Team:Evry/Notebook/CellCharacterization/Antibiotic test

From 2014.igem.org

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<h6> <u>Tests of antibiotics' stocks</u> </h6>
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Six plates of LB agar were made.
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Five of them contained one of those antibiotics in the dilution 1:1000:
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<li> Chloramphénicol
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<li> Kanamycin
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<li> Erythromycin
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<li> Ampicilin
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<li> Tetracyclin
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(The last one was the control of the growth of our bacteria without antibiotics)
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We sowed 10µL of a liquid LB culture of Bl21, and we let them incubate overnight at 37°C.
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<h6> <u>Survivability tests</u> </h6>
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Two plates of MB 1X and M9 1X were made and divised in two parts.
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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.
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<h6> <u>Pre-cultures</u> </h6>
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Cultures in MB 1X and M9 1X of Pseudovibrio were passed by a 1/10 dilution and let incubate overnight at 30°C.
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New cultures of different E.Coli were also started from glycerol or plate and let incubated overnight at 37°C.
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<u>From glycerol stocks:</u>
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<li>Bl21
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  <h2 class=title2> <b> Antibiotic test </b></h2>
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<li>Top10
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<li>DH5a
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<u>From plates:</u>
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<li>DH5a tranformed with pCB1C3
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<li>Top10 transformed with pQexp
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<li>DH5a pyr tranformed with pMK2
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Bacteria tranformed with plasmid were cultivated with the corresponding antibiotic for the selection.
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For each medium we make a negative contrôle without bacteria.
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<h4>08-17-2014</h4>
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Revision as of 17:01, 23 August 2014

Picture

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

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