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http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/TSS_trans
Team:Lyon-INSA-ENS/Realisation/Protocols/TSS trans
2012-01-10T10:45:24Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> TSS chemical transformation </font> </p><br />
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<p style="line-height : 1.5em"><br />
This protocol aims at inserting plasmids notably into NM522 strains, or any other E.Coli strain<br />
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<p style="line-height : 1.5em"><br />
It has been found more efficient than the CaCl2 transformation protocol, thus we recommend using it.<br />
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<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
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<p style="line-height : 1.5em"><br />
<b>1.</b> Monitor the OD600 of a 5mL culture of NM522 cells ( or the strain you want to transform ) in LB medium at 37°C. Proceed to the next step when it reaches 0.3-0.4 ( not higher than 0.6 ), which takes approximately 3 hours.<br />
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<b>2.</b> Take 1 mL from the previous culture and centrifuge at 6000rpm for 2mn .<br />
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<b>3.</b> Resuspend into 100µL of TSS. Starting from this step, keep the bacteria on ice.<br />
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<b>4.</b> Incubate on ice for 5-10mn.<br />
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<b>5.</b> Add 5µL of the DNA to be transformed.<br />
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<b>6.</b> Incubate on ice for 10mn.<br />
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<b>7.</b> Heat shock the bacteria by heating them at 42°C for 50s.<br />
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<b>8.</b> Incubate on ice for 2mn.<br />
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<b>9.</b> Add 900µL LB and mix by inverting the tube.<br />
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<b>10.</b> Incubate for 1h at 37°C.<br />
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<b>11.</b> Plate 200µL on LB medium with the appropriate antibiotic.<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Storage
Team:Lyon-INSA-ENS/Realisation/Protocols/Storage
2012-01-10T10:45:02Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Storage of strains</font> </p><br />
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<p style="line-height : 1.5em"><br />
This protocol aims at storing strains at -20°C for long-time conservation. <br />
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<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
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<b>1.</b> In a 1.5mL eppendorf tube, add 750µL of overnight LB culture and 250µL glycerol 80%.<br />
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<b>2.</b> Store in a -20°C freezer.<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Miniprep
Team:Lyon-INSA-ENS/Realisation/Protocols/Miniprep
2012-01-10T10:44:44Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> <br />
Plasmid DNA purification using QIAprep® Spin Miniprep Kit </font> </p><br />
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<p style="line-height : 1.5em"><br />
This protocol is designed for purification of up to 20 µg of high-copy plasmid DNA from 1-5mL overnight cultures in LB mediul.<br />
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<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
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<p style="line-height : 1.5em"><br />
<b>1.</b> Pellet 1-5mL bacterial overnight culture by centrifugation at >8000 rpm ( we used 13000 ) for 3mn at room temperature<br />
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<b>2.</b> Resuspend pelleted bacterial cells in 250µL buffer P1 and transfer to a microcentrifuge tube <br />
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<b>3.</b> Add 250µL buffer P2 and mix thoroughly by inverting the tybe 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5mn.<br />
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<b>4.</b> Add 350µL buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times.<br />
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<b>5.</b> Centrifuge for 10mn at 13000 rpm in a table-top microcentrifuge<br />
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<b>6.</b> Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60s and discard the flow-through.<br />
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<b>7.Recommended if the strain is endA+</b> Wash the QIAprep spin column by adding 0.5mL buffer PB. Centrifuge for 30-60s and discard the flow-through.<br />
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<b>8.</b> Wash the QIAprep spin column by adding 0.75 mL buffer PE. Centrifuge for 30-60s and discard the flow-through.<br />
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<b>9.</b> Centrifuge for 1mn to remove residual wash buffer<br />
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<b>10.</b> Place the QIAprep column in a clean 1.5mL microcentrifuge tube. To elute DNA, add 50 µL buffer EB ( 10mM Tris.Cl, pH 8.5 ), let stand for 1mn and centrifuge for 1mn.<br />
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<p style = "text-align : center"><br />
<small> This protocol is extracted from "QIAprep Miniprep Handbook" by <B>QIAGEN </B>. </small><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/DNA-purification
Team:Lyon-INSA-ENS/Realisation/Protocols/DNA-purification
2012-01-10T10:44:21Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Plasmid or Cosmid DNA Purification Using QIAGEN HiSpeed Plasmid Midi Kits </font> </p><br />
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<p style="line-height : 1.5em"><br />
This protocol is for preparation of up to 200µg of high- or low-copy plasmid or cosmid DNA using the QIAGEN HiSpeed Plasmid Midi Kit.<br />
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<p style="line-height : 1.5em"><br />
A final DNA concentration of up to 0.4µg/µL can be expected, if eluting a high-copy plasmid with 500µL of Buffer TE. <br />
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<font color = "green">Things to do before starting:</font><br />
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<li> <p style="text-align:justify"> Add the provided RNase A solution to Buffer P1 before use. Use one vial of RNase A (centrifuge briefly before use) per bottle of Buffer P1, to give a final concentration of 100µg/mL. </p><br><br />
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<li> <p style="text-align:justify"> Check Buffer P2 for SDS precipitation due to low storage temperatures. If necessary, dissolve the SDS by warming to 37°C. </p><br><br />
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<li> <p style="text-align:justify"> Pre-chill Buffer P3 at 4°C.</p><br><br />
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<li> <p style="text-align:justify"> Pre-warm the adequate volume of Buffer QF and Buffer TE at approx. 50°C.</p><br><br />
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<li> <p style="text-align:justify"> Grow bacterial culture during approximately 16 hours, which typically is the transition from logarithmic into stationary growth phase. </p><br><br />
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<li> <p style="text-align:justify"> Optional: Add LyseBlue reagent to Buffer P1. </p><br />
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<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
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<b>1.</b> Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2-5mL LB medium containing the appropriate selective antibiotic. Incubate for approx. 8h at 37°C with vigorous shaking (approx. 300 rpm).<br />
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<b>2.</b> Dilute the starter 1/500 to 1/1000 into selective LB medium. For high-copy plasmids, inoculate 50mL medium. Grow at 37°C for 12-16h with vigorous shaking (approx. 300 rpm). <br />
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<b>3.</b> Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.<br />
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<b>4.</b> Resuspend the bacterial pellet in 6 mL Buffer P1.<br />
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<b>5.</b> Add 6 mL Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4-6 times, and incubate at room temperature (15-25°C) for 5 min. <br />
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During the incubation prepare the QIAfilter Cartridge. Screw the cap onto the outlet nozzle of the QIAfilter Midi Cartridge. Place the filter into a convenient tube or a QIArack.<br />
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<b>6.</b> Add 6mL of chilled Buffer P3 to the lysate, and mix immediately and thoroughly by vigorously inverting 4-6 times. Do not incubate the lysate on ice.<br />
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<b>7.</b> Pour the lysate into the barrel of the QIAfilter Cartridge. Incubate at room temperature for 10 min. Do not insert the plunger !<br />
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<b>8.</b> Equilibrate a HiSpeed Midi Tip by applying 4 mL Buffer QBT and allow the column to empty by gravity flow.<br />
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<b>9.</b> Remove the cap from the QIAfilter outlet nozzle. Gently insert the plunger into the QIAfilter Midi Cartridge and filter the cell lysate into the previously equilibrated HiSpeed Tip.<br />
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<b>10.</b> Allow the cleared lysate to enter the resin by gravity flow.<br />
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<b>11.</b> Wash the HiSpeed Midi Tip with 20 mL of Buffer QC.<br />
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<b>12.</b> Elute DNA with 5 mL of Buffer QF.<br />
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<b>13.</b> Precipitate DNA by adding 3,5mL of room-temperature isopropanol to the eluted DNA. Mix and incubate at room-temperature during 5 min.<br />
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<b>14.</b> During the incubation remove the plunger from a 20 mL syringe and attach the QIAprecipitator Midi Module onto the outlet nozzle. <br />
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<b>15.</b> Place the QIAprecipitator over a waste bottle, transfer the eluate/isopropanol mixture into the 20<br />
mL syringe, and insert the plunger. Filter the eluate/ isopropanol mixture through the QIA precipitator using constant pressure.<br />
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<b>16.</b> Remove the QIAprecipitator from the 20 mL syringe and pull out the plunger. Re-attach the QIAprecipitator and add 2mL 70% ethanol to the syringe. Wash the DNA by inserting the plunger and pressing the ethanol through the QIAprecipitaor using constant pressure.<br />
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<b>17.</b> Remove the QIAprecipitator from the 20 mL syringe and pull out the plunger. Attach the QIAprecipitator to the 20 mL syringe again, insert the plunger, and dry the membrane by pressing air through the QIAprecipitaot quickly and forcefully. Repeat this step.<br />
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<b>18.</b> Dry the outlet nozzle of the QIAprecipitator with absorbent paper to prevent ethanol carryover.<br />
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<b>19.</b> Remove the plunger from a new 5 mL syringe and attach the QIAprecipitator onto the outlet nozzle. Hold the outlet of the QIAprecipitator over a 1,5 mL collection tube. Add 1 mL of Buffer TE to the 5 mL syringe. Insert the plunger and elute the DNA into the collection tube using constant pressure.<br />
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<b>20.</b> Remove the QIAprecipitator from the 5 mL syringe, pull out the plunger and reattach the QIAprecipitator to the 5 mL syringe.<br />
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<b>21.</b> Transfer the eluate from step 19 to the 5 mL syringe and eluate for a second time into the seme 1,5 mL tube.<br />
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Determination of yield.<br />
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Agarose gel analysis.<br />
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<p style = "text-align : center"><br />
<small> This protocol is extracted from "HiSpeed Plasmid Purification Handbook" by <B>QIAGEN </B>. </small><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Pouring
Team:Lyon-INSA-ENS/Realisation/Protocols/Pouring
2012-01-10T10:21:08Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Pouring of plates</font> </p><br />
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<p style="line-height : 1.5em"><br />
This protocol aims at creating your own plates with desired antibiotics. It is more efficient than plating an antibiotic on an already-made plate. With 125mL of LB 2X and agar, this should allow to create about 8-10 plates. All must be sterile.<br />
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<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
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<b>1.</b> Melt the agar completely in a water bath.<br />
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<b>2.</b> Allow it to cool down until you can hold it easily in bare hands. If too hot, the antibiotics would be damaged.<br />
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<b>3.</b> Meanwhile, add 2.5mL of each desired antibiotic (100X) into the LB 2X bottle<br />
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<b>4.</b> Mix the bottles in a sterile Erlen and pour immediately into sterile Petri dishes. All the surface should be covered but it doesn't need to be very thick.<br />
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<b>5.</b> Allow them to cool down and solidify and store them at 4°C.<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Ozyme
Team:Lyon-INSA-ENS/Realisation/Protocols/Ozyme
2012-01-10T10:20:48Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Ozyme digestion </font> </p><br />
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This protocol aims at cutting plasmids on specific restriction sites using restriction enzymes.<br />
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<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
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<b>1.</b> In an eppendorf tube, add the following solutions in any order :<br/><br />
-11µL water<br/><br />
-10µL DNA<br/><br />
-2.5µL NE Buffer 2<br/><br />
-0.5µL BSA X50<br/><br />
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<p style="line-height : 1.5em"><br />
<b>2.</b> Add 0.5µL of each one of the desired enzymes.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Incubate at 37°C for 10mn<br />
</p><br />
<br />
<br/> <br />
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<p style="line-height : 1.5em"><br />
<b>4.</b> Inactivate the enzymes by a 20mn incubation at 80°C<br />
</p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Microscopy
Team:Lyon-INSA-ENS/Realisation/Protocols/Microscopy
2012-01-10T10:20:30Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Microscopy test</font> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
This protocol aims at preparing glass slides for observation of biofilms with a fluorescent microscope.<br />
<br />
</p><br />
<br />
<br/> <br/><br />
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<br />
<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>1.</b> In empty sterile plates, introduce 10mL of M63G medium and 100µL of appropriate antibiotic.<br />
<p/><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>2.</b> Add 100µL of bacteria from a saturated liquid culture. <br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Finally, add 3 or 4 sterile glass slides with a sterile tweezer. <br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>4.</b> Incubate at 30°C overnight. <br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>5.</b> Observe the slides with a fluorescent microscope.<br />
</p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Isolation
Team:Lyon-INSA-ENS/Realisation/Protocols/Isolation
2012-01-10T10:20:05Z
<p>Suxiaohui: </p>
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<br />
<p style="text-align : center"> <font color="green" size="5"> NucleoSpin Plasmid QuickPure : Isolation of high-copy plasmid DNA from E. coli<br />
</font> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
