Team:Evry/Notebook/CellCharacterization/Antibiotic test

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<h4 class=title4> <FONT COLOR="blue">Preparation of antibiotic stocks </FONT></h4>
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<h4 class=title4> <u> Preparation of antibiotic stocks </u> </h4>
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   <td> <b>Antibiotic </b> </td>
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   <td> <b><FONT COLOR=#000033>Antibiotic</font> </b> </td>
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   <td> <b>Stock concentration </b> </td>
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   <td> <b><FONT COLOR=#000033>Stock concentration</font> </b> </td>
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   <td class= table> <b>Protocol </b> </td>
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   <td class= table> <b><FONT COLOR=#000033>Protocol </font></b> </td>
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   <td> Amplicilin </td>
   <td> Amplicilin </td>
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   <td class= conc> 50 mg/mL </td>
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   <td class= conc> 100 mg/mL </td>
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   <td class= proto> Weight 1g of Amplicilin powder, solubilization into 40mL of miliQ water. <br>Vortex 5min and filtration with a 200nm filter. </br>  Stock : -20°C (aliquots of 1mL) <br> </br> </td>
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   <td class= proto> Weight 1g of Amplicilin powder, solubilization into 20mL of miliQ water. <br>Vortex 5min and filtration with a 200nm filter. </br>  Stock : -20°C (aliquots of 1mL) <br> </br> </td>
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<h6>08-16-2014</h6>
 
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<h4> <u>Tests of antibiotics' stocks</u> </h4>
 
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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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<ul>
 
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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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</ul>
 
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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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<h4> <u>Survivability tests</u> </h4>
 
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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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<h4> <u>Pre-cultures</u> </h4>
 
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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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<ul>
 
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<li>Bl21
 
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<li>Top10
 
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<li>DH5a
 
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</ul>
 
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<u>From plates:</u>
 
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<ul>
 
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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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</ul>
 
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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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<h6>08-17-2014</h6>
 
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<h4> <u>Tests of antibiotics' stocks - Results</u> </h4>
 
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<table border="1">
 
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<tr >
 
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  <td> <b>Plates </b> </td>
 
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  <td>LB Agar only </td>
 
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  <td>LB+Cam </td>
 
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  <td>LB+Kan </td>
 
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  <td>LB+Amp </td>
 
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  <td>LB+Erm </td>
 
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  <td>LB+Tet </td>
 
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</tr>
 
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<tr>
 
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  <td> <b>E.coli</b> </td>
 
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  <td>X  </td>
 
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  <td>0  </td>
 
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  <td>X  </td>
 
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  <td>0  </td>
 
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  <td>X  </td>
 
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  <td>0  </td>
 
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</tr>
 
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</table>
 
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There was a problem with the stock of Kanamycine so we made a new one at 25mg/mL.
 
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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
 
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<h4> <u>Survivability tests - Results</u> </h4>
 
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<table border="1">
 
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<tr >
 
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  <td> <b>liquid cultures/Plates </b> </td>
 
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  <td> <b>MB </b> </td>
 
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  <td> <b>M9 </b> </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>MB </b> </td>
 
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  <td> <b>++++ </b> </td>
 
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  <td> <b>++ </b> </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>M9 </b> </td>
 
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  <td> <b>++ </b> </td>
 
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  <td> <b>+ </b> </td>
 
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</tr>
 
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</table>
 
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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.
 
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<h6>08-18-2014</h6>
 
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<h4> <u>Tests of antibiotics' stocks - Results(2)</u> </h4>
 
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<table border="1">
 
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<tr >
 
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  <td> <b>Plates </b> </td>
 
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  <td>LB Agar only </td>
 
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  <td>LB+Kan </td>
 
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  <td>LB+Erm (1:100)</td>
 
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</tr>
 
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<tr>
 
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  <td> <b>E.coli</b> </td>
 
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  <td>X  </td>
 
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  <td>0  </td>
 
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  <td>0  </td>
 
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</tr>
 
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</table>
 
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New stocks are ready for antibiotics' tests on Pseudovribrio
 
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<h4> <u>Antibiotics's tests in liquid M9 1X</u> </h4>
 
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For each tested antibiotic we made the following range of concentrations:
 
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<table border="1">
 
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<tr >
 
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  <td> <b>0 </b> </td>
 
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  <td> <b>1:500 </b> </td>
 
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  <td> <b>1:1000 </b> </td>
 
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  <td> <b>1:50 000 </b> </td>
 
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  <td> <b>1:100 000 </b> </td>
 
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</tr>
 
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</table>
 
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We made these range two time to have a replicate and we added a controle tube without antibiotic for each bacteria.
 
