Revision as of 17:32, 25 August 2014

IGEM Evry 2014

Notebook - Cell Characterization

Preparation of antibiotic stocks

Antibiotic	Stock concentration	Protocol
Kanamycin	25 mg/mL	Weight 0.56g of Kanamycin powder, solubilization into 20mL of miliQ water. Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL)
Streptomycin	100 mg/mL	Weight 1g of Streptomycin powder, solubilization into 20mL of miliQ water. Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL)
Amplicilin	100 mg/mL	Weight 1g of Amplicilin powder, solubilization into 20mL of miliQ water. Vortex 5min and filtration with a 200nm filter. Stock : -20°C (aliquots of 1mL)
Tetracyclin	15 mg/mL	Weight 0.6g of Tetracyclin powder, solubilization into 20mL of ethanol 50%. Vortex 5min and filtration with a 200nm filter. Stock : -20°C and hiden from light(aliquots of 1mL)
Chloramphenicol	34 mg/mL	Weight 0.34g of Chloramphenicol powder, solubilization into 10mL of ethanol 100%. Vortex 5min and filtration with a 200nm filter. Stock : -20°C and hiden from light(aliquots of 1mL)

Tests of antibiotics' stocks - Results

Plates	LB Agar only	LB+Cam	LB+Kan	LB+Amp	LB+Erm	LB+Tet
E.coli	X	0	X	0	X	0

There was a problem with the stock of Kanamycine so we made a new one at 25mg/mL. For the erythrompycine, we learn that E. Coli is not really sensitive to this antibiotic and that it's better to use it with a dilution at 1:100.

Survivability tests - Results

liquid cultures/Plates	MB	M9
MB	++++	++
M9	++	+

Any liquid culture can be plated and will grow up. Of course, it works better on MB medium, wich is a rich medium than on M9.

Aug 17

Tests of antibiotics' stocks
Six plates of LB agar were made. Five of them contained one of those antibiotics in the dilution 1:1000:

Chloramphénicol

Kanamycin

Erythromycin

Ampicilin

Tetracyclin
(The last one was the control of the growth of our bacteria without antibiotics) We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.

Survivability tests
Two plates of MB 1X and M9 1X were made and divised in two parts. Each plate was sowed with 5µL of a liquid MB 1X culture of Pseudovibrio denitrificans(dated 12th August) in a part and with 5µL of a liquid M9 1X culture Pseudovibrio denitrificans (dated 12th August) in the other part.

Pre-cultures
Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C. New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C.

From glycerol stocks:

Bl21

Top10

DH5a

From plates:

DH5a tranformed with pCB1C3 (CamR)

Top10 transformed with pQexp (ErmR)

DH5a pyr tranformed with pMK2 (KanR)

Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection. For each medium we make a negative contrôle without bacteria.

Aug 16

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