Team:Evry/Notebook/CellCharacterization/Antibiotic test

From 2014.igem.org

(Difference between revisions)
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New stocks are ready for antibiotics' tests on Pseudovribrio
New stocks are ready for antibiotics' tests on Pseudovribrio
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<h4> <u>Plates for antibiotics's tests</u> </h4>
 
-
For each tested antibiotic we made the following range of concentrations:
+
<h4> <u>Antibiotics's tests in liquid M9 1X</u> </h4>
 +
For each tested antibiotic we made the following range of concentrations than in liquid.
<table border="1">
<table border="1">
<tr >
<tr >
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</tr>
</tr>
</table>
</table>
-
We 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.
+
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):
 +
<table border="1">
 +
<tr >
 +
  <td> <b>/ </b> </td>
 +
  <td> <b>0 </b> </td>
 +
  <td> <b>1:500 </b> </td>
 +
  <td> <b>1:1000 </b> </td>
 +
  <td> <b>1:50 000 </b> </td>
 +
  <td> <b>1:100 000 </b> </td>
 +
</tr>
 +
 
 +
<tr >
 +
  <td> <b>Medium+agar </b> </td>
 +
  <td>3mL </td>
 +
  <td>3mL </td>
 +
  <td>3mL </td> 
 +
  <td>3mL </td>
 +
  <td>3mL </td>
 +
</tr>
 +
 
 +
<tr >
 +
  <td> <b>Antibiotic </b> </td>
 +
  <td>0 </td>
 +
  <td>6µL of stock</td>
 +
  <td>3µL of stock</td>
 +
  <td>6µL of stock diluted 1/100</td>
 +
  <td>3µL of stock diluted 1/100</td>
 +
</tr>
 +
 
 +
</table>
 +
 
 +
 
 +
 
 +
<h4> <u>Plates for antibiotics's tests</u> </h4>
 +
 
 +
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.
We made these same ranges of tested antibiotic's concentrations for three media:
We made these same ranges of tested antibiotic's concentrations for three media:
<ul>
<ul>
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And plates are organised like shown in the following picture:
And plates are organised like shown in the following picture:
<img src="https://static.igem.org/mediawiki/2014/b/bf/EVRYSch%C3%A9maplates2.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
<img src="https://static.igem.org/mediawiki/2014/b/bf/EVRYSch%C3%A9maplates2.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
 +
 +
<table border="1">
 +
<tr >
 +
  <td> <b>A </b> </td>
 +
  <td> <b>B </b> </td>
 +
  <td> <b>C </b> </td>
 +
  <td> <b>C </b> </td>
 +
</tr>
 +
 +
<tr >
 +
  <td>Pseudovibrio denitrificans </td>
 +
  <td>Pseudovribrio denitrificans* </td>
 +
  <td>E. Coli</td>
 +
  <td>E. Coli**</td>
 +
</tr>
 +
 +
</table>
 +
 +
*Bacteria cultivated in media with antibiotic
 +
**Bacteria tranformed with a plasmid containing a resistance cassette for the antibiotic

Revision as of 10:56, 25 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

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

Antibiotics's tests in liquid M9 1X

For each tested antibiotic we made the following range of concentrations than in liquid.
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

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. 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
      And plates are organised like shown in the following picture: GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE
      A B C C
      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