This protocol is for a preparation of up to 15µg of high-copy plasmid or DNA using the Macherey-Nagel Nucleospin plasmid QuickPure Kit.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>1.</b>Use 1-5mL ( we used 1.5mL ) of a saturated E.Coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at<br />
11,000 x g. Discard the supernatant and remove as much of the liquid as possible.<br />
<p/><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>2.</b> Add 250 μL Buffer A1. Resuspend the cell pellet<br />
completely by vortexing or pipetting up and down. Make<br />
sure no cell clumps remain before addition of Buffer A2!<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Check Buffer A2 for precipitated SDS prior to<br />
use. If a white precipitate is visible, warm the buffer for<br />
several minutes at 30 – 40 °C until precipitate is dissolved<br />
completely. Cool buffer down to room temperature<br />
(18 – 25 °C).<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>4.</b> Add 250 μL Buffer A2. Mix gently by inverting the tube<br />
6 – 8 times. Do not vortex to avoid shearing of genomic<br />
DNA. Incubate at room temperature for up to 5 min or<br />
until lysate appears clear.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>5.</b> Add 300 μL Buffer A3. Mix thoroughly by inverting the<br />
tube 6 – 8 times. Do not vortex to avoid shearing of<br />
genomic DNA!<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>6.</b> Centrifuge for 5 min at 11,000 x g at room temperature.<br />
Repeat this step in case the supernatant is not clear!<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>7.</b> Place a NucleoSpin Plasmid Column<br />
in a Collection Tube (2 mL) and decant the supernatant<br />
from step 3 or pipette a maximum of 750 μL of the<br />
supernatant onto the column. Centrifuge for 1 min<br />
at 11,000 x g. Discard flow-through and place the<br />
NucleoSpin Plasmid Column back into<br />
the collection tube.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>8.</b> Add 600 μL Buffer A4 (supplemented with ethanol ). Centrifuge for 1 min at 11,000 x g.<br />
Discard flow-through and place the NucleoSpin<br />
Plasmid Column back into the empty<br />
collection tube.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>9.</b> Centrifuge for 2 min at 11,000 x g and discard the<br />
collection tube.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>10.</b> Place the NucleoSpin Plasmid Column<br />
in a 1.5 mL tube and<br />
add 50 μL Buffer AE. Incubate for 1 min at room<br />
temperature. Centrifuge for 1 min at 11,000 x g.<br />
</p><br />
<br />
<br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style = "text-align : center"><br />
<small> This protocol is extracted from "Plasmid DNA purification user manual" by <B>Macherey-Nagel </B>. </small><br />
</p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Ligation
Team:Lyon-INSA-ENS/Realisation/Protocols/Ligation
2012-01-10T10:19:45Z
<p>Suxiaohui: </p>
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<br />
<p style="text-align : center"> <font color="green" size="5"> Ligation </font> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
This protocol aims at ligating compatible, previously cut restriction sites on plasmids. Volumes of DNA are given for similar concentrations. You may want to adapt these volumes if your concentrations are significantly different. Enzymes and buffers were supplied by Fermentas.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>1. Standard ligation</b> In an eppendorf tube, add the following solutions :<br/><br />
- 6 µL insert<br/><br />
- 4 µL vector<br/><br />
- 2 µL T4 DNA ligase buffer<br/><br />
- 6 µL water<br/><br />
<br />
<br/> <br />
<br />
<br />
<b>Or 1. 3A ligation</b> In an eppendorf tube, add the following solutions :<br/><br />
- 2 µL of each DNA solution <br/><br />
- 2 µL T4 DNA ligase buffer <br/><br />
- 10 µL water<br/><br />
<br />
<p/><br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>2.</b> Add 2µL of ligase.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Incubate at room temperature for 3h<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>4.</b> Incubate at 70°C for 5 mn to inactivate the ligase.<br />
</p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/Fermentas
Team:Lyon-INSA-ENS/Realisation/Protocols/Fermentas
2012-01-10T10:19:20Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Fermentas digestion </font> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
This protocol aims at cutting plasmids on specific restriction sites using restriction enzymes. <br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>1.</b> In an eppendorf tube, add the following solutions in any order :<br/><br />
-250 ng DNA ( if the concentration is unknown, use 5µL )<br/><br />
-2.5µL (1X) or 5µL (2X) 10X tango buffer ( this depends on the enzymes used )<br/><br />
-water to get a volume of 25µL after addition of the enzymes<br />
<p/><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>2.</b> Add 1µL of each one of the desired enzymes.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Incubate at 37°C for 1h30<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>4.</b> Incubate at 70°C for 10 mn to inactivate the restriction enzymes.<br />
</p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/CaCl2_trans
Team:Lyon-INSA-ENS/Realisation/Protocols/CaCl2 trans
2012-01-10T10:18:49Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> CaCl2 chemical transformation </font> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
This protocol aims at inserting plasmids notably into NM522 strains, or any other E.Coli strain<br />
</p><br />
<br />
<p style="line-height : 1.5em"><br />
It has been found less efficient than the TSS transformation protocol, thus we do not recommend using it.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>1.</b> Monitor the OD600 of a 50mL culture of NM522 cells ( or the strain you want to transform ) in LB medium at 37°C. Proceed to the next step when it reaches 0.2-0.4 ( not higher than 0.6 ), which takes approximately 3 hours.<br />
<p/><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>2.</b> Aliquot the culture into fractions of the desired volume V ( 10 or 15 mL usually ).<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Incubate on ice for 10mn<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>4.</b> Centrifuge at 4°C, 10mn, 5000rpm<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>5.</b> Empty the supernatant<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>6.</b> Add V/2 mL cold and sterile CaCl2 100mM<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>7.</b> Incubate for 20mn on ice<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>8.</b> Centrifuge at 4°C, 5mn, 5000rpm<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>9.</b> Empty the supernatant and resuspend ( do not vortex ) in V/15 mL CaCl2 and V/30 mL glycerol 40%<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>10.</b> Aliquot the bacteria in 200 µL fractions and keep on ice. These bacteria should be used within the day.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>11.</b> Add the desired quantity of DNA to the fractions. For positive control, we used Puc18 plasmid. For negative control, we used water.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>12.</b> Incubate for 30mn on ice<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>13.</b> Heat shock at 42°C for 2mn<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>14.</b> Incubate for 5mn on ice<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>15.</b> Add 800µL of LB medium and incubate for 1h at 37°C<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>16.Optional</b> Concentrate 10X by centrifuging for 2mn at 11000 rpm, eliminate the supernatant and resuspend in 100µL LB.<br />
</p><br />
<br />
<br/> <br />
<br />
<p style="line-height : 1.5em"><br />
<b>17.</b> Plate 100µL of bacteria on LB medium with the appropriate antibiotic resistance and incubate at 37°C.<br />
</p><br />
<br />
<br />
<p style="line-height : 1.5em"><br />
<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/BiofilmQuant
Team:Lyon-INSA-ENS/Realisation/Protocols/BiofilmQuant
2012-01-10T10:17:49Z
<p>Suxiaohui: </p>
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<p style="text-align : center"> <font color="green" size="5"> Biofilm quantification </font> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
This protocol aims at quantifying the adherence of any given strain in a 24 well plate.<br/> <br/><br />
<br />
All solutions used need to be sterile. EDTA and CoCl2 solutions were sterilized by filtration on a 0.2µm filter.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p style="text-align : center"> <font color="green" size="5"> Procedure </font> </p><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>1.</b> Prepare 10 mL of the desired media in 10mL falcon tubes. For LB/2, dilute 25mL sterile LB with 25mL sterile water and aliquot this mother solution. For M63G, prepare 100mL M63 + 1mL glucose 20%+ 1mL LB (all sterile) and aliquot 10mL of this mother solution. Each 10mL tube will be used to fill one column of 4 wells.<br />
<p/><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>2.</b> Grow 5mL cultures of the desired strains overnight with the appropriate antibiotic if the strain has a resistance (50µL) in the same media as the ones that are going to be used for the test.<br />
<p/><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>3.</b> Add any other required solution to the 10mL tubes ( 100 µL antibiotic, CoCl2 to obtain the desired concentration... ). <br />
<p/><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>4.</b> Measure the OD600 of the 5mL bacterial cultures and add an appropriate volume of bacteria to the 10 mL tubes to obtain a concentration of 10⁷ bacteria/mL ( 0.6 OD units is roughly equivalent to 2.10⁸ bacteria/mL )<br />
<p/><br />
<br />
<br/> <br/><br />
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<b>5.</b> Wash the 24 well plate with sterile EDTA 4mM, empty the plate, wash with sterile water, empty the plate. This step allows the capture of metals that are on the plate and which could interfere with the test.<br />
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<b>6.</b> Fill the wells with 2mL each from the 10mL tubes. <br />
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<b>7.</b> Incubate at 30°C for 24 to 48 hours, depending on the medium ( M63 takes longer than LB ) <br />
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<b>8. OD600 method</b> Transfer the 2mL supernatant into small test tubes (S) <br/><br />
Gently wash the biofilm with 1mL medium ( the same as the one used for the culture ) and add this to the S tubes. <br/><br />
Add 1mL medium and scratch the biofilm. Pour the biofilm with the medium into a test tube (B) and vortex for 20s.<br/><br />
Measure the OD600 of each tube. The % of adherence is 100*B/(B+3S)<br/><br />
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<b>8. Methyl violet method</b>Eliminate the supernatant<br/><br />
Gently wash the biofilm with 1mL medium and eliminate the wash liquid <br/><br />
Heat the plate at 80°C for 1h. Add 0.2mL of a methyl violet solution and wait for 2mn. Methyl violet is toxic and should be manipulated under a hood with gloves.<br/><br />
Eliminate the dye solution and wash with water. Dry the plate.<br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Protocols/BiofilmQuant
Team:Lyon-INSA-ENS/Realisation/Protocols/BiofilmQuant
2012-01-10T10:17:28Z
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<p style="text-align : center"> <font color="green" size="5"> Biofilm quantification </font> </p><br />
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<p style="line-height : 1.5em"><br />
This protocol aims at quantifying the adherence of any given strain in a 24 well plate.<br/> <br/><br />
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All solutions used need to be sterile. EDTA and CoCl2 solutions were sterilized by filtration on a 0.2µm filter.<br />
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<b>1.</b> Prepare 10 mL of the desired media in 10mL falcon tubes. For LB/2, dilute 25mL sterile LB with 25mL sterile water and aliquot this mother solution. For M63G, prepare 100mL M63 + 1mL glucose 20%+ 1mL LB (all sterile) and aliquot 10mL of this mother solution. Each 10mL tube will be used to fill one column of 4 wells.<br />
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<p style="line-height : 1.5em"><br />
<b>2.</b> Grow 5mL cultures of the desired strains overnight with the appropriate antibiotic if the strain has a resistance (50µL) in the same media as the ones that are going to be used for the test.<br />
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<b>3.</b> Add any other required solution to the 10mL tubes ( 100 µL antibiotic, CoCl2 to obtain the desired concentration... ). <br />
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<p style="line-height : 1.5em"><br />
<b>4.</b> Measure the OD600 of the 5mL bacterial cultures and add an appropriate volume of bacteria to the 10 mL tubes to obtain a concentration of 10⁷ bacteria/mL ( 0.6 OD units is roughly equivalent to 2.10⁸ bacteria/mL )<br />
<p/><br />
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<br/> <br/><br />
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<p style="line-height : 1.5em"><br />
<b>5.</b> Wash the 24 well plate with sterile EDTA 4mM, empty the plate, wash with sterile water, empty the plate. This step allows the capture of metals that are on the plate and which could interfere with the test.<br />
<p/><br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>6.</b> Fill the wells with 2mL each from the 10mL tubes. <br />
<p/><br />
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<p style="line-height : 1.5em"><br />
<b>7.</b> Incubate at 30°C for 24 to 48 hours, depending on the medium ( M63 takes longer than LB ) <br />
<p/><br />
<br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>8. OD600 method</b> Transfer the 2mL supernatant into small test tubes (S) <br/><br />
Gently wash the biofilm with 1mL medium ( the same as the one used for the culture ) and add this to the S tubes. <br/><br />
Add 1mL medium and scratch the biofilm. Pour the biofilm with the medium into a test tube (B) and vortex for 20s.<br/><br />
Measure the OD600 of each tube. The % of adherence is 100*B/(B+3S)<br/><br />
<p/><br />
<br />
<br />
<br/> <br/><br />
<br />
<p style="line-height : 1.5em"><br />
<b>8. Methyl violet method</b>Eliminate the supernatant<br/><br />
Gently wash the biofilm with 1mL medium and eliminate the wash liquid <br/><br />
Heat the plate at 80°C for 1h. Add 0.2mL of a methyl violet solution and wait for 2mn. Methyl violet is toxic and should be manipulated under a hood with gloves.<br/><br />
Eliminate the dye solution and wash with water. Dry the plate.<br/><br />
<p/><br />
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{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week19
Team:Lyon-INSA-ENS/Realisation/Week19
2011-10-27T15:27:53Z
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<h1 style="color: white;"> Week 19 </h1> <br />
</div><br />
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<p style="text-align : center"> <small> From Monday the 24th of October to Friday the 28th of October 2011 </small> </p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week19
Team:Lyon-INSA-ENS/Realisation/Week19
2011-10-27T15:27:08Z
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<h1 style="color: white;"> Week 19 </h1> <br />
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<p style="text-align : center"> <small> From Monday the 24th of October to Friday the 28th of October 2011 </small> </p><br />
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<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week18"/><font color="grey"><b>Previous Week</b></font></a><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week18
Team:Lyon-INSA-ENS/Realisation/Week18
2011-10-27T15:25:59Z
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<h1 style="color: white;"> Week 18 </h1> <br />
</div><br />
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<p style="text-align : center"> <small> From Monday the 17th of October to Sunday the 23rd of October 2011 </small> </p><br />
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http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week18
Team:Lyon-INSA-ENS/Realisation/Week18
2011-10-27T15:22:23Z
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<h1 style="color: white;"> Week 18 </h1> <br />
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<p style="text-align : center"> <small> From Monday the 17th of October to Sunday the 23rd of October 2011 </small> </p><br />
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Suxiaohui
http://2011.igem.org/Template:Lyon-INSA-ENS/menuNotebookVertical
