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| - | <div class=" cd-timeline-block"> | + | <div align="center"> |
| - | </html>
| + | <h4 class=title4> <FONT COLOR="blue">Preparation of antibiotic stocks </FONT></h4> |
| - | {{:Team:Evry/Notebook/CellCharacterization/Antibiotic/TopImage}}
| + | </div> |
| - | <html>
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| - | <div class="cd-timeline-content">
| + | |
| - | <h4 class=title4> <u> Preparation of antibiotic stocks </u> </h4> | + | |
| | <p> | | <p> |
| - | <table> | + | <table border="1"> |
| | <tr > | | <tr > |
| - | <td> <b>Antibiotic </b> </td> | + | <td> <b><FONT COLOR=#000033>Antibiotic</font> </b> </td> |
| - | <td> <b>Stock concentration </b> </td> | + | <td> <b><FONT COLOR=#000033>Stock concentration</font> </b> </td> |
| - | <td class= table> <b>Protocol </b> </td> | + | <td class= table> <b><FONT COLOR=#000033>Protocol </font></b> </td> |
| | </tr> | | </tr> |
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| | <tr> | | <tr> |
| | <td> Amplicilin </td> | | <td> Amplicilin </td> |
| - | <td class= conc> 50 mg/mL </td> | + | <td class= conc> 100 mg/mL </td> |
| - | <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> | + | <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> |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
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| | </p> | | </p> |
| | | | |
| - | <p>
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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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| - |
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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:
| |
| - | <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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| - |
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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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| - |
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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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| - |
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| - |
| |
| - | <table border="1">
| |
| - | <tr >
| |
| - | <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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| - |
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| - |
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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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| - |
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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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| - |
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| - | <h4> <u>Survivability tests - Results</u> </h4>
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| - |
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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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| - |
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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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| - |
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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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| - |
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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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| - |
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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">
| |
| - | <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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| - |
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| - |
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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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| - |
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| - | New stocks are ready for antibiotics' tests on Pseudovribrio
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| - |
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| - | <h4> <u>Launch of antibiotics's tests in liquid M9 1X</u> </h4>
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| - |
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| - | For each tested antibiotic we made the following range of concentrations:
| |
| - | <table border="1">
| |
| - | <tr >
| |
| - | <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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| - |
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| - | Each tube is made like in the following table in Falcon tubes (15mL):
| |
| - | <table border="1">
| |
| - | <tr >
| |
| - | <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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| - |
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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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| - |
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| - | </table>
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| - |
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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">
| |
| - | <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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| - |
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| - | <h4> <u>Plates for antibiotics's tests</u> </h4>
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| - |
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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. Plates will be checked during three days.
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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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| - |
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| - | Each plate is made like in the following table:
| |
| - | <table border="1">
| |
| - | <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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| - |
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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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| - |
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| - | </table>
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| - |
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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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| - |
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| - | <ul>
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| - | <li>Chloramphenicol
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| - | <img src="https://static.igem.org/mediawiki/2014/2/29/Chloramphenicol.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - |
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| - | <li>Kanamycin
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| - | <img src="https://static.igem.org/mediawiki/2014/f/f9/Kanamycine_M9_J1.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - | Because of technical problems, the tests on E.Coli+KanR are launched with a gap of few days.
