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Week 1: 17^th June - 23^rd June

Tuesday, 19th June

As a start for the labwork, we first decided to make competent cells. We grew from glycerol samples three E. coli strains in 2 mL of LB medium.
The chemically competent strains chosen are as follows:

BL21
DH5α
TOP10

In a 15 mL tube, add 2 mL of LB medium and inoculate cells from glycerol

Thursday, 20th June

We started to make 200 mL of BL21 and DH5α E. coli strains and 400 mL of TOP10 to make competent cells.

First prepare the following solutions required to make competent cells:
Solution for 1M Cacl2:
Add 14,30g of CaCl2 into 100 ml desalted water

Solution for 0,1M Cacl2:
Add 50 mL of CaCl2 1M solution into 450 ml of desalted water

Solution for 0,1M Cacl2 + 15% glycerol:
Add 50 mL of CaCl2 1M solution and 75 mL of glycerol 100% into 450 ml of desalted water

For 200 ml LB medium, add 400 µL of strain sample.
Let the bacteria grow until it reaches an OD between 0,3 and 0,35.
Once it reached the right OD, put the medium on ice for 30 minutes to slow down growth.
Split the 200 mL into 4x50 mL tubes then centrifuge at 3000 rpm for 5 minutes at 4°C and suppress supernatant afterwards.
Resuspend the cells with 5 mL of Cacl2 at 0,1M for each 50 mL tube.
Again, put the medium on ice for 30 minutes.
Centrifuge at 3000 rpm for 5 minutes at 4°C then suppress supernatant.
Resuspend the cells with 1 mL of Cacl2 at 0,1M + 15% glycerol for each 50 mL tube.
Split the total 4 mL into 40 tubes containing each 100 µL of concentrated cell solution. This step should be executed fast enough and on ice.

Friday, 21st June

To check the quality of our work, each strain has been plated on LB medium with ampicillin, kanamycin or chloramphenicol in order to evaluate if it contaminated or not. Furthermore, to evaluate wether our strains are competent or not, we also transformed our bacteria with a pSB1A3 plasmid (red colonies) and plated them on LB medium with ampicillin only.

For 100 µL of chemical competent cells, add 1 µL of plasmidic DNA.
Let 30 minutes on ice.
Let the cells at 42°C for exactly 50 seconds.
Let 5 minutes on ice.
Resuspend the cells with 1 mL of LB spread on a petri dish then let it at 37°C.

Week 2: 24^th June - 30^th June

Monday, June 24th

Our competents cells made on friday the 21st were plated and incubated the whole week-end at 30°C. Today, we analyzed the plates and came to the conclusion that the medium was contaminated due to poor protocol execution. As a consequence, we decided to make new LB medium and recalculate the antibiotic concentrations.

Add 20 g of LB broth into 1000 mL of desalted water and autoclave.

Kanamycin is used as an effective concentration of 10-50 µg/ml and at a storage concentration of 10 mg/ml
Carbenicillin is used as an effective concentration of 20-60 µg/ml and at a storage concentration of 50 mg/ml
Chloramphenicol is used as an effective concentration of 25-170 µg/ml and at a storage concentration of 34 mg/ml (ethanol)
Tetracyclin is used as an effective concentration of 10-50 and at a storage concentration of 5 mg/ml (ethanol)

Tuesday, June 25th

New competent cells have been made with the same bacterial strains (BL21, DH5α and TOP10). We thought that the contamination of our cells was due to the CaCl2 solution that may not have been sterile and/or incorrect manipulated. As a consequence, we prepared new CaCl2 solutions made sure to autoclave them before usage. We ran the same tests as monday the 24th to evaluate the quality of our work and check for potential contamination.

Wednesday, June 26th

The results of the competent cells protocol is as follows:
DH5α strain was not contaminated but competent
TOP10 strain was contaminated only on the Kanamycin plate but competent
BL21 strain was completely contaminated and, thus, not usable.

Thurdsay, June 27th

We finished designing are first two plasmids and constructed the primers to extract the natural promoters from the genomic DNA from E. coli and the different parts for Golden Gate assembly.

Friday, June 28th

We want to obtain reasonnable competent cells for our oncoming transformation for newt week. Thus, we decided to plate the three strains on LB-Agar medium in order to isolate one and only one colony for monday when we'll start over the whole competent process.

Week 3: 1^st July - 7^th July

Week 4: 8^th July - 14^th July

Week 5: 15^th July - 21^st July

Week 6: 22^nd July - 28^th July

Week 7: 29^th July - 4^th August

Week 8: 5^th August - 11^th August

Week 9: 12^th August - 18^th August

Week 10: 19^th August - 25^th August

Week 11: 26^th August - 1^st September

Week 12: 2^nd September - 8^th September

Week 13: 9^th September - 15^th September

Week 14: 16^th September - 22^nd September

Week 15: 23^rd September - 29^th September

Week 16: 30^th September - 6^th October

Week 17: 7^th October - 13^th October

Week 18: 14^th October - 20^th October

Week 19: 21^st October - 27^th October

Week 20: 28^th October - 3^rd November

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