Template:Lyon-INSA-ENS/menuNotebookVertical
2011-10-27T15:08:54Z
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week19
Team:Lyon-INSA-ENS/Realisation/Week19
2011-10-27T15:08:28Z
<p>Suxiaohui: Created page with "{{Lyon-INSA-ENS/header}} {{Lyon-INSA-ENS/blocStyle}} {{INSA-Lyon/styletestaurelie}} {{Lyon-INSA-ENS/menuhorizontal}} {{Lyon-INSA-ENS/menuNotebookVertical|Week 19 = actif}} <htm..."</p>
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<p style="text-align : center"> <small> From Monday the 24th of October to Friday the 28th of October 2011 </small> </p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week18
Team:Lyon-INSA-ENS/Realisation/Week18
2011-10-27T15:07:53Z
<p>Suxiaohui: Created page with "{{Lyon-INSA-ENS/header}} {{Lyon-INSA-ENS/blocStyle}} {{INSA-Lyon/styletestaurelie}} {{Lyon-INSA-ENS/menuhorizontal}} {{Lyon-INSA-ENS/menuNotebookVertical|Week 18 = actif}} <htm..."</p>
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<h1 style="color: white;"> Week 18 </h1> <br />
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<p style="text-align : center"> <small> From Monday the 17th of October to Sunday the 23rd of October 2011 </small> </p><br />
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Suxiaohui
http://2011.igem.org/Template:Lyon-INSA-ENS/menuNotebookVertical
Template:Lyon-INSA-ENS/menuNotebookVertical
2011-10-27T15:06:49Z
<p>Suxiaohui: </p>
<hr />
<div><div id="menu"><br />
<br />
<ul class="niveau1"> <br />
<br />
<li class ="{{{BeforeStarting|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Notebook/BeforeStarting|Before Starting]] </li> <br />
<br />
<li class ="{{{Protocols|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Protocols|Protocols]] </li> <br />
<br />
<li class ="{{{Week 1|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Notebook/Week1|Week 1]] </li> <br />
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<li class ="{{{Week 2|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week2|Week 2]] </li> <br />
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<li class ="{{{Week 3|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week3|Week 3]] </li> <br />
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<li class ="{{{Week 4|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week4|Week 4]] </li> <br />
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<li class ="{{{Week 5|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week5|Week 5]] </li><br />
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<li class ="{{{Week 6|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week6|Week 6]] </li> <br />
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<li class ="{{{Week 7|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week7|Week 7]] </li> <br />
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<li class ="{{{Week 8|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week8|Week 8]] </li> <br />
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<li class ="{{{Week 9|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week9|Week 9]] </li> <br />
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<li class ="{{{Week 10|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week10|Week 10]] </li> <br />
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<li class ="{{{Week 11|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week11|Week 11]] </li> <br />
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<li class ="{{{Week 12|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week12|Week 12]] </li> <br />
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<li class ="{{{Week 13|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week13|Week 13]] </li> <br />
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<li class ="{{{Week 14|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week14|Week 14]] </li><br />
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<li class ="{{{Week 17|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week17|Week 17]] </li><br />
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<li class ="{{{Week 18|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week18|Week 18]] </li><br />
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<li class ="{{{Week 19|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week19|Week 19]] </li><br />
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<li class ="{{{Collection|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Collection|Collection]] </li> <br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week17
Team:Lyon-INSA-ENS/Realisation/Week17
2011-10-27T15:05:18Z
<p>Suxiaohui: Created page with "{{Lyon-INSA-ENS/header}} {{Lyon-INSA-ENS/blocStyle}} {{INSA-Lyon/styletestaurelie}} {{Lyon-INSA-ENS/menuhorizontal}} {{Lyon-INSA-ENS/menuNotebookVertical|Week 17 = actif}} <htm..."</p>
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<a href="https://2011.igem.org/Main_Page" ><br />
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<img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week14Fr">Version Fran&ccedil;aise</a><br />
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<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
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<h1 style="color: white;"> Week 17 </h1> <br />
</div><br />
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<p style="text-align : center"> <small> From Monday the 10th of October to Sunday the 16th of October 2011 </small> </p><br />
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Suxiaohui
http://2011.igem.org/Template:Lyon-INSA-ENS/menuNotebookVertical
Template:Lyon-INSA-ENS/menuNotebookVertical
2011-10-27T15:03:34Z
<p>Suxiaohui: </p>
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<ul class="niveau1"> <br />
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<li class ="{{{BeforeStarting|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Notebook/BeforeStarting|Before Starting]] </li> <br />
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<li class ="{{{Protocols|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Protocols|Protocols]] </li> <br />
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<li class ="{{{Week 1|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Notebook/Week1|Week 1]] </li> <br />
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<li class ="{{{Week 2|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week2|Week 2]] </li> <br />
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<li class ="{{{Week 3|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week3|Week 3]] </li> <br />
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<li class ="{{{Week 4|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week4|Week 4]] </li> <br />
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<li class ="{{{Week 5|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week5|Week 5]] </li><br />
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<li class ="{{{Week 6|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week6|Week 6]] </li> <br />
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<li class ="{{{Week 7|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week7|Week 7]] </li> <br />
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<li class ="{{{Week 8|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week8|Week 8]] </li> <br />
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<li class ="{{{Week 9|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week9|Week 9]] </li> <br />
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<li class ="{{{Week 10|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week10|Week 10]] </li> <br />
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<li class ="{{{Week 11|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week11|Week 11]] </li> <br />
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<li class ="{{{Week 12|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week12|Week 12]] </li> <br />
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<li class ="{{{Week 13|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week13|Week 13]] </li> <br />
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<li class ="{{{Week 14|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week14|Week 14]] </li><br />
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<li class ="{{{Week 17|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Week17|Week 17]] </li><br />
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<li class ="{{{Collection|inactif}}}"> [[Team:Lyon-INSA-ENS/Realisation/Collection|Collection]] </li> <br />
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</ul><br />
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</div></div>
Suxiaohui
http://2011.igem.org/World_Championship_Jamboree/Schedule/Practice_Sessions
World Championship Jamboree/Schedule/Practice Sessions
2011-10-27T12:32:05Z
<p>Suxiaohui: </p>
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<!--BEGIN REGONLINE LINK CODE!--><br />
<table style="position: absolute; margin-top:127px; margin-left:15px; z-index: 10;" align="center" class='pbrROL'><tr><td><div class='pbrROL-04' ><div class='ROLbtn'><ul><li><a href='http://www.regonline.com/igem2011worldchampionship' target='_blank' title='iGEM 2011 World Championship Jamboree'><span id='regLink'>Register Now!</span></a></li></ul></div></div></td></tr></table><link rel="stylesheet" href="https://www.regonline.com/styles/ClientButton.css" type="text/css"><br />
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<a href="https://2011.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a><br />
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<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Schedule">Schedule</a><br />
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<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Campus_Map">Campus Map</a><br />
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<td class="menu" ><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Program">Jamboree Program</a><br />
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<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Workshops">Workshops</a><br />
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<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Press">Press Kits</a><br />
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<td colspan="3" valign="top"><br />
<div class="main_item"> <!-- World Jamboree Schedule Goes Here--> <br />
<div class="title">Presentation Practice: Friday November 4th 2011</div><br />
<br />
Use this sign-up sheet to sign up for a slot on Friday night (November 4th) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. <br><br> <br />
<br />
There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment. Please keep in mind that there will be teams waiting to use the room after you, so make sure that your practice finishes on time.<br><br><br />
<br />
Also, on Friday, November 4th, there will also be pre-registration available <!-- beginning at <strong>1pm at Compton Lounge</strong> -->. Conference services will be on-site to pass out team registration boxes (see the <a href="https://2011.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a>). <br><br><br />
<br />
<strong>Note</strong>: Use the wiki edit button to add your team to the schedule (the markup is located at the bottom of the page). Room numbers and locations will be updated as soon as possible. Additional rooms may be added in the coming weeks.<br />
<br />
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</td><br />
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</body><br />
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<html> <!--Practice Session Sign-Up Sheet - ADD YOUR TEAM NAME HERE!--><br />
<table class="calendar" align="center"><h2 class="date"><a name="Friday Practice">Friday, November 4</a></h2><br />
<thead><br />
<tr><br />
<th style="width:100px;">Time</th><br />
<th>RM 56-162 </th><br />
<th>RM 34-101 </th><br />
<th>RM 56-154 </th><br />
<th>RM 32-144 </th><br />
<th>RM 10-250 </th><br />
<th>RM 32-123 </th><br />
<th>RM 56-180 </th><br />
<th>RM 56-114 </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>6:00p - 6:30p</th><br />
<td>Washington</td><br />
<td>Dundee</td><br />
<td>TU München</td><br />
<td>A4</td><br />
<td>Lyon-INSA-ENS</td><br />
<td>MIT</td><br />
<td>Fatih Turkey</td><br />
<td>A8</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30p - 7:00p</th><br />
<td>IIT Madras</td><br />
<td>B2</td><br />
<td>B3</td><br />
<td>B4</td><br />
<td>B5</td><br />
<td>B5</td><br />
<td>B5</td><br />
<td>B5</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00p - 7:30p</th><br />
<td>NYMU-Taipei</td><br />
<td>C2</td><br />
<td>Paris Bettencourt</td><br />
<td>C4</td><br />
<td>Calgary</td><br />
<td>C6</td><br />
<td>C7</td><br />
<td>C8</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30p - 8:00p</th><br />
<td>Tokyo Tech</td><br />
<td>SYSU-China</td><br />
<td>D3</td><br />
<td>D4</td><br />
<td>D5</td><br />
<td>D6</td><br />
<td>D7</td><br />
<td>D8</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00p - 8:30p</th><br />
<td>UNICAMP-EMSE</td><br />
<td>USTC-China</td><br />
<td>E3</td><br />
<td>E4</td><br />
<td>Bielefeld-Germany</td><br />
<td>EPF-Lausanne</td><br />
<td>E7</td><br />
<td>E8</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30p - 9:00p</th><br />
<td>ZJU-China</td><br />
<td>USTC-Software</td><br />
<td>Tsinghua</td><br />
<td>F4</td><br />
<td>Osaka</td><br />
<td>F6</td><br />
<td>F7</td><br />
<td>F8</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00p - 9:30p</th><br />
<td>WHU-China</td><br />
<td>uOttawa</td><br />
<td>G3</td><br />
<td>K.U.Leuven</td><br />
<td>HKUST-Hong_Kong</td><br />
<td>UNITS_Trieste</td><br />
<td>Imperial College London</td><br />
<td>G8</td><br />
</tr><br />
<tr class="even"><br />
<th>9:30p - 10:00p</th><br />
<td>Berkeley</td><br />
<td>Queens_Canada</td><br />
<td>UPO-Sevilla</td><br />
<td>Wisconsin-Madison</td><br />
<td>Tokyo-NoKoGen</td><br />
<td>Harvard</td><br />
<td>Johns Hopkins</td><br />
<td>ETH Zurich</td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
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<html><br />
<table><br><br><br />
<center><br />
<iframe width="425" height="350" frameborder="0" scrolling="no" marginheight="0" marginwidth="0" src="http://www.google.com/maps/ms?vpsrc=6&amp;ctz=240&amp;ie=UTF8&amp;msa=0&amp;t=m&amp;msid=208072632249057954752.0004b024a38f4f8325221&amp;source=embed&amp;ll=42.359617,-71.091839&amp;spn=0.000808,0.002135&amp;output=embed"></iframe><br /><small>View <a href="http://www.google.com/maps/ms?vpsrc=6&amp;ctz=240&amp;ie=UTF8&amp;msa=0&amp;t=m&amp;msid=208072632249057954752.0004b024a38f4f8325221&amp;source=embed&amp;ll=42.359617,-71.091839&amp;spn=0.000808,0.002135" style="color:#0000FF;text-align:left">My Saved Places</a> in a larger map</small><br />
</center><br />
</table><br />
</html></div>
Suxiaohui
http://2011.igem.org/World_Championship_Jamboree/Schedule/Practice_Sessions
World Championship Jamboree/Schedule/Practice Sessions
2011-10-27T12:29:28Z
<p>Suxiaohui: </p>
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<td rowspan="6" style="width: 200px;" valign="top"><br />
<div id="imgleft"><br />
<img src="https://static.igem.org/mediawiki/2011/4/44/2011_WC_Main_Left1.jpg"><br />
</div><br />
<!--BEGIN REGONLINE LINK CODE!--><br />
<table style="position: absolute; margin-top:127px; margin-left:15px; z-index: 10;" align="center" class='pbrROL'><tr><td><div class='pbrROL-04' ><div class='ROLbtn'><ul><li><a href='http://www.regonline.com/igem2011worldchampionship' target='_blank' title='iGEM 2011 World Championship Jamboree'><span id='regLink'>Register Now!</span></a></li></ul></div></div></td></tr></table><link rel="stylesheet" href="https://www.regonline.com/styles/ClientButton.css" type="text/css"><br />
<!--END REGONLINE LINK CODE!--><br />
</td><br />
<br />
<td rowspan="6"><br />
<div id="imgcenter"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree"><img src="https://static.igem.org/mediawiki/2011/5/55/2011_WC_Main1.jpg"></a><br />
</div><br />
</td><br />
<br />
<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a><br />
</td><br />
</tr><br />
<tr><br />
<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Schedule">Schedule</a><br />
</td><br />
</tr><br />
<tr><br />
<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Campus_Map">Campus Map</a><br />
</td><br />
</tr><br />
<tr><br />
<td class="menu" ><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Program">Jamboree Program</a><br />
</td><br />
</tr><br />
<tr><br />
<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Workshops">Workshops</a><br />
</td><br />
</tr><br />
<tr><br />
<td class="menu"><br />