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Each tube is made like in the following table in Falcon tubes (15mL):
 
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<table border="1">
 
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<tr >
 
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  <td> <b>/ </b> </td>
 
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  <td> <b>0 </b> </td>
 
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  <td> <b>1:500 </b> </td>
 
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  <td> <b>1:1000 </b> </td>
 
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  <td> <b>1:50 000 </b> </td>
 
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  <td> <b>1:100 000 </b> </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>Medium+agar </b> </td>
 
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  <td>3mL </td>
 
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  <td>3mL </td>
 
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  <td>3mL </td> 
 
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  <td>3mL </td>
 
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  <td>3mL </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>Antibiotic </b> </td>
 
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  <td>0 </td>
 
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  <td>6µL of stock</td>
 
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  <td>3µL of stock</td>
 
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  <td>6µL of stock diluted 1/100</td>
 
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  <td>3µL of stock diluted 1/100</td>
 
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</tr>
 
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<tr >
 
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  <td> <b>Pre-culture </b> </td>
 
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  <td>300µL</td>
 
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  <td>300µL</td>
 
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  <td>300µL</td>
 
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  <td>300µL</td>
 
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  <td>300µL</td>
 
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</tr>
 
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</table>
 
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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)
 
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The initial OD at 600nm of each bacteria has been mesured to after be able to calculate the ΔOD at 600nm, during three days.
 
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<table border="1">
 
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<tr >
 
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  <td> <b>/ </b> </td>
 
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  <td> <b>Pseudo </b> </td>
 
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  <td> <b>Bl21 </b> </td>
 
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  <td> <b>DH5a </b> </td>
 
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  <td> <b>Top 10 </b> </td>
 
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  <td> <b>DH5a pyr+KanR </b> </td>
 
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  <td> <b>DH5a+CamR </b> </td>
 
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  <td> <b>Top 10+ErmR </b> </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>OD init </b> </td>
 
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  <td> <b>0.02 </b> </td>
 
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  <td> <b>0.795 </b> </td>
 
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  <td> <b>0.873 </b> </td>
 
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  <td> <b>0.693 </b> </td>
 
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  <td> <b>0.408 </b> </td>
 
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  <td> <b>0.687 </b> </td>
 
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  <td> <b>0.319 </b> </td>
 
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</tr>
 
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</table>
 
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<h4> <u>Plates for antibiotics's tests</u> </h4>
 
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For each tested antibiotic we made the same range of concentrations than in liquid.
 
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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.
 
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We made these same ranges of tested antibiotic's concentrations for three media:
 
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<ul>
 
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<li>MB agar 1X
 
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<li>MB agar 0.5X
 
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<li>M9 agar 1X
 
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<ul>
 
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Each plate is made like in the following table:
 
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<table border="1">
 
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<tr >
 
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  <td> <b>/ </b> </td>
 
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  <td> <b>0 </b> </td>
 
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  <td> <b>1:500 </b> </td>
 
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  <td> <b>1:1000 </b> </td>
 
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  <td> <b>1:50 000 </b> </td>
 
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  <td> <b>1:100 000 </b> </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>Medium+agar </b> </td>
 
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  <td>20mL </td>
 
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  <td>20mL </td>
 
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  <td>20mL </td> 
 
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  <td>20mL </td>
 
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  <td>20mL </td>
 
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</tr>
 
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<tr >
 
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  <td> <b>Antibiotic </b> </td>
 
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  <td>0 </td>
 
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  <td>40µL of stock</td>
 
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  <td>20µL of stock</td>
 
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  <td>40µL of stock diluted 1/100</td>
 
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  <td>20µL of stock diluted 1/100</td>
 
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</tr>
 
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</table>
 
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And plates are organised like shown in the following picture:
 
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<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" />
 
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<table border="1">
 
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<tr >
 
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  <td> <b>A </b> </td>
 
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  <td> <b>B </b> </td>
 
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  <td> <b>C </b> </td>
 
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  <td> <b>D </b> </td>
 
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</tr>
 
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<tr >
 
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  <td>Pseudovibrio denitrificans </td>
 
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  <td>Pseudovribrio denitrificans* </td>
 
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  <td>E. Coli</td>
 
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  <td>E. Coli**</td>
 
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</tr>
 
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</table>
 
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*Bacteria cultivated in media with antibiotic
 
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**Bacteria tranformed with a plasmid containing a resistance cassette for the antibiotic
 
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Plates will be checked during three days.
 
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<h6>08-19-2014</h6>
 
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<h4> <u>Antibiotics' tests in liquid M9 1X - Results Day1</u> </h4>
 
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Latest revision as of 10:40, 17 October 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 100 mg/mL Weight 1g of Amplicilin powder, solubilization into 20mL 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)

Retrieved from "http://2014.igem.org/Team:Evry/Notebook/CellCharacterization/Antibiotic_test"