Team:Evry/Notebook/CellCharacterization/Antibiotic test

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	50 mg/mL	Weight 1g of Amplicilin powder, solubilization into 40mL 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)

08-16-2014

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
• Top10 transformed with pQexp
• DH5a pyr tranformed with pMK2

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

08-17-2014

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.

08-18-2014

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

08-19-2014

Antibiotics' tests in liquid M9 1X - Results Day1

• Chloramphenicol
• Kanamycin Because of technical problems, the tests on E.Coli+KanR are launched with a gap of few days.
• Ampicilin
• Tetracyclin
• Erythromycin

  Launch of antibiotics' tests on plates

  Plates are showed and organised like shown in the following picture, and showed with 1µL of each culture:
  

  A	B	C	D
  Pseudovibrio denitrificans	Pseudovribrio denitrificans*	E. Coli	E. Coli**

  *Bacteria cultivated in media with antibiotic **Bacteria tranformed with a plasmid containing a resistance cassette for the antibiotic

  08-19-2014

  Antibiotics' tests in liquid M9 1X - Results Day2

• Chloramphenicol
• Kanamycin
• Ampicilin
• Tetracyclin
• Erythromycin

  Antibiotics' tests on MB 1X plates - Results Day1

  Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
• Chloramphenicol
• Kanamycin
• Ampicilin
• Tetracyclin
• Erythromycin

  Antibiotics' tests on MB 03.5X plates - Results Day1

  Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
• Chloramphenicol
• Kanamycin
• Ampicilin
• Tetracyclin
• Erythromycin

  Antibiotics' tests on M9 1X plates - Results Day1

  Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case. Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
• Chloramphenicol
• Kanamycin No readable results
• Ampicilin No readable results
• Tetracyclin No readable results
• Erythromycin No readable results