<a href="https://2011.igem.org/World_Championship_Jamboree/Press">Press Kits</a><br />
<div><br />
</td><br />
</tr><br />
<td colspan="3" valign="top"><br />
<div class="main_item"> <!-- World Jamboree Schedule Goes Here--> <br />
<div class="title">Presentation Practice: Friday November 4th 2011</div><br />
<br />
Use this sign-up sheet to sign up for a slot on Friday night (November 4th) to practice your talk. Note that there will NOT be any A/V (audio/visual) support on staff. All classrooms will be unlocked and you should use them and leave them as you found them. <br><br> <br />
<br />
There are a limited number of time slots available on a first-come first-serve basis so please only choose one slot. We cannot match the room that you will ultimately give your presentation in with the practice room. This should, however, give you a chance to practice your talk in a new environment. Please keep in mind that there will be teams waiting to use the room after you, so make sure that your practice finishes on time.<br><br><br />
<br />
Also, on Friday, November 4th, there will also be pre-registration available <!-- beginning at <strong>1pm at Compton Lounge</strong> -->. Conference services will be on-site to pass out team registration boxes (see the <a href="https://2011.igem.org/World_Championship_Jamboree/Handbook">Jamboree Handbook</a>). <br><br><br />
<br />
<strong>Note</strong>: Use the wiki edit button to add your team to the schedule (the markup is located at the bottom of the page). Room numbers and locations will be updated as soon as possible. Additional rooms may be added in the coming weeks.<br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</body><br />
</html><br />
<br />
<html> <!--Practice Session Sign-Up Sheet - ADD YOUR TEAM NAME HERE!--><br />
<table class="calendar" align="center"><h2 class="date"><a name="Friday Practice">Friday, November 4</a></h2><br />
<thead><br />
<tr><br />
<th style="width:100px;">Time</th><br />
<th>RM 56-162 </th><br />
<th>RM 34-101 </th><br />
<th>RM 56-154 </th><br />
<th>RM 32-144 </th><br />
<th>RM 10-250 </th><br />
<th>RM 32-123 </th><br />
<th>RM 56-180 </th><br />
<th>RM 56-114 </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="even"><br />
<th>6:00p - 6:30p</th><br />
<td>Washington</td><br />
<td>Dundee</td><br />
<td>TU München</td><br />
<td>Lyon-INSA-ENS</td><br />
<td>A5</td><br />
<td>MIT</td><br />
<td>Fatih Turkey</td><br />
<td>A8</td><br />
</tr><br />
<tr class="odd"><br />
<th>6:30p - 7:00p</th><br />
<td>IIT Madras</td><br />
<td>B2</td><br />
<td>B3</td><br />
<td>B4</td><br />
<td>B5</td><br />
<td>B5</td><br />
<td>B5</td><br />
<td>B5</td><br />
</tr><br />
<tr class="even"><br />
<th>7:00p - 7:30p</th><br />
<td>NYMU-Taipei</td><br />
<td>C2</td><br />
<td>Paris Bettencourt</td><br />
<td>C4</td><br />
<td>Calgary</td><br />
<td>C6</td><br />
<td>C7</td><br />
<td>C8</td><br />
</tr><br />
<tr class="even"><br />
<th>7:30p - 8:00p</th><br />
<td>Tokyo Tech</td><br />
<td>SYSU-China</td><br />
<td>D3</td><br />
<td>D4</td><br />
<td>D5</td><br />
<td>D6</td><br />
<td>D7</td><br />
<td>D8</td><br />
</tr><br />
<tr class="odd"><br />
<th>8:00p - 8:30p</th><br />
<td>UNICAMP-EMSE</td><br />
<td>USTC-China</td><br />
<td>E3</td><br />
<td>E4</td><br />
<td>Bielefeld-Germany</td><br />
<td>EPF-Lausanne</td><br />
<td>E7</td><br />
<td>E8</td><br />
</tr><br />
<tr class="even"><br />
<th>8:30p - 9:00p</th><br />
<td>ZJU-China</td><br />
<td>USTC-Software</td><br />
<td>Tsinghua</td><br />
<td>F4</td><br />
<td>Osaka</td><br />
<td>F6</td><br />
<td>F7</td><br />
<td>F8</td><br />
</tr><br />
<tr class="odd"><br />
<th>9:00p - 9:30p</th><br />
<td>WHU-China</td><br />
<td>uOttawa</td><br />
<td>G3</td><br />
<td>K.U.Leuven</td><br />
<td>HKUST-Hong_Kong</td><br />
<td>UNITS_Trieste</td><br />
<td>Imperial College London</td><br />
<td>G8</td><br />
</tr><br />
<tr class="even"><br />
<th>9:30p - 10:00p</th><br />
<td>Berkeley</td><br />
<td>Queens_Canada</td><br />
<td>UPO-Sevilla</td><br />
<td>Wisconsin-Madison</td><br />
<td>Tokyo-NoKoGen</td><br />
<td>Harvard</td><br />
<td>Johns Hopkins</td><br />
<td>ETH Zurich</td><br />
</tr><br />
</tbody><br />
</table><br />
</html><br />
<br />
<html><br />
<table><br><br><br />
<center><br />
<iframe width="425" height="350" frameborder="0" scrolling="no" marginheight="0" marginwidth="0" src="http://www.google.com/maps/ms?vpsrc=6&amp;ctz=240&amp;ie=UTF8&amp;msa=0&amp;t=m&amp;msid=208072632249057954752.0004b024a38f4f8325221&amp;source=embed&amp;ll=42.359617,-71.091839&amp;spn=0.000808,0.002135&amp;output=embed"></iframe><br /><small>View <a href="http://www.google.com/maps/ms?vpsrc=6&amp;ctz=240&amp;ie=UTF8&amp;msa=0&amp;t=m&amp;msid=208072632249057954752.0004b024a38f4f8325221&amp;source=embed&amp;ll=42.359617,-71.091839&amp;spn=0.000808,0.002135" style="color:#0000FF;text-align:left">My Saved Places</a> in a larger map</small><br />
</center><br />
</table><br />
</html></div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Communication/EventsFr
Team:Lyon-INSA-ENS/Communication/EventsFr
2011-09-21T23:49:57Z
<p>Suxiaohui: </p>
<hr />
<div><!-- create template--><br />
{{Lyon-INSA-ENS/header}}<br />
{{Lyon-INSA-ENS/blocStyle}}<br />
{{INSA-Lyon/styletestaurelie}}<br />
{{Lyon-INSA-ENS/menuhorizontalFrComm}}<br />
<!-- {{Lyon-INSA-ENS/menuCommFrEvents}} --><br />
{{Lyon-INSA-ENS/menuHumanPracticeVerticalFr|Events = actif}}<br />
<br />
<br />
<html><br />
<br />
<style type="text/css"><br />
.cadre{ <br />
text-align:center;<br />
width : 600px;<br />
height:auto;<br />
margin-left : 230px ; <br />
margin-top : 20px;<br />
border: 4px solid green;<br />
-moz-border-radius: 10px;<br />
-webkit-border-radius: 10px; <br />
-goog-ms-border-radius: 10px; <br />
border-radius: 10px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-webkit-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-goog-ms-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
}<br />
</style><br />
<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events">English version </a> <br />
</div><br />
<br />
<br><br><br />
<br><br />
<br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Visites </h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/68/P1010017.JPG" width="240px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Visite d'une centrale nucléaire</strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 25 mai, l'équipe s'est rendue sur le site de la centrale nucléaire française du Tricastin : nous avons pu poser nos questions et visiter les installations. Ainsi, cette journée a été pour nous une belle opportunité pour comprendre le contexte dans lequel s'inscrit notre projet.<br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_tricastinFr"/>Plus de photos</a><br />
<br/> <br/><br />
</p><br />
</div><br />
<br />
<!-- centraco --><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/d/db/Visit_centraco_gallery.jpg" width="240px"; style="float:right; " /> <br />
</div> <br />
<br />
<p style="text-align:center;"><br />
<strong>Visite de Centraco </strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 11 juillet, l'équipe s'est rendu à Centraco, une usine des traitements des déchets nucléaires. <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_centracoFr"/>Plus de photos</a><br />
</p><br />
<br/> <br/><br />
</div><br />
<br />
<br><br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Activités en dehors du projet</h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Dragon_boat.jpg" width="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Dragon boat</strong> <br><br><br />
<br/> <br/><br />
L'équipe s'est vaillamment défendue contre une équipe de biologistes le 27 juillet 2011.<br />
</p><br />
<br />
<br />
<br/> <br/><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/DragonBoat"/>Plus de photos</a><br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
</div><br />
<br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/Photo_presentation_tournage.jpg" width="240px"; height="180px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Tournage de la vidéo de présentation de l'équipe</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Organisée par nos intraitables metteurs en scène Margaux et Clémence, toute l'équipe s'est réunie pour un grand moment. Après une répétition rapide, nous avons tourné la vidéo. Chacun a fait de son mieux pour produire une superbe vidéo ; l'un de nos courageux acteurs a même déclaré "J'ai quelques courbatures de cette montée des marches" <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/ShootingFr"/>Plus de photos</a><br />
</p><br />
<br/><br />
<br/><br />
</div><br />
<br />
<br/> <br/><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Cross_igem.jpg" width="300px"; height="250px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>L'équipe iGEM est dans la course</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Le <b>CROSS de l'INSA de Lyon</b> réunit pas moins de 600 coureurs sur deux circuits (de 4 ou 6 km) <br><br><br />
Ce qui rend cet évènement si original est que la plupart des personnes, en équipe de 4, se déguisent pour la course. Les costumes les plus originaux sont alors récompensés. <br><br><br />
Cette année, environ 10 participants ont représenté le projet iGEM parmis lequels les remarquables "CobaltBuster" et les "Pili-Pili". <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/CrossFr"/>Plus de photos</a><br />
</p><br />
<br/><br />
</div><br />
<br />
<br />
<p><br />
<br/><br/><br />
<br/><br/><br/><br />
</p><br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Communication/EventsFr
Team:Lyon-INSA-ENS/Communication/EventsFr
2011-09-21T23:48:59Z
<p>Suxiaohui: </p>
<hr />
<div><!-- create template--><br />
{{Lyon-INSA-ENS/header}}<br />
{{Lyon-INSA-ENS/blocStyle}}<br />
{{INSA-Lyon/styletestaurelie}}<br />
{{Lyon-INSA-ENS/menuhorizontalFrComm}}<br />
<!-- {{Lyon-INSA-ENS/menuCommFrEvents}} --><br />
{{Lyon-INSA-ENS/menuHumanPracticeVerticalFr|Events = actif}}<br />
<br />
<br />
<html><br />
<br />
<style type="text/css"><br />
.cadre{ <br />
text-align:center;<br />
width : 600px;<br />
height:auto;<br />
margin-left : 230px ; <br />
margin-top : 20px;<br />
border: 4px solid green;<br />
-moz-border-radius: 10px;<br />
-webkit-border-radius: 10px; <br />
-goog-ms-border-radius: 10px; <br />
border-radius: 10px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-webkit-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-goog-ms-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
}<br />
</style><br />
<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events">English version </a> <br />
</div><br />
<br />
<br><br><br />
<br><br />
<br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Visites </h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/68/P1010017.JPG" width="240px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Visite d'une centrale nucléaire</strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 25 mai, l'équipe s'est rendue sur le site de la centrale nucléaire française du Tricastin : nous avons pu poser nos questions et visiter les installations. Ainsi, cette journée a été pour nous une belle opportunité pour comprendre le contexte dans lequel s'inscrit notre projet.<br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_tricastinFr"/>Plus de photos</a><br />
<br/> <br/><br />
</p><br />
</div><br />
<br />
<!-- centraco --><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/d/db/Visit_centraco_gallery.jpg" width="240px"; style="float:right; " /> <br />
</div> <br />
<br />
<p style="text-align:center;"><br />
<strong>Visite de Centraco </strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 11 juillet, l'équipe s'est rendu à Centraco, une usine des traitements des déchets nucléaires. <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_centracoFr"/>Plus de photos</a><br />
</p><br />
<br/> <br/><br />
</div><br />
<br />
<br><br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Activités en dehors du projet</h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Dragon_boat.jpg" width="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Dragon boat</strong> <br><br><br />
<br/> <br/><br />
L'équipe s'est vaillamment défendue contre une équipe de biologistes le 27 juillet 2011.<br />
</p><br />
<br />
<br />
<br/> <br/><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/DragonBoat"/>Plus de photos</a><br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
</div><br />
<br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/Photo_presentation_tournage.jpg" width="240px"; height="180px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Tournage de la vidéo de présentation de l'équipe</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Organisée par nos intraitables metteurs en scène Margaux et Clémence, toute l'équipe s'est réunie pour un grand moment. Après une répétition rapide, nous avons tourné la vidéo. Chacun a fait de son mieux pour produire une superbe vidéo ; l'un de nos courageux acteurs a même déclaré "J'ai quelques courbatures de cette montée des marches" <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/ShootingFr"/>Plus de photos</a><br />
</p><br />
<br/><br />
<br/><br />
</div><br />
<br />
<br/> <br/><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Cross_igem.jpg" width="250px"; height="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>L'équipe iGEM est dans la course</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Le <b>CROSS de l'INSA de Lyon</b> réunit pas moins de 600 coureurs sur deux circuits (de 4 ou 6 km) <br><br><br />
Ce qui rend cet évènement si original est que la plupart des personnes, en équipe de 4, se déguisent pour la course. Les costumes les plus originaux sont alors récompensés. <br><br><br />
Cette année, environ 10 participants ont représenté le projet iGEM parmis lequels les remarquables "CobaltBuster" et les "Pili-Pili". <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/CrossFr"/>Plus de photos</a><br />
</p><br />
<br/><br />
</div><br />
<br />
<br />
<p><br />
<br/><br/><br />
<br/><br/><br/><br />
</p><br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Communication/EventsFr
Team:Lyon-INSA-ENS/Communication/EventsFr
2011-09-21T23:48:15Z
<p>Suxiaohui: </p>
<hr />
<div><!-- create template--><br />
{{Lyon-INSA-ENS/header}}<br />
{{Lyon-INSA-ENS/blocStyle}}<br />
{{INSA-Lyon/styletestaurelie}}<br />
{{Lyon-INSA-ENS/menuhorizontalFrComm}}<br />
<!-- {{Lyon-INSA-ENS/menuCommFrEvents}} --><br />
{{Lyon-INSA-ENS/menuHumanPracticeVerticalFr|Events = actif}}<br />
<br />
<br />
<html><br />
<br />
<style type="text/css"><br />
.cadre{ <br />
text-align:center;<br />
width : 600px;<br />
height:auto;<br />
margin-left : 230px ; <br />
margin-top : 20px;<br />
border: 4px solid green;<br />
-moz-border-radius: 10px;<br />
-webkit-border-radius: 10px; <br />
-goog-ms-border-radius: 10px; <br />
border-radius: 10px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-webkit-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-goog-ms-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
}<br />
</style><br />
<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events">English version </a> <br />
</div><br />
<br />
<br><br><br />
<br><br />
<br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Visites </h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/68/P1010017.JPG" width="240px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Visite d'une centrale nucléaire</strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 25 mai, l'équipe s'est rendue sur le site de la centrale nucléaire française du Tricastin : nous avons pu poser nos questions et visiter les installations. Ainsi, cette journée a été pour nous une belle opportunité pour comprendre le contexte dans lequel s'inscrit notre projet.<br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_tricastinFr"/>Plus de photos</a><br />
<br/> <br/><br />
</p><br />
</div><br />
<br />
<!-- centraco --><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/d/db/Visit_centraco_gallery.jpg" width="240px"; style="float:right; " /> <br />
</div> <br />
<br />
<p style="text-align:center;"><br />
<strong>Visite de Centraco </strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 11 juillet, l'équipe s'est rendu à Centraco, une usine des traitements des déchets nucléaires. <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_centracoFr"/>Plus de photos</a><br />
</p><br />
<br/> <br/><br />
</div><br />
<br />
<br><br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Activités en dehors du projet</h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Dragon_boat.jpg" width="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Dragon boat</strong> <br><br><br />
<br/> <br/><br />
L'équipe s'est vaillamment défendue contre une équipe de biologistes le 27 juillet 2011.<br />
