng.µL -1 / 1.89 / 2.11 Gel avec Plasmide non digéré (digestion totale ?), Plasmide + insert (dig-lig), Plasmide RFP dig (Enzyme ok ?) Transformation dans DH5α : 3 conditions : (plasmide + insert) dig-lig (Gautier) / (Plasmide)dig-lig (V4) / (Plasmide)dig (V4) Q5 PCR sur MdlB pour refaire le stock + clean up + nanodrop + gel

26/06/18 Observation à la MagicBox des DH5α Dig-Lig RFP-MDLB cPCR sur les colonies supposées bonnes pour MDLB Digestion de nouveau de MdlB + clean up [MdlB dig] = 31.40 ng.µL (dilué dans 30 µL) Gel avec produit de la digestion et cPCR Q5 PCR pour refaire les stocks + gel

27/06/18 Ligation de MDLB + RFP (tout deux digérés) à 37°C pendant 3H Transformation du produit de la ligation dans les V4

28/06/18 Boîtes transformés de la veille → pas de colonies blanches le matin mais apparition de colonies blanches le soir. Il ne s’agit pas de DH5α mais d’une autre Bactérie qui est compétente de base.

HYD1 : 26/06/18 Réception de Hyd1 + mise en eau dans 10 µL de PPI Q5 PCR pour l’amplifier + Gel (problème de taille → Migration à relancer) 27/06/18 Gel de Hyd1 qui a été amplifié par Q5 PCR la veille (ce n’est pas Hyd1 qui a été amplifié) Amplification de Hyd1 par Q5 PCR [Hyd1]= 37 ng.µL Migration de Hyd1 → Obtention d’une bande beaucoup trop grande pour qu’elle corresponde à Hyd1. Hypothèses : ™ ne correspond pas, la durée d’élongation est beaucoup trop longue ! 1min = 1kb !! PCR avec Hyd1 en utilisant la Taq DNA Polymérase + ™ 60°C et Élongation 30 secondes à 72°C + Final élongation 5 min. Gel Seconde PCR mais avec un gradient de T°. 28/06/18 Hyd1 clean up [Hyd1 dig]= 8 ng.µL-1 Gel

29/06/18

Ligation + transformation de Hyd1 dans DH5α