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 | ||
- | |||
- | + | <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
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
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
- DH5a tranformed with pCB1C3
- Top10 transformed with pQexp
- DH5a pyr tranformed with pMK2
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 |
Survivability tests - Results
liquid cultures/Plates | MB | M9 |
MB | ++++ | ++ |
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 |
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 |
/ | 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 A B C C Pseudovibrio denitrificans Pseudovribrio denitrificans* E. Coli E. Coli**