</p><br />
<br />
<br />
<br/> <br/><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/DragonBoat"/>Plus de photos</a><br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
</div><br />
<br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/Photo_presentation_tournage.jpg" width="240px"; height="180px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Tournage de la vidéo de présentation de l'équipe</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Organisée par nos intraitables metteurs en scène Margaux et Clémence, toute l'équipe s'est réunie pour un grand moment. Après une répétition rapide, nous avons tourné la vidéo. Chacun a fait de son mieux pour produire une superbe vidéo ; l'un de nos courageux acteurs a même déclaré "J'ai quelques courbatures de cette montée des marches" <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/ShootingFr"/>Plus de photos</a><br />
</p><br />
<br/><br />
<br/><br />
</div><br />
<br />
<br/> <br/><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Cross_igem.jpg" width="250px"; height="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>L'équipe iGEM est dans la course</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Le <b>CROSS de l'INSA de Lyon</b> réunit pas moins de 600 coureurs sur deux circuits (de 4 ou 6 km) <br><br><br />
Ce qui rend cet évènement si original est que la plupart des personnes, en équipe de 4, se déguisent pour la course. Les costumes les plus originaux sont alors récompensés. <br><br><br />
Cette année, environ 10 participants ont représenté le projet iGEM parmis lequels les remarquables "CobaltBuster" et les "Pili-Pili". <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/CrossFr"/>Plus de photos</a><br />
</p><br />
<br/><br />
</div><br />
<br />
<br />
<p><br />
<br/><br/><br />
<br/><br/><br/><br />
</p><br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Communication/EventsFr
Team:Lyon-INSA-ENS/Communication/EventsFr
2011-09-21T23:47:17Z
<p>Suxiaohui: </p>
<hr />
<div><!-- create template--><br />
{{Lyon-INSA-ENS/header}}<br />
{{Lyon-INSA-ENS/blocStyle}}<br />
{{INSA-Lyon/styletestaurelie}}<br />
{{Lyon-INSA-ENS/menuhorizontalFrComm}}<br />
<!-- {{Lyon-INSA-ENS/menuCommFrEvents}} --><br />
{{Lyon-INSA-ENS/menuHumanPracticeVerticalFr|Events = actif}}<br />
<br />
<br />
<html><br />
<br />
<style type="text/css"><br />
.cadre{ <br />
text-align:center;<br />
width : 600px;<br />
height:auto;<br />
margin-left : 230px ; <br />
margin-top : 20px;<br />
border: 4px solid green;<br />
-moz-border-radius: 10px;<br />
-webkit-border-radius: 10px; <br />
-goog-ms-border-radius: 10px; <br />
border-radius: 10px;<br />
-moz-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-webkit-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
-goog-ms-box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
box-shadow: rgba(0, 0, 0, 0.3) 0px 0px 12px 7px;<br />
}<br />
</style><br />
<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events">English version </a> <br />
</div><br />
<br />
<br><br><br />
<br><br />
<br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Visites </h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/68/P1010017.JPG" width="240px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Visite d'une centrale nucléaire</strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 25 mai, l'équipe s'est rendue sur le site de la centrale nucléaire française du Tricastin : nous avons pu poser nos questions et visiter les installations. Ainsi, cette journée a été pour nous une belle opportunité pour comprendre le contexte dans lequel s'inscrit notre projet.<br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_tricastinFr"/>Plus de photos</a><br />
<br/> <br/><br />
</p><br />
</div><br />
<br />
<!-- centraco --><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/d/db/Visit_centraco_gallery.jpg" width="240px"; style="float:right; " /> <br />
</div> <br />
<br />
<p style="text-align:center;"><br />
<strong>Visite de Centraco </strong> <br />
</p><br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;"><br />
Le 11 juillet, l'équipe s'est rendu à Centraco, une usine des traitements des déchets nucléaires. <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/photos_visite_centracoFr"/>Plus de photos</a><br />
</p><br />
<br/> <br/><br />
</div><br />
<br />
<br><br />
<div class="cadre" ; style="background-color:green;" ><br />
<br/><h1 style="color: white;"> Activities around the project </h1> <br />
</div><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/6/63/Dragon_boat.jpg" width="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Dragon boat</strong> <br><br><br />
<br/> <br/><br />
L'équipe s'est vaillamment défendue contre une équipe de biologistes le 27 juillet 2011.<br />
</p><br />
<br />
<br />
<br/> <br/><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/wiki/index.php?title=Team:Lyon-INSA-ENS/Team/DragonBoat"/>Plus de photos</a><br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
</div><br />
<br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/b/bc/Photo_presentation_tournage.jpg" width="240px"; height="180px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>Tournage de la vidéo de présentation de l'équipe</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Organisée par nos intraitables metteurs en scène Margaux et Clémence, toute l'équipe s'est réunie pour un grand moment. Après une répétition rapide, nous avons tourné la vidéo. Chacun a fait de son mieux pour produire une superbe vidéo ; l'un de nos courageux acteurs a même déclaré "J'ai quelques courbatures de cette montée des marches" <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/ShootingFr"/>En savoir plus sur cet événement</a><br />
</p><br />
<br/><br />
<br/><br />
</div><br />
<br />
<br/> <br/><br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="float:right;margin-right:2%"><br />
<img src="https://static.igem.org/mediawiki/2011/2/22/Cross_igem.jpg" width="250px"; height="200px"; style="float:right; " /> <br />
</div><br />
<br />
<p style="text-align:center;"><br />
<strong>L'équipe iGEM est dans la course</strong> <br />
</p><br />
<br />
<p style="margin-top:5%;margin-right:45%;margin-left:5%;"><br />
Le <b>CROSS de l'INSA de Lyon</b> réunit pas moins de 600 coureurs sur deux circuits (de 4 ou 6 km) <br><br><br />
Ce qui rend cet évènement si original est que la plupart des personnes, en équipe de 4, se déguisent pour la course. Les costumes les plus originaux sont alors récompensés. <br><br><br />
Cette année, environ 10 participants ont représenté le projet iGEM parmis lequels les remarquables "CobaltBuster" et les "Pili-Pili". <br />
</p><br />
<br />
<p style="text-align:center;"><br />
<br/><br/><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Events/CrossFr"/>En savoir plus sur cet événement</a><br />
</p><br />
<br/><br />
</div><br />
<br />
<br />
<p><br />
<br/><br/><br />
<br/><br/><br/><br />
</p><br />
<br />
<br />
<br />
<br />
</html><br />
<br />
{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Communication/PressReviewFr
Team:Lyon-INSA-ENS/Communication/PressReviewFr
2011-09-21T23:45:39Z
<p>Suxiaohui: </p>
<hr />
<div>{{Lyon-INSA-ENS/header}}<br />
{{Lyon-INSA-ENS/blocStyle}}<br />
{{INSA-Lyon/styletestaurelie}}<br />
{{Lyon-INSA-ENS/menuhorizontalFrComm}}<br />
<!-- {{Lyon-INSA-ENS/menuCommFrPress}} --><br />
{{Lyon-INSA-ENS/menuHumanPracticeVerticalFr|PressReview = actif}}<br />
<br />
<br />
<html><br />
<body><br />
<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Communication/PressReview">English version </a> <br />
</div><br />
<br />
<div class="contenugrand2";><br />
<br><br><br />
<br><br><br />
<br />
<h5> Site internet</h5><br />
<br />
<div class="cadre" ><br />
<br/> <br/> <br />
<br />
<p style="text-align: center; line-height : 1.5em"><br />
On parle de nous dans <a href="http://envue.insa-lyon.fr/2011mai/art2_0511.php#igem">la newsletter et sur le site de l'INSA</a>, mais aussi sur <a href="http://biologie.ens-lyon.fr/l3/participation-igem-2011-1"> le site de l'ENS-Lyon</a>.<br />
<br/> <br/> <br />
<br />
<a href="http://insa-lyon.fr/fr/concours-igem-2011"><br />
<img src="https://static.igem.org/mediawiki/2011/2/23/INSA_LOGO.png"; width=170px;" style="margin-right: 10%;"><br />
</a><br />
<a href="http://biologie.ens-lyon.fr/l3/participation-igem-2011-1?set_language=fr&cl=fr"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d5/Logo_ens.jpg"; width=170px;"><br />
</a><br />
</p><br />
<br/> <br/> <br />
<br />
<br/> <br/> <br />
</div><br />
<br />
<br />
<div class="cadre" ; ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<a href="https://static.igem.org/mediawiki/2011/0/0b/Com-viva-villeurbanne-2.png" title="Details Viva"/><br />
<img src="https://static.igem.org/mediawiki/2011/a/ad/Com-viva-villeurbanne.jpg" width="240px"; style="float:right; " /> <br />
</a><br />
</div><br />
<br />
<br/> <br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;line-height : 1.5em"><br />
On parle de nous sur le site de la ville de Villeurbanne, entre l'article sur la fête nationale et celui sur l'équipe de basket ASVEL. <br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br />
<br />
</div><br />
<br />
<br><br><br />
<br><br><br />
<br />
<br />
<h5> Journaux </h5><br />
<br />
<div class="cadre" ><br />
<br/><br />
<div style="border: 1px solid black; float:right;margin-right:2%"><br />
<a href="https://static.igem.org/mediawiki/2011/8/8d/LeProgres.png" title="Details LeProgres"/><br />
<img src="https://static.igem.org/mediawiki/2011/8/8f/LeProgres2.png" width="240px"; style="float:right; " /> <br />
</a><br />
</div><br />
<br />
<br/><br/><br/><br/><br/><br/> <br/> <br />
<br />
<p style="margin-top:8%;margin-right:45%;margin-left:5%;line-height : 1.5em"><br />
On parle également de nous dans le Progrès (édition de Lyon et de Villeurbanne).<br />
</p><br />
<br />
<br/> <br/><br/><br/><br/><br/><br/><br/><br/><br/> <br/><br/><br />
<br />
</div><br />
<br />
<br/><br/><br />
<br/><br/><br />
<br />
<br />
<h5> A la radio </h5><br />
<br />
<div class="cadre" ><br />
<br/><br />
<div style=" float:right;margin-right:7% ; width : 500px;"><br />
<iframe frameborder="0" width="480" height="270" src="http://www.dailymotion.com/embed/video/koJjRara6qaU422ihsj"></iframe><br /><br />
<a href="http://www.dailymotion.com/video/xk6svj_radiorcf_tech" target="_blank">radioRCF</a> <i>par <a href="http://www.dailymotion.com/iGEM_Lyon_2011" target="_blank">iGEM_Lyon_2011</a></i> <br />
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<p style="margin-top:8%;text-align :center"><br />
Nous avons été interviewé par la radio <a href="http://www.rcf.fr/rubrique.php3?id_rubrique=18" target="_blank"> RCF </a> (Radio Chrétienne Française),<br />
le 21 juillet. <br />
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<br><h5>A la télévision </h5><br/><br />
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<div class="cadre" ><br />
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<iframe frameborder="0" width="260" height="180" src="http://www.dailymotion.com/embed/video/xl2937"></iframe><br />
<br /><br />
<a href="http://www.dailymotion.com/video/xl2937_f3-cobalt-buster-vob_tech" target="_blank">On FRANCE 3</a><br />
<i>par <a href="http://www.dailymotion.com/iGEM_Lyon_2011" target="_blank">iGEM_Lyon_2011</a></i><br />
<br/> <br />
La chaîne régionale <a href="http://rhone-alpes.france3.fr/" target="_blank"> FRANCE 3 </a> nous a filmé le mardi 6 Septembre, et le reportage a été diffusé au journal télévisé du soir.<br><br><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Project/AchievementFr
Team:Lyon-INSA-ENS/Project/AchievementFr
2011-09-21T23:43:13Z
<p>Suxiaohui: </p>
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</style><br />
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<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Project/Achievement">English version </a> <br />
</div><br />
<br />
<div class="contenugrand2";><br />
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<br />
<br><br><br/><br/><br />
<p id="top"> <font color="green" size="6"><br />
Objectifs atteints<br><HR><br />
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</font><br />
</p><br />
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<br><br> <br />
<p style="line-height:1.5em;text-indent:0%;"><br />
<ul style="list-style-image:url('https://static.igem.org/mediawiki/2011/4/4d/Chek.JPG'); margin-left:50px;"><br />
<br />
<li>Nous avons vécu une <b>belle expérience humaine</b>, rencontré de nouvelles personnes et <b> on en a bien profité </b>!!</li><br><br><br />
<li>Nous avons acquis de <b>nouvelles connaissances et compétences </b> en ce qui concerne les problèmes de sciences et de logistique.</li><br><br><br />
<li>Nous avons déposé <b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Project/PartFr">2 parts documentées</a></b> dans la registry et montré qu'elles fonctionnent comme prévu.</li><br><br><br />
<li>Nous avons caractérisé une <b><a href="http://partsregistry.org/Part:BBa_K342003"> part pré-existante</a></b>.</li><br><br><br />
<li>Nous avons <b><a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Sponsors/CollaborationFr">collaboré</a></b> avec une autre équipe iGEM: TU Delft.</li><br><br><br />
<li><p style="line-height:1.5em;text-indent:0%;"><br />
Nous avons <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/MainFr">présenté notre projet ainsi que la Biologie de Synthèse</a> sous différentes formes : dans différents <b>sites web</b>,<br/> dans des <b>journaux</b>, à la <b>radio</b>, à la <b>Télévision</b>, … et même <b>réalisé une vidéo du projet en musique</b>; tout ceci ayant pour but d'informer le publique <br/>de manières aussi variées que possible.<br />
</p></li><br><br><br />
<li>Nous avons<b> fait en sorte que notre projet colle au plus près de la réalité </b> en rencontrant des experts afin de prendre en considération <b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Project/IndustrializationFr">la partie industrialisation</a></b>.</li><br/><br/><br />
<li>Nous avons aussi exploré <b><a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Safety/IntroFr"> l'aspect sécuritaire </a></b> de notre projet dans son ensemble.</li><br/><br />
<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Project/AchievementFr
Team:Lyon-INSA-ENS/Project/AchievementFr
2011-09-21T23:42:44Z
<p>Suxiaohui: </p>
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<style type="text/css";><br />
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</style><br />
<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Project/Achievement">English version </a> <br />
</div><br />
<br />
<div class="contenugrand2";><br />
<br />
<br />
<br><br><br/><br/><br />
<p id="top"> <font color="green" size="6"><br />
Objectifs atteints<br><HR><br />
<br/><br />
</font><br />
</p><br />
<br />
<br><br> <br />
<p style="line-height:1.5em;text-indent:0%;"><br />
<ul style="list-style-image:url('https://static.igem.org/mediawiki/2011/4/4d/Chek.JPG'); margin-left:50px;"><br />
<br />
<li>Nous avons vécu une <b>belle expérience humaine</b>, rencontré de nouvelles personnes et <b> on en a bien profité </b>!!</li><br><br><br />
<li>Nous avons acquis de <b>nouvelles connaissances et compétences </b> en ce qui concerne les problèmes de sciences et de logistique.</li><br><br><br />
<li>Nous avons déposé <b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Project/PartsFr">2 parts documentées</a></b> dans la registry et montré qu'elles fonctionnent comme prévu.</li><br><br><br />
<li>Nous avons caractérisé une <b><a href="http://partsregistry.org/Part:BBa_K342003"> part pré-existante</a></b>.</li><br><br><br />
<li>Nous avons <b><a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Sponsors/CollaborationFr">collaboré</a></b> avec une autre équipe iGEM: TU Delft.</li><br><br><br />
<li><p style="line-height:1.5em;text-indent:0%;"><br />
Nous avons <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/MainFr">présenté notre projet ainsi que la Biologie de Synthèse</a> sous différentes formes : dans différents <b>sites web</b>,<br/> dans des <b>journaux</b>, à la <b>radio</b>, à la <b>Télévision</b>, … et même <b>réalisé une vidéo du projet en musique</b>; tout ceci ayant pour but d'informer le publique <br/>de manières aussi variées que possible.<br />
</p></li><br><br><br />
<li>Nous avons<b> fait en sorte que notre projet colle au plus près de la réalité </b> en rencontrant des experts afin de prendre en considération <b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Project/IndustrializationFr">la partie industrialisation</a></b>.</li><br/><br/><br />
<li>Nous avons aussi exploré <b><a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Safety/IntroFr"> l'aspect sécuritaire </a></b> de notre projet dans son ensemble.</li><br/><br />
<br />