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| - |
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| - | <li>Ampicilin
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| - | <img src="https://static.igem.org/mediawiki/2014/6/69/Ampiciline_M9_J1.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - |
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| - | <li>Tetracyclin
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| - | <img src="https://static.igem.org/mediawiki/2014/8/8c/Tetracycline_M9_J1.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - |
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| - | <li>Erythromycin
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| - | <img src="https://static.igem.org/mediawiki/2014/0/0c/Erythromycine_M9_J1.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - |
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| - | <img src="https://static.igem.org/mediawiki/2014/2/2a/Graphe_J1.jpg" alt="GROSSE NAZE T'AS PAS REUSSI A METTRE L'IMAGE" />
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| - |
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| - |
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| - | <h6>08-20-2014</h6>
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| - |
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| - | <h4> <u>Antibiotics' tests in liquid M9 1X - Results Day2</u> </h4>
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| - |
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| - |
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| - | <li>Chloramphenicol
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| - | <img src="https://static.igem.org/mediawiki/2014/7/73/Cloram_M9_J2_Graphe.jpg" alt="Cam" />
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| - |
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| - | <li>Kanamycin
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| - | <img src="https://static.igem.org/mediawiki/2014/d/d6/Kan_M9_J2_Graphe.jpg" alt="Kan" />
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| - |
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| - | <li>Ampicilin
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| - | <img src="https://static.igem.org/mediawiki/2014/f/f5/Amp_M9_J2_Graphe.jpg" alt="Amp" />
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| - |
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| - | <li>Tetracyclin
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| - | <img src="https://static.igem.org/mediawiki/2014/3/34/Tet_M9_J2_Graphe.jpg" alt="Tet" />
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| - |
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| - | <li>Erythromycin
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| - | <img src="https://static.igem.org/mediawiki/2014/0/0e/Erm_M9_J2_Graphe.jpg" alt="Erm" />
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| - |
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| - | <img src="https://static.igem.org/mediawiki/2014/7/7f/GraphJ2.jpg" alt="Graphs" />
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| - |
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| - |
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| - | <h4> <u>Launch of antibiotics' tests on plates</u> </h4>
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| - |
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| - | Plates are showed and organised like shown in the following picture, and showed with 1µL of each culture:
| |
| - | <ul>
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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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| - | </ul>
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| - |
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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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| - |
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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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| - |
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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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| - |
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| - |
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| - | <h6>08-21-2014</h6>
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| - | <h4> <u>Antibiotics' tests on MB 1X plates - Results Day1</u> </h4>
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| - |
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| - | Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case.
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| - | Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
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| - | <li>Chloramphenicol
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| - | <img src="https://static.igem.org/mediawiki/2014/c/cb/Cam_MB_J1%28v%29_Graphe.jpg" alt="Cam" />
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| - |
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| - | <li>Kanamycin
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| - | <img src="https://static.igem.org/mediawiki/2014/8/8f/Kan_MB_J1%28v%29_Graphe.jpg" alt="Kan" />
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| - |
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| - | <li>Ampicilin
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| - | <img src="https://static.igem.org/mediawiki/2014/8/80/Amp_MB_J1%28v%29_Graphe.jpg" alt="Amp" />
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| - |
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| - | <li>Tetracyclin
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| - | <img src="https://static.igem.org/mediawiki/2014/7/7e/Tet_MB_J1%28v%29_Graphe.jpg" alt="Tet" />
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| - |
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| - | <li>Erythromycin
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| - | <img src="https://static.igem.org/mediawiki/2014/e/e7/Erm_MB_J1%28v%29_Graphe.jpg" alt="Erm" />
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| - |
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| - | <img src="https://static.igem.org/mediawiki/2014/8/85/Graphe_J1-MB1X.jpg" alt="Graphs" />
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| - |
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| - | <h4> <u>Antibiotics' tests on MB 03.5X plates - Results Day1</u> </h4>
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| - |
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| - | Results of antibiotics' tests on plate are calculated as percentage of bacteria's growth by compared with the controle case.
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| - | Pseudovibrio coming from culture with antibiotics (part B on plates), are taken from antibiotics' range in M9 at Day2.
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| - | <li>Chloramphenicol
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| - | <img src="https://static.igem.org/mediawiki/2014/6/6b/Cam_MB05%28v%29_J1.jpg" alt="Cam" />
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| - |
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| - | <li>Kanamycin
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| - | <img src="https://static.igem.org/mediawiki/2014/4/41/Kan_MB05_J1.jpg" alt="Kan" />
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| - |
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| - | <li>Ampicilin
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| - | <img src="https://static.igem.org/mediawiki/2014/9/91/Amp_MB05_J1.jpg" alt="Amp" />
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| - |
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| - | <li>Tetracyclin
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| - | <img src="https://static.igem.org/mediawiki/2014/2/2c/Tetra_MB05_J1.jpg" alt="Tet" />
| |
| - |
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| - | <li>Erythromycin
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| - | <img src="https://static.igem.org/mediawiki/2014/f/f3/Erythromycine_MB05_J1.jpg" alt="Erm" />
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| - |
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| - | <img src="https://static.igem.org/mediawiki/2014/8/8d/Graphe_J1-MB05X.jpg" alt="Graphs" />
| |
| - |
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| - | <h4> <u>Antibiotics' tests on M9 1X plates - Results Day1</u> </h4>
| |
| - |
| |
| - | 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.
