Team:Evry/Notebook/CellCharacterization/Antibiotic test/08-18-2014

From 2014.igem.org

Tests of antibiotics' stocks - Results(2)

Plates	LB Agar only	LB+Kan	LB+Erm (1:100)
E.coli	X	0	0

New stocks are ready for antibiotics' tests on Pseudovribrio

Launch of antibiotics's tests in liquid M9 1X
For each tested antibiotic we made the following range of concentrations:

0

1:500

1:1000

1:50 000

1:100 000

We made these range two time to have a replicate and we added a controle tube without antibiotic for each bacteria. Each tube is made like in the following table in Falcon tubes (15mL):

/	0	1:500	1:1000	1:50 000	1:100 000
Medium+agar	3mL	3mL	3mL	3mL	3mL
Antibiotic	0	6µL of stock	3µL of stock	6µL of stock diluted 1/100	3µL of stock diluted 1/100
Pre-culture	300µL	300µL	300µL	300µL	300µL

For each range, we test Pseudovibrio denitrificans but also E.Coli and E.Coli tranformed with a speicific resistance cassette (NM:The E.Coli strain for the control case is the same strain that is tranformed for the cassette efficiency test). The initial OD at 600nm of each bacteria has been mesured to after be able to calculate the ΔOD at 600nm, during three days.

/	Pseudo	Bl21	DH5a	Top 10	DH5a pyr+KanR	DH5a+CamR	Top 10+ErmR
OD init	0.02	0.795	0.873	0.693	0.408	0.687	0.319

Plates for antibiotics's tests
For each tested antibiotic we made the same range of concentrations than in liquid. We also made these range two time to have a replicate and we added a controle plate without antibiotic on which we showed all our tested bacteria. Plates will be checked during three days. We made these same ranges of tested antibiotic's concentrations for three media:

MB agar 1X
MB agar 0.5X
M9 agar 1X

Each plate is made like in the following table:

/	0	1:500	1:1000	1:50 000	1:100 000
Medium+agar	20mL	20mL	20mL	20mL	20mL
Antibiotic	0	40µL of stock	20µL of stock	40µL of stock diluted 1/100	20µL of stock diluted 1/100

Aug 18