</ul></p><br />
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{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Project/StategyFr
Team:Lyon-INSA-ENS/Project/StategyFr
2011-09-21T23:38:35Z
<p>Suxiaohui: </p>
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.firstHeading{background-image: url(https://static.igem.org/mediawiki/2011/0/0a/Banniere_centrale_nucleaire.jpg);background-repeat: no repeat;}<br />
</style><br />
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<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/e/ef/Drapeau-anglais.gif"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Project/Ethics">English version </a> <br />
</div><br />
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<div class="contenugrand2"><br />
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<h1><font color="green"> Une course entre deux stratégies différentes afin d'obtenir la première part </font></h1> <HR><br />
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<p style="line-height:1.5em; margin-right : 5%"><br />
Chez <i>E. coli</i> le système de production de curli est organisé en deux opérons divergents avec les gènes csgA, csgB et csgC d'un côté et csgD, csgE, csgF et csgG de l'autre. La propriété recherchée, l'adhésion par l'intermédiaire de curli, peut être réalisée par l'intermédiaire de deux voies différentes: la création d'une voie de synthèse indépendante de curlis (première option), ou en activant la voie de synthèse des curlis existante présente dans la plupart des souches de laboratoire <i>E. coli</i> (deuxième en option).<br />
<br/> <br/><br />
</p><br />
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<p style="line-height:1.5em; margin-right : 5%">Pour atteindre la première option consistant en la création d'un seul opéron curli indépendante, nous avons essayé deux méthodes en parallèle: une approche complètement synthétique, et une méthode classique impliquant une étape de mutagenèse afin de se débarrasser de trois sites de restriction internes à la part et non conformes au règlement iGEM, suivie d'une étapes de PCR puis de ligations.<br />
<br/><br />
</p><br />
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<ul style="list-style-type:circle;margin-left:5%;"> <br />
<li style="line-height : 1.5em; margin-right : 5%"> <b>La première approche</b> consiste à commander toute la part à une entreprise privée (GeneCust).</li><br />
<br/><br />
<li style="line-height : 1.5em; margin-right : 5%"> <b>La seconde approche</b> consiste à faire directement la synthèse sur paillace, cette approche comporte trois étapes: premièrement une amplification par PCR de chacune des sous-parts, puis une étape de mutagenèse pour supprimer tous les sites naturels EcoRI ou Pst1 présents dans les sous-parts, et enfin une étape de ligation de ces parts.</li><br />
<br/><br />
</ul><br />
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<p style="line-height:1.5em; margin-right : 5%"><br />
Les deux approches ont été initiées en même temps. La seconde nous a permis d'obtenir une amplifications par PCR de taille correcte et une construction complète de Prcn-csgBAEFG, malheureusement l'analyse du séquençage a révélé des mutations inattendues qui n'ont pas été enlevées avant la réception de l'ensemble de la part synthétisée par GeneCust. En bref, la stratégie "clic et commande" a gagné la course!<br />
</p><br />
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<p style="line-height:1.5em; margin-right : 5%"><br />
<p> <font color="green" size="5"><br />
Amélioration de la Souche<br><HR><br />
</font><br />
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<p style="line-height:1.5em; margin-right : 10%"><br />
<p> <font color="green" size="3"><br />
Rendre notre souche auxotrophe<br><br />
</font><br />
</p><br />
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<p style="line-height:1.5em; margin-right : 5%"><br />
Afin d'éviter la dispersion des souches et les problèmes que les OGM peuvent engendrer, nous visons à rendre notre souche dépendante de certaines conditions de culture. Nous avons décidé de rendre notre souche auxotrophe, c'est à dire déléter un gène de biosynthèse d'un acide aminé. Grâce à un "knock out", la souche aurait besoin d'un milieu de culture contenant l'acide aminé manquant et mourrait en l'absence de ce dernier.<br />
<br/><br />
En pratique, nous aurions utilisé le kit "Quick & Easy <i>E.coli</i> Gene Deletion Kit". Nous aurions conçu une séquence avec un gène de résistance aux antibiotiques tels que la Tétracycline, certains accessoires fournis par le kit et une séquence homologue du gène que nous voulons inactiver. Par recombinaison homologue, la séquence entière aurait été insérée dans le gène. Enfin par excision, la résistance pourrait être enlevée afin d'éviter des problèmes éthiques. <br/><br />
Ces modifications auraient nécessité plusieurs semaines de travail, elles restent donc à l'état de projet pour le moment.<br />
<br />
<br/> <br/><br />
</p><br />
<p style="line-height:1.5em; margin-right : 10%"><br />
<p> <font color="green" size="3"><br />
Insertion d'un transporteur de gène directement dans le gène de la pompe d'efflux<br><br />
</font><br />
</p><br />
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<p style="line-height:1.5em; margin-right : 5%"><br />
Nous pourrions avoir deux problèmes avec notre souche: <br />
</p><br />
<br/><br />
<br />
<ul style="list-style-type:circle;margin-left:5%;"> <br />
<li style="line-height : 1.5em; margin-right : 5%">Les caractéristiques du transporteur sont situés sur un plasmide qui pourrait ne pas être stable </li><br/><br />
<li style="line-height : 1.5em; margin-right : 5%"> Il y a un gène de résistance à la kanamycine dans le chromosome (inactivant le gène de la pompe d'efflux).</li><br/><br />
</ul><br />
<br />
<p style="line-height:1.5em; margin-right : 5%"><br />
Une seule solution unique à ces deux problèmes: il faudrait insérer les fonctionnalités du transporteur dans le gène de la pompe d'efflux.<br/><br />
Nous utiliserions le même Kit que précédemment mentionné, mais nous n’utiliserions pas un gène de résistance à un antibiotique, que nous retirerions après coup, mais directement le gène du transporteur NiCoT. Les souches répondant au cobalt (Co) seraient alors sélectionnées.<br />
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<img src = "https://static.igem.org/mediawiki/2011/3/3c/Design_Auxotrophie.jpg"; width=80%><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Sponsors/Collaboration
Team:Lyon-INSA-ENS/Sponsors/Collaboration
2011-09-21T23:37:40Z
<p>Suxiaohui: </p>
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<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
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<img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Sponsors/CollaborationFr">Version Fran&ccedil;aise</a><br />
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The <a href="https://2011.igem.org/Team:TU-Delft"> TU Delft team </a> also works on a part which can provide stickiness to E. coli. Thus, we have decided to collaborate to improve the results and characterization of our respective parts.<br />
Their project is about mussel glue and protein responsible of the attachement of mussel to rocks. The strength of this adherence, transposed to bacteria, could be an efficient way to fix bacteria on a support for various purposes, like the production of biofuels.<br />
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<p style="line-height : 1.5em; margin-right: 5%; margin-left: 3%"><br />
<br/><br />
After an exchange of e-mails and a visioconference, we were able to find different ways to help each other.<br/><br/><br />
<br />
We offered to reproduce each other's experiments, to validate the repeatability of our characterization tests. But also...<br/><br/><br />
<br />
</p><br />
<br />
<ul style="list-style-type:circle;margin-left:10%;margin-right: 5%"> <br />
<b>What we did for them</b><br/><br/><br />
<br />
<li style="line-height : 1.5em"> Both team had issues with a promoter existing in the registry, we have decided to synthesise a strong promoter (trc promoter) and to provide it to TU Delft team. </li><br />
<br/><br />
<li style="line-height : 1.5em"> We have shared our stickiness characterization protocol in 24 well plate and by violet crystal in order to help them characterize their part. </li><br />
<br/><br />
<br />
<b>What they did for us</b><br/><br/><br />
<br />
<br />
<br />
<br />
<li style="line-height : 1.5em"> <br />
With these protocols, they are able to repeat our experiment to confirm our results. </li><br />
<li style="line-height : 1.5em"> They provided us an access to their confocal microscope to get some detailed pictures of our sticky strain. </li> <br />
<li style="line-height : 1.5em"> They offered us to use their model of adherence. However, after examining it, we realized this model was not adapted to our project. </li> <br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Sponsors/Acknowledgements
Team:Lyon-INSA-ENS/Sponsors/Acknowledgements
2011-09-21T23:37:29Z
<p>Suxiaohui: </p>
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Special thanks for special people </font><br><HR><br />
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First, thanks to our patners, without them the project wouldn't have been possible. <br/><br/><br />
We would also like to thank all the person who helped us with our project : <br/><br/><br />
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<ul style="margin-left:10%; line-height : 1.5em;list-style-type:circle;list-style-position: outside;"><br />
<li><p style="line-height:1.5em;"><u>UMR5240 CNRS-UCBL-INSA-Bayer Crop Sciences</u>: Microbiologie, Adaptation et Pathogénie, particularly, <i>Véronique, Sylvie, Julien and Agnès </i> for their help.</p></li> <br/><br />
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<li><p style="line-height : 1.5em;"><u>The University Claude Bernard</u>, and especially the <i>Laboratory of microbial ecology </i></li> <br/><br />
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<li><p style="line-height : 1.5em;"><u>UMR203 INRA-INSA</u>: Biologie Fonctionnelle, Insectes et Interactions.</li> <br/><br />
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<li><p style="line-height:1.5em;"> <u>The Direction of Communication of the INSA de Lyon</u>, especially <i>Amélie Fioretta</i> and <i>Nathalie Falloni</i> for the communication relative to INSA.</li> <br/><br />
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<li><p style="line-height:1.5em;"><i>Joelle Pornin</i> <u>charged of the communications at the ENS</u> for the communications relative to the ENS.</li> <br/><br />
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<li> <p style="line-height : 1.5em;"><i>Olivier Brette</i>, from <u>Humanity Department of INSA de Lyon</u>, for his advices in the economic analysis of the project.</li> <br/><br />
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<li> <p style="line-height : 1.5em;"><i>Jean-Francois Pageaux</i>, from <u>Biosciences Department of INSA de Lyon</u>, for his advices regarding statistics.</li> <br/><br />
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<li><p style="line-height : 1.5em;"><i>Nathalie Taillendier</i>, who makes the order of all the products we use.</li> <br/><br />
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<li><p style="line-height : 1.5em;"><i>Bruno Cedat</i> from the <u>Rovaltain Center</u>, technopole implied in the environmental protection.</li> <br/> <br />
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<li><p style="line-height:1.5em;"><i>Chun Chau Sze</i> from the <u>NTU Laboratory</u>, who gave us a fluorescent strain (MG1655) and allowed us to use it for our experiment in the iGEM competition.(<a href="http://www.ncbi.nlm.nih.gov/pubmed/18926860">J Microbiol Methods. 2009 Feb;76(2):109-19. Epub 2008 Sep 24 </a>)</p><br />
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<p style="margin-left:5%; line-height : 1.5em;"><br />
For some aspects of our communication, thanks to <i>Amanda Lo Van</i> for editing the video of the "CROSS de l'INSA de LYON" and <i>France3 Rhône-Alpes, Le Progrès, Radio RCF, Viva,</i>... for broadcasting our project. <br/><br/><br />
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Thank to <a href="https://2011.igem.org/Team:TU-Delft">TU Delft</a> for their collaboration and especially <i>Krijn Warringa</i> for making it possible.<br/><br/><br />
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Then, our project, close to industrial purposes, led us to deal with people from firms connected to the nuclear power plant industry. We are very grateful to the <i>Tricastin nuclear power plant, Centraco, Michel from the Bugey Nuclear Power Station and Assystem </i> for answering our questions and helping us to get our project closer to reality.<br/><br/><br />
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To conclude, we have to dedicate a really big thanks to the team INSA-LYon iGEM (2010)(check their <a href="https://2010.igem.org/Team:INSA-Lyon"> wiki</a>). They engaged for the first time Lyon and its prestigious schools into the iGEM competition. Without them, the Lyon Biosciences team wouldn't exist. <br/><br />
Using their experience, we learned a lot and understood all the benefits to collaborate wih other people.<br />
In this aim, we create a partnership between the INSA de Lyon and the ENS de Lyon, everyone contributing by different ways to an unique project. <br/><br />
But the gathering between these two schools wouldn’t have been possible without the work of some of our supervisors! So, as students and advisors, we would like first to thank them for all the efforts! This collaboration is really rewarding for all the members of the team, as on the scientific side than the human side. <br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week14
Team:Lyon-INSA-ENS/Realisation/Week14
2011-09-21T23:36:54Z
<p>Suxiaohui: </p>
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<h1 style="color: white;"> Week 14 </h1> <br />
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<p style="text-align : center"> <small> From Monday the 19th of September to Wednesday the 23th of September 2011 </small> </p><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week13
Team:Lyon-INSA-ENS/Realisation/Week13
2011-09-21T23:36:42Z
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<p style="text-align : center"> <small> From Monday the 12th of September to Sunday the 18th of September 2011 </small> </p><br />
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<h6 style="text-align :left"> Monday </h6> <HR><br />
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/><br />
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Extraction of PHL1414+pIG3, PHL1414+pIG16 (clone 1,2,3) , MC4100+piG34 200µl (clone 1,2), MC4100+pIG34 800µl (clone 1, 2).<br/><br/><br />
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Digestion by EcoRI and PstI.<br/><br/><br />
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Gel electrophoresis to verify the ligations. Inserts have the good length for pIG3, pIG16 so the transformation went well.<br />
Inserts of pIG34 200µl clone 1 and pIG34 800µl clone 1 are good so the ligation went well. We’re keeping in collection the pIG34 200µl clone 1 as pIG34 in the plasmids’ collection and MC400+pIG34 as S32 in the strains’ collection.<br/><br/><br />
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Cloning and transformation of rcn-ompR234 + pSB1C3 in MC4100.<br/><br/><br/><br />
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<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/><br />
Plating of S17 from the collection to extract a bigger quantity of pIG25 (pSB1C3 containing rcn-csgBAEFG).<br/><br/><br />
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Storage of the correct clone of:<br/><br />
PHL1414 + pIG3 (S30)<br/><br />
PHL1414+pIG16 (S31) <br/><br />
MC4100 + pIG34 (S32) <br/><br/><br />
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Storage of rcn-OmpR in psB1K3 (pIG34) from the previous miniprep. <br/><br/><br/><br />
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<FONT COLOR="purple"> <b> Microscopy Test </b> </FONT> <br/><br/><br />
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<b>Preparation</b><br/><br />
Sterilization of microscope cover slips in ethanol during a few minutes, then rinsed in sterilized water. <br/><br/><br />
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<b>Test</b> <br/><br />
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Preparation of plates for the following strains (one repetition):<br/><br />
-PHL1414/piG3 AmpR = negative control without Co and with Co 25µM<br/><br />
-PHL1414/piG16 AmpR without Co, Co 10µM, Co 25µM, Co 50µM and Co 100µM<br/><br/><br />