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| - | <li>Chloramphenicol:
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| - | <b>No readable results</b>
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| - |
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| - | <li>Kanamycin:
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| - | <b>No readable results</b>
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| - |
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| - | <li>Ampicilin:
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| - | <b>No readable results</b>
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| - |
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| - | <li>Tetracyclin:
| |
| - | <b>No readable results</b>
| |
| - |
| |
| - | <li>Erythromycin:
| |
| - | <b>No readable results</b>
| |
| - |
| |
| - |
| |
| - | <h4> <u>Antibiotics' tests in liquid M9 1X - Results Day3</u> </h4>
| |
| - |
| |
| - | <li>Chloramphenicol
| |
| - | <img src="" alt="Cam" />
| |
| - |
| |
| - | <li>Kanamycin
| |
| - | <img src="" alt="Kan" />
| |
| - |
| |
| - | <li>Ampicilin
| |
| - | <img src="" alt="Amp" />
| |
| - |
| |
| - | <li>Tetracyclin
| |
| - | <img src="" alt="Tet" />
| |
| - |
| |
| - | <li>Erythromycin
| |
| - | <img src="" alt="Erm" />
| |
| - |
| |
| - | <img src="" alt="Graphs" />
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - |
| |
| - | <h6>08-21-2014</h6>
| |
| - | <h4> <u>Antibiotics' tests in liquid M9 1X - Results Day3</u> </h4>
| |
| - | <h4> <u>Antibiotics' tests on MB 1X plates - Results Day2</u> </h4>
| |
| - |
| |
| - | 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.
| |
| - | <li>Chloramphenicol
| |
| - | <img src="https://static.igem.org/mediawiki/2014/3/30/Cloram_MB_J1_Graphe.jpg" alt="Cam" />
| |
| - |
| |
| - | <li>Kanamycin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/8/8f/Kan_MB_J1%28v%29_Graphe.jpg" alt="Kan" />
| |
| - |
| |
| - | <li>Ampicilin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/a/aa/Amp_MB_J1_Graphe.jpg" alt="Amp" />
| |
| - |
| |
| - | <li>Tetracyclin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/1/10/Tet_MB_J1_Graphe.jpg" alt="Tet" />
| |
| - |
| |
| - | <li>Erythromycin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/e/e8/Erm_MB_J1_Graphe.jpg" alt="Erm" />
| |
| - |
| |
| - | <img src="" alt="Graphs" />
| |
| - |
| |
| - | <h4> <u>Antibiotics' tests on MB 0.5X plates - Results Day2</u> </h4>
| |
| - | <li>Chloramphenicol
| |
| - | <img src="https://static.igem.org/mediawiki/2014/6/64/Cam_MB05_J2.jpg" alt="Cam" />
| |
| - |
| |
| - | <li>Kanamycin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/6/63/Kan_MB05_J2.jpg" alt="Kan" />
| |
| - |
| |
| - | <li>Ampicilin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/2/2e/Amp_MB05_J2.jpg" alt="Amp" />
| |
| - |
| |
| - | <li>Tetracyclin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/d/da/Tetra_MB05_J2.jpg" alt="Tet" />
| |
| - |
| |
| - | <li>Erythromycin
| |
| - | <img src="https://static.igem.org/mediawiki/2014/d/d2/Erythromycine_MB05_J2.jpg" alt="Erm" />
| |
| - |
| |
| - | <img src="" alt="Graphs" />
| |
| - |
| |
| - | <h4> <u>Antibiotics' tests on M9 1X plates - Results Day2</u> </h4>
| |
| - |
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| - |
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| - |
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| - |
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| - | </p>
| |
| - | <span class="cd-date"></span>
| |
| - | </div>
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| - | </div>
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| | </html> | | </html> |
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