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Incubation at 30°C overnight (during 16 hours).<br/><br/><br/><br />
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<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
Pouring of 9 LB+Amp plates<br/><br/><br />
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Dilution of 5µL pIG25 (371.4 ng/µL) into 25µL water to obtain 3 additional tubes at concentration of about 50ng/µL for sequencing. <br/><br />
Sequencing (pIG25 and pIG27) sent. <br/><br/><br />
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<h6 style="text-align :left"> Tuesday </h6> <HR><br />
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/><br />
Purification and culture of 2 clones from each Petri dish of the transformation of rcn-ompR234 + pSB1C3 in MC4100.<br/><br />
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Extraction of piG33 800µl 1 and 2, and pIG33 200µl 1 and 2.<br/><br />
Digestion by EcoR1, Pst1 and gel electrophoresis for verification.<br/><br/><br/><br />
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<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/><br />
Start of a 5mL liquid culture of S17 in LB+Cm medium.<br/><br />
Minipreps of this culture to extract pIG25 in larger quantity. Digestion (E+P) and electrophoresis for control : the insert of pIG25 is not good so there was a mistake the first time it was controlled. More or less 500bp are missing. Back to the lab, let’s try to built it again to have it on time.<br/><br />
The inserts pIG33 800µl 1 and 200µl 2 are good. pIG33 800µl 1 becomes pIG33 and is put in the plasmids’ collection.<br/><br />
MC4100+pIG33 = S33 is stored.<br />
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<FONT COLOR="red"> <b>Strain construction </b> </FONT> <br/><br/><br />
The Ptrc strong promoter has been received from Genecust in pBluescriptIISK+: transformation in MC4100 and plating on LB+Amp medium.<br/><br/><br />
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Digestion by E+P of this plasmid and ligation into pSB1C3 overnight<br/><br/><br />
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Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3 (pIG25) <br/><br />
Plating of PHL916 on LB ( sureE strain that should not recombine DNA ) in case the transformation of pIG25 into MC4100 fails. <br/><br/><br />
Digestion of pIG20 with E and P.<br/><br />
Gel electrophoresis for control of the insert → good<br/><br/><br />
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Cloning of rcn-csgBAEFG in pSB1C3, transformation in pSB1C3.<br/><br/><br/><br />
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<FONT COLOR="purple"><b> Adherence Test - pRcn-OmpR234 Characterization </b></FONT> <br/><br/><br />
Start of a rcn-OmpR234 24 well plate with :<br/><br />
S27 ( rcn in pSB1C3 ) without cobalt and with cobalt (25µM). <br/><br />
S33 ( rcn-OmpR234 in pSB1C3 ) with increasing concentration of cobalt (0, 10, 25 and 100 µM).<br/><br/><br/><br />
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<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
Plating of PHL644, PHL1256, PHL628 to be sent to other research teams, on request.<br />
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<h6 style="text-align :left"> Thursday </h6> <HR><br />
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<FONT COLOR="red"> <b>Strain construction </b> </FONT> <br/><br/><br />
None of the transformation of the day before has grown. Let’s try it again.<br/><br/><br/><br />
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<FONT COLOR="purple"><b> Fluorescence Test - rcn Characterization </b></FONT> <br/><br/><br />
Characterization of rcn-gfp with the kinetic studie.<br/><br />
3 people are repeating the same 4 erlen of culture with the M63G medium:<br/><br />
- NM522/p157 + spc + 25µM Co<br/><br />
- NM522/p157<br/><br />
- NM522/p115 + spc + 25µM Co<br/><br />
- NM522/p115 + spc<br/><br />
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1 ml of spc (spectinomycine) is added<br/><br />
The culture is sed by 0,5mL of a overnight preculture.<br/><br/><br />
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<FONT COLOR="purple"><b> Adherence Test - 18A-0mpR234 Characterization</b></FONT> <br/><br/><br />
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Start a 24 well plates in M63G medium + Amp with PHL1414/piG3 (negative control) and PHL1414/piG16.<br/><br />
Incubation at 30°C for 48h.<br/><br/><br />
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<h6 style="text-align :left"> Friday </h6><HR><br/><br/><br />
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/><br />
The transformations from Wednesday have grown : selection of 5 clones for pIG36 and Ptrc +Cm, 10 for pIG25.<br/><br/><br />
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Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of all clones for pIG25, 4 for pIG36, 2 for Ptrc-Cm ( the other cultures had not grown ). One correct clone is found for pIG25 and pIG36, none is correct for Ptrc-Cm.<br/><br/><br />
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<FONT COLOR="purple"> <b> Adherence Test - pRcn-OmpR234 Characterization </b> </FONT> <br/><br/><br />
Revealing of the plate : no effect from rcn-ompR234 is visible compared to the controls. <br/><br/><br />
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<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
Dissolution of 0.11g of Ampiciline sodium salt ( Sigma A0166-5G) into 10mL water and filtration to obtain a solution of Amp at 10mg/mL.<br />
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<h6 style="text-align :left"> Saturday </h6><HR><br/><br/><br />
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<FONT COLOR="blue"><b>Plasmid and strain Collection</b></FONT> <br/><br/><br />
Storage of the correct clones and extracted plasmids from the previous day : S34 (NM522 + pIG25), S35 (NM522+pIG36), pIG25, pIG37 (=pIG36 after transformation and extraction ).<br/><br/><br />
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<FONT COLOR="purple"> <b> Adherence Test - 18A-OmpR234 Characterization </b> </FONT> <br/><br/><br />
Revealing of the plate : ompR234 doubles the adherence compared to negative controls.<br/><br/><br />
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<h6 style="text-align :left"> Sunday </h6><HR><br />
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/><br />
Miniprep (QIAGen), digestion (Fermentas E+P) and electrophoresis of the last 3 clones for Ptrc-Cm.<br/><br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week12
Team:Lyon-INSA-ENS/Realisation/Week12
2011-09-21T23:36:31Z
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<h1 style="color: white;"> Week 12 </h1> <br />
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<p style="text-align : center"> <small> From Monday the 5th of September to Friday the 9th of September 2011 </small> </p><br />
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<h6 style="text-align :left"> Monday </h6> <HR><br />
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<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<b>Miniprep</b> of three individual clones of MC4100/piG6 and MC4100/piG2 isolated on 09/02 in order to chek the presence of the right plasmid.<br/><br />
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<FONT COLOR="purple"> <b>Results of Flocculation Test and New Test</b> </FONT> <br/><br/><br />
Crystal Violet coloration for the series of 09/02.<br/><br/><br />
<br />
Start of a fourth test in M63G medium with :<br/><br />
-MC4100/piG6 (AmpR) without Co and with Co 10µM (negative controls)<br/><br />
-MC4100/piG2 (AmpR) without Co and with Co 10µM, 25µM, 50µM, 100µM.<br/><br />
(three repetitions)<br/><br/><br />
<br />
These liquid cultures are let for two days at 30°C and low stirring (75).<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Adherence Test – pRcn-csgBAEFG Characterization</b> </FONT> <br/><br/><br />
Start of three replica of 24 well plates in M63G medium + Amp with MC4100/piG6 without Co and with Co 10µM<br/> (negative control) and MC4100/piG2 without Co and with Co 10µM, 25µM, 50µM, 100µM.<br/><br />
Incubation at 30°C for 48h.<br/><br/><br />
<br />
<br />
Revealing of the well plates started on 09/02.<br/><br />
Measuring OD600 and Crystal Violet coloration.<br/><br />
<br />
<br />
<br/> <br/><br />
<br />
</p><br />
<br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/> <br/> <br />
<br />
<br />
<br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/3/37/2011-03-17_10-26-29.jpg" width="180px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em;margin-left:30%"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<b>TSS transformation</b> of MC4100 with pIG27 (Cm) or pIG16 (Amp).<br/><br/><br />
<br />
<b>Digestion</b> and <b>electrophoresis</b> of the previously extracted DNA of MC4100+pIG6 clones and MC4100+pIG2 clones. Comparison made with the parts put in the collection.--> Verification is OK.<br/><br/><br/><br />
<br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"> <b>Strain Collection</b> </FONT> <br/><br/><br />
Storage of the strains : <br/><br />
-<b>S25 = MC4100/piG6</b> (clone #3)<br/><br />
-<b>S26 = MC4100/piG2</b> (clone #3)<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Microscopy Test</b> </FONT> <br/><br/><br />
<b>Preparation</b><br/><br />
Start of 5mL liquid cultures of PHL1414/piG2 (AmpR), PHL1414/piG6 (AmpR) from solid cultures (09/02)<br/><br />
and S19/p127 (KanR/AmpR), S19/p116 (KanR/AmpR) from solid cultures (08/23).<br/><br/><br />
<br />
<b>Test</b><br/> <br />
Preparation of plates for the following strains (two repetitions):<br/><br />
-S19/p127 KanR/AmpR<br/><br />
-S19/p116 KanR/AmpR = negative control<br/><br />
(18A-OmpR234 Characterization)<br/><br />
-PHL1414/piG6 AmpR = negative control without Co and with Co 10µM<br/><br />
-PHL1414/piG2 AmpR without Co and with Co 10µM, Co 25µM, Co 50µM, Co 100µM<br/><br />
(pRcn-csgBAEFG Characterization)<br/><br />
Incubation at 30°C overnight (for 15 hours).<br/><br/><br/><br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/><br />
Liquid culture of strains for the test.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/><br />
Culture of S22 in LB with Cm (NM522/pIG33) for conservation.<br />
</p><br />
<br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Results of tuesday transformations : no clone on the negative controls, several on the tests. Isolation of 4 clones per transformation in solid and liquid culture.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Results of Microscopy Test</b> </FONT> <br/><br/><br />
Observation of the different slides with a fluorescent microscope.<br/><br />
There is a problem with S19/p127 and S19/p116 so we start again the same test for these two strains.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Results of Adherence Test - pRcn-csgBAEFG Characterization</b> </FONT> <br/><br/><br />
<br />
Revealing of the well plates started on Monday.<br/><br />
Measuring OD600 and Crystal Violet coloration.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/><br />
CFA test (4 plates) to check stickiness of strains with PHL1414, MC4100, NM522, S18, S20, S21, S23, S24, S25, S26 (10µL of overnight culture)<br/><br/><br/><br />
</p><br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/><br />
Pouring of Amp plates.br/><br/><br />
No growth of S22 (Too much antibiotic), new culture with the right dose of antibiotics.<br />
<br />
</p><br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Extraction with Qiagen’s kit of:<br/><br />
- MC4100 pIG27 clones 1, 2, 3 and 4<br/><br />
- MC4100 pIG16 clones 1, 2, 3 and 4<br/><br />
- MC4100 pIG33 clones 1, 2 and 3<br/><br />
<br />
E+P <b>digestion (Fermentas) and electrophoresis</b> of all the isolated clones for the following transformations : MC4100+pIG27, MC4100 + pIG16, MC4100 + rcn-ompR234.<br/><br />
All clones are correct for pIG27 and pIG16. No clone is correct for rcn-ompR234 -> detection of an error in the ligation. <br/><br/><br />
<br />
<div style="border:1px solid black;margin-left:7%; width : 600px"><br />
<img src="https://static.igem.org/mediawiki/2011/3/3e/Gelwiki12.jpg" width="600px"/> <br />
</div><br />
</p><br />
<br />
<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"> <b>Plasmid Collection</b> </FONT> <br/><br/><br />
Storage of clone 1 for pIG27 and pIG16.<br/> <br />
Plating of those same clones on LB+antibiotic (Cm and Amp respectively ).<br/><br/><br/> <br />
</p> <br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Results of Microscopy Test</b> </FONT> <br/><br/><br />
Observation of the different slides with a fluorescent microscope.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Result of Flocculation Test</b> </FONT> <br/><br/><br />
Test started on 09/05.<br/><br />
Crystal Violet coloration for the three series.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>CFA Test</b> </FONT> <br/><br/><br />
No significative results with this test<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/><br />
No growth of S22 again. Culture of S22 with and without antibiotics.<br />
</p><br />
<br />
<br />
<br />
<br/><br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Friday </h6><HR><br />
<br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/><br />
Construction of the composite part rcn+ompR234 in the chassis MC4100. Because both part are in the plasmid pSB1C3, the ligation 3A is done with the plasmid Kanamycine :<br/><br />
pIG27 (ES) + pIG11 (XP) + pSB1K3 (EP)<br/><br/><br />
<br />
An electrophoresis gel is done to verifie the digestion. Everything is ok.<br/><br />
<br />
A transformation following the TSS transformation protocol is done and the Petri dishes are incubated overnight.<br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"> <b>Sequencing</b> </FONT> <br/><br/><br />
<b>Nanodrop quantification</b> of the previous pIG27 miniprep : 48.0 ng/µL.<br/><br />
Preparation of tubes for sequencing (30µL at approximately 50-100 ng/µL).<br/><br />
The pIG27 miniprep is used as such.<br/> <br />
pIG25 is diluted from 5µL of the collection (371.4 ng/µL) with 25µL water.<br/><br/><br/><br />
</p> <br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/><br />
No growth of S22 neither with antibiotic nor without antibiotic. We drop the conservation of the plasmid and restart the construction of pIG33 (fusion between promoter Rcn and ompR234).<br />
<br />
</p><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Saturday </h6><HR><br />
<br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br/><br/><br />
Results of the ligation from the 09/09/2011<br/><br />
- Petri Dish 800µl E.coli → 40 clones<br/><br />
- Petri dish 200µl E.coli → 12 clones<br/><br/><br />
<br />
4 clones of each dish are purified.<br/><br/><br />
<br />
Results of the transformation of pIG3 in PHL1414:<br/><br />
- Witness - (PHL1414 alone) → nothing<br/><br />
- PHL1414 + pIG3 → 1 clone<br/><br />
- PHL1414 +pIG16 → a lot of clones<br/><br/><br />
<br />
Purification of the clone PHL1414+pIG3 and purification of 4 clones of PHL1414+pIG16<br />
</p><br />
<br />
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<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week11
Team:Lyon-INSA-ENS/Realisation/Week11
2011-09-21T23:36:18Z
<p>Suxiaohui: </p>
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<img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week11Fr">Version Fran&ccedil;aise</a><br />
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<div class="contenugrand2";><br />
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<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 11 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 29th of August to Friday the 2nd of September 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<br />
<b>Nanodrop quantification</b> of several midipreps :<br/><br />
p116 : 726.3 ng/µL<br/><br />
piG30 : 251.6 ng/µL<br/><br />
p115: 488.4 ng/µL<br/><br />
p10 : 276.2 ng/µL<br/><br />
p127 : 673.5 ng/µL<br/><br/><br />
<br />
<b>Digestion</b> and electrophoresis of p115 (from S19 and NM522), p116(from S19 and NM522),<br/><br />
p127(from S19 and NM522), p10(from NM522), p157 (from S19 and NM522), 2 clones each.<br/> <br/><br />
<br />
Start of solid cultures of S18 and S21 from the collection<br/><br/><br />
<br />
<b>Plating</b> of PHL1414 from the collection <br/> <br/><br/><br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Results of Flocculation Test</b> </FONT> <br/><br/><br />
</p><br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/8/8a/Wikiweek11.jpg" width="180px"/> <br />
</div><br />
<br />
<br/><br/><br/><br />
<p style=" line-height : 1.5em;margin-left :30%; text-indent:0%"><br />
Test started on 08/26.<br/><br />
No result in the LB/2 medium.<br/><br />
In the M63G medium, a biofilm is obtained for MC4100/piG2 and there is no biofilm for the negative control. For this series, Crystal Violet coloration.<br/><br/><br/><br/><br/><br/><br />
<br />
<br />
</p><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Adherence Test Preparation</b> </FONT> <br/><br/><br />
Start of 5mL liquid cultures in M63G medium of PHL818, MC4100, MC4100/piG2 (AmpR)<br/> <br />
and MC4100/piG6 (AmpR).<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Results of Adherence Test</b> </FONT> <br/><br/><br />
Revealing of the 24 well plates started on Friday.<br/><br />
Measuring OD600 (only for the M63 well plate because the results of the other plate weren't exploitable) and Crystal Violet coloration.<br/><br/><br/><br />
</p><br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Microscopy Test Preparation</b> </FONT> <br/><br/><br />
Sterilization of glass slides at 180°C for 4 hours.<br/><br/><br/><br />
</p><br />
<br />
<br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/> <br/> <br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Start of 5mL liquid cultures for transformations : PHL1414, MC4100, MC4100+pIG2<br/><br/><br />
<b>TSS Transformation</b> and plating of PHL1414 with pIG2, MC4100 with R1 and R1+p150,<br/>MC4100+pIG2 with p150.<br/><br/><br/><br />
<br />
<br />
</p><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Flocculation Test</b> </FONT> <br/><br/><br />
<b>Preparation:</b><br/><br />
Start of 5mL liquid cultures in M63G medium + Amp of S18 and S21 from solids cultures (08/29).<br/><br/><br />
<br />
<b>Test:</b><br/><br />
Start of a second test in M63G medium with:<br/><br />
-MC4100/piG6 (AmpR) = negative control without Co and with Co 10µM.<br/><br />
-MC4100/piG2 (AmpR) without Co, with Co 10µM and with Co 100µM.<br/><br />
-S21 (AmpR) = negative control without Co and with Co 10µM.<br/><br />
-S18 (AmpR) without Co, with Co 10µM and with Co 100µM.<br/><br />
These liquid cultures are let over the weekend at 30°C and low stirring (85).<br />
<br />
<br />
<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Adherence Test</b> </FONT> <br/><br/><br />
Start a repetition of two 24 well plates in M63G medium with PHL818 (positive control),<br/><br />
MC4100 (negative control), MC4100/piG6 (pUC18) without Co and with Co 10µM,<br/><br />
MC4100/piG2 (rcn - csgBAEFG in pUC18) without Co and with Co 10µM.<br/><br />
Incubation at 30°C for 48h.<br />
<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Fluorescence Test</b> </FONT> <br/><br/><br />
Measuring the spectro fluorescence and the OD600 for the tubes from Thursday 08.25.<br/><br />
</p><br />
<br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Obtention of a few clones on all previous transformations except on MC4100+R1 ( too many clones, suspicious )<br/><br/><br />
<br />
<br />
<b>TSS Transformation</b> and plating of PHL1414 with pIG6 (Amp), MC4100 with pIG6+p150(Amp+Kan),R1(Cm),<br/> <br />
pIG26(Cm) and pIG26+p150(Cm+Kan).<br/> <br />
Negative controls : PHL1414 and MC4100 on every type of antibiotic previously used. <br/><br/><br />
</p><br />
<br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
The negative control for the transformations of MC4100 on Cm grew : start of several tests (plating on solid medium with Cm, liquid cultures in LB+Cm) to identify the origin of the failure of the negative control.<br/><br />
Isolation of 4 individual clones on other antibiotic selections.<br/><br/><br />
<br />
The tests for the failure of the negative transformation control showed that our starting MC4100 strain was resistant to Cm -> all transformations using this strain are eliminated. <br/><br/><br />
<br />
Because of this problem, new <b>transformation and plating</b> of a new strain MC4100 with piG6 (Amp)<br/><br />
and piG2 (Amp). Negative control : MC4100 on Amp.<br/><br/><br />
<br />
Start of 2.5mL cultures for extraction and checking of the individual clones of PHL1414+piG6 and PHL1414+piG2.<br/><br/><br />
<br />
<b>Miniprep</b> of PHL1414+pIG2 and PHL1414+pIG6 (4 clones each).<br/><br/><br/><br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"> <b> Collaboration </b> </FONT> <br/><br/><br />
<b>Plating</b> of PHL1414+pIG6 and PHL1414+pIG2 on LB+Amp for TUdelft collaboration <br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence Test Preparation </b></FONT> <br/><br/><br />
Start of 5mL cultures for 24 well plates in M63G medium, with 50µL antibiotic if appropriate, from 10µL of saturated cultures : PHL818, NM522, NM522+pIG3 (Amp), NM522+pIG16(Amp).<br/><br/><br/><br />
</p><br />
<br />
<br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/5/50/Plaque_14.jpg" width="180px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em; margin-left:30%"><br/><br />
<FONT COLOR="purple"> <b> Results of Adherence Test – pRcn-csgBAEFG Characterization</b> </FONT> <br/><br/><br />
Revealing of the 24 well plates started on Tuesday.<br/><br />
Measuring OD600 and Crystal Violet coloration.<br/><br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Microscopy Test </b> </FONT> <br/><br/><br />
<br />
Preparation of plates for the following strains (two repetitions):<br/><br />
-S19p127 KanR/AmpR<br/><br />
-S19p116 KanR/AmpR = negative control<br/><br />
(18A-OmpR234 Characterization)<br/><br />
-MC4100/piG2/p150 KanR/AmpR<br/><br />
-MC4100/piG6/p150 KanR/AmpR = negative control<br/><br />
-PHL1414/piG2 AmpR<br/><br />
-PHL1414/piG6 AmpR = negative control<br/><br />
(pRcn-csgBAEFG Characterization)<br/><br/><br />
<br />
Incubation at 30°C overnight (for 23 hours).<br/><br/><br/><br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p> <br />
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<br />
<h6 style="text-align :left"> Friday </h6><HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<b>Digestion</b> of the previous minipreps by E and electrophoresis -> all clones are correct <br/><br/><br />
<br />
<br />
Isolation of three individual clones from the transformations of MC4100/piG6 and MC4100/piG2 (09/01) for a future Adherence Test.<br/><br/><br/><br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"> <b> Plasmid Collection</b> </FONT> <br/><br/><br />
<b>Storage</b> of clone 3 from PHL1414+pIG2 (S23) and clone 2 from PHL1414+pIG6(S24) <br/><br />
Plating of those same clones on LB+Amp medium.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Flocculation Test</b> </FONT> <br/><br/><br />
Start of a third test in M63G medium with :<br/><br />
-MC4100/piG6 (AmpR) without Co and with Co 10µM (negative controls)<br/><br />
-MC4100/piG2 (AmpR) without Co and with Co 10µM, 50µM, 100µM.<br/><br />
(two repetitions)<br/><br />
<br />
-S21 (AmpR) without Co and with Co 10µM (negative controls)<br/><br />
-S18 (AmpR) without Co and with Co 10µM, 50µM, 100µM.<br/><br/><br />
<br />
These liquid cultures are let over the weekend at 30°C and low stirring (75).<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence Test </b></FONT> <br/><br/><br />
Start of <b>24 well plates </b> in M63G : PHL818 (positive control), NM522 (negative control), NM522+piG3(Amp), NM522+piG16(Amp). Two same plates are done by two different operators.<br />
<br/><br/><br />
<br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b> Results of Microscopy Test</b> </FONT> <br/><br/><br />
</p><br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 300px"><br />
<img src="https://static.igem.org/mediawiki/2011/f/fc/Microwiki11.jpg" width="300px"/> <br />
</div><br />
<br />
<br/><br />
<p style=" line-height : 1.5em;margin-left :47%; text-indent:0%"><br />
Observation of the different slides with<br/> <br />
a fluorescent microscope.<br/><br />
Good results obtained for PHL1414/piG6<br/><br />
(negative control)and PHL1414/piG2.<br/><br/><br/><br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
</p> <br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week10
Team:Lyon-INSA-ENS/Realisation/Week10
2011-09-21T23:36:06Z
<p>Suxiaohui: </p>
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<br/><br />
<h1 style="color: white;"> Week 10 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 22th of August to Friday the 26th of August 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<b>QIAGen extraction</b> of NM522 and NM522+pIG4 and electrophoresis : no plasmid is in the strains. <br><br><br />
<br />
A new NM522 strain is found in another lab.<br/><br />
Plating of 10µL of this new NM522 on LB <br/><br/><br />
<br />
<b>Transformation and plating</b> of NM522 on LB with : pUC18 (=pIG6) (3 µL) + Amp, pIG25 (3 µL) + Cm<br/><br />
<b>Transformation and plating</b> of S19 on LB and the antibiotic Amp with : p157 (3 µL) + SpcR, p115 (3 µL) + SpcR, p127 (3 µL) + Kn, p116 (3 µL) + Kn <br/><br/><br/> <br />
<br />
</p><br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Results of Adherence Test - Result of Flocculation Test </b></FONT> <br/><br/><br />
Revealing of 24 well plates and flocculation test from 08.19.11. The (2) has failed. The others plates are quite good.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br><br />
Digested the PCR result with both EcoRI and PstI.<br><br />
Cloned the resulting DNA in pSB1C3.<br><br />
Transformed the result in NM522 strain.<br><br />
<br />
</p> <br />
<br/><br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#5C656F><b>Creating the rcn-ompR234 part</b></FONT> <br><br><br />
Digested both our rcn part and the ompR part from last year's INSA iGEM team with respectively EcoRI, SpeI and XbaI, PstI.<br><br />
Digested pB1C3 with both EcoRI and PstI.<br><br />
3A ligation on those construct.<br><br />
Transformation of a NM522 strain with the result of the ligation.<br><br />
<br />
</p> <br />
<br/><br/><br/><br />
<br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
Both NM522 and NM522/pIG24 grew on LB+Amp ( liquid and solid ) : antibiotic concentration in liquid was not sufficient, a more concentrated solution of Amp (10mg/mL which corresponds to 100X) is made and sterilized by filtration.<br/><br/><br />
Creation of our own LB+Amp plates by plating 200µL of Amp on a LB plate.<br/><br/><br />
Plating of several AmpS strains on the previous plates : PHL818, MC4100, NM522+pIG4, NM522 from the collection, NM522 from the previous LB+Amp plate, a new NM522 found in another lab.<br/><br/><br />
Dissolution of 0.08g of Spectinomycin ( C14H24N2O7 + 2 HCl + 5 H20 ) into 5mL water and filtration to obtain a solution at 10mg/mL.<br/><br/><br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p> <br />
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<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/> <br/> <br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Start of 5mL liquid cultures of two clones for each plates obtained by the previous transformations, in order to check the presence of the right plasmids and start fluorescence tests.<br/><br/><br />
</p><br />
<br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/0/0d/Petri_transfo_24.08.jpg" width="180px"/> <br />
</div><br />
<br />
<br />
<p style=" line-height : 1.5em; margin-left : 32%"><br />
<br/><br />
<b>Transformation and plating</b> of NM522 on LB with : p157 (2 µL) + Spc, p115 (2 µL) + Spc, p56 (2 µL) + Amp, p10 (3 µL) + Amp, pIG30 (2 µL) + Cm, p127 (2 µL) + Kn, p116 (5 µL) + Kn <br> <br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"><b>Strain Collection</b></FONT> <br/><br/><br />
Storage of <b>S20</b>. <br/><br />
<br />
Plating of S20 on home-made LB+Amp dishes and start of 5mL liquid cultures in LB+Amp. <br/><br/><br />
<br />
Start of 50mL liquid culture of NM522/piG6 for a future extraction.<br/><br />
Storage of NM522/piG6 in the collection as <b>S21</b>.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence Test Preparation </b></FONT> <br/><br/><br />
Start of 5mL cultures for 24 well plates in M63G medium, with 50µL antibiotic if appropriate, from 10µL of saturated cultures : PHL818, NM522, NM522+pUC18 (Amp), S18(Amp).<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br><br />
Miniprep on the NM522 growing overnight, results are unclear.<br />
<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#5C656F><b>Creating the rcn-ompR234 part</b></FONT> <br><br><br />
Miniprep on 3 different clones, analysed on gel, one seems to be correct.<br><br />
Supposedly correct clone, and DNA extracted from it stored in collection.<br><br />
<br />
</p> <br />
<br/><br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
Results of the LB+Amp tests from the previous day : MC4100, NM522+piG4, PHL818, the new NM522 don't grow.<br/><br />
NM522 and NM522 from the collection produce a few colonies -> the collection was contaminated by an AmpR strain.<br/><br />
NM522 in the collection (S11) is replaced by the new NM522. <br/><br/><br />
<br />
Test of 2 other batches of LB+Amp dishes by plating the new NM522 on them.<br/><br/><br />
<br />
Plating of NMYO on LB+Kan from an old solid culture.<br/><br />
<br />
</p><br />
<br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Start of 5mL liquid cultures of two clones for each plates obtained by the previous transformations, in order to check the presence of the right plasmids.<br/><br/><br />
<br />
Plating of MC4100 on LB from an old solid culture (07/26) for future transformations.<br/><br/><br />
<br />
<b>Miniprep</b> of : <br/><br />
- S19/p157<br/><br />
- S19/p115<br/><br />
- S19/p127<br/><br />
- S19/p116<br/><br />
<b>Digestion</b> by E and electrophoresis to control the presence of the two plasmids : the plasmid piG16 doesn’t seem to be in these strains!!<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"><b>Plasmid Collection</b></FONT> <br/><br/><br />
<b>Midiprep</b> of pIG6 (=pUC18)<br/><br />
<b>Digestion</b> by E and electrophoresis to control the plasmid : one band at 2.5-3kb which is coherent. <br/><br />
<b>Nanodrop</b> quantification : c=83ng/µL, 260/280=2.02 <br/><br />
<b>Storage</b> of 300µL of the plasmid at -20°C.<br/><br/><br />
<br />
Start of 50mL cultures (500µL antibiotic if necessary and 100µL bacteria ) of NM522 + p10 (Amp),NM522 + p127 (Kan),NM522 + p56 (Amp),NM522 + p157 (Spc),NM522 + p115 (Spc)for future extractions.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence Test - pRcn-csgBAEFG Characterization </b></FONT> <br/><br/><br />
Start of 24 well plate adherence test in M63G medium with PHL818 (positive control), NM522 (negative control), S21+Amp without Co, S21+Amp+Co 0,1mM, S18+Amp without Co, S18+Amp+Co 0,1mM.<br/><br />
Incubation at 30°C for 48h. <br/><br/><br/></p><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Fluorescence Tests </b></FONT> <br/><br/><br />
Measuring the fluorescence and the OD600 of the tubes from the transformation of tuesday.<br/><br/><br/> <br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br><br />
New minipreps on new NM522 clones results are still unclear. Transformation might not have worked.<br />
<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b> Others </b></FONT> <br><br><br />
Result of the test of the 2 batches of LB+Amp : NM522 doesn't grow so they seem efficient.<br><br></p><br />
<br />
Start of a 5mL preculture of NMYO from the previous dish <br><br><br />
<br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Start of 5mL liquid culture of MC4100 from solid culture (08/24).<br/><br/><br />
Start of 5mL liquid culture for fluorescence of : S19/p127, S19/p116, NM522/p127, NM522/p116 in LB/2 and M63G : 30°C and slow agitation<br/><br />
Start of 5mL liquid culture for fluorescence of : S19/p115, S19/p157, NM522/p115, NM522/p157 in LB/2 and M63G : 37°C and fast agitation. For each strain, there are 4 tubes without Cobalt and with 3 concentrations of Cobalt<br/><br/><br />
<br />
<br />
<b>Transformations and plating</b> of MC4100 on LB+Amp with : piG6 (3 µL), piG2 (3µL) and sterile water (3µL) for negative control. <br/><br/><br/> <br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"><b>Plasmid Collection</b></FONT> <br/><br/><br />
<b>Midiprep</b> of p56 and p157.<br/><br />
<b>Digestion</b> by E and electrophoresis to control the plasmid : the obtained bands are coherent for each plasmid.<br/><br />
<b>Nanodrop quantification</b> : <br/><br />
-p56: c = 15,1 ng/µL<br/><br />
-p157: c = 19,3 ng/µL<br/><br />
<b>Storage</b> of 300µL of the plasmid at -20°C.<br/><br />
<b>p56 = piG31</b><br/><br />
<b>p157 = piG32</b><br/><br/><br />
<br />
Start of 50mL liquid cultures (500µL antibiotic if necessary and 100µL saturated cultures (08/24)) of NM522/piG30 (Cm), NM522/p127 (Kan), NM522/p116 (Kan) for future extractions.<br/><br/><br/><br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
<br />
Pouring of Cm+Amp dishes<br/><br/><br />
<br />
Preparation of new Spc at 10mg/mL from 0.15g ( C14H24N2O7 + 2 HCl + 5 H20 ) into 10mL water. <br/><br/><br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Friday </h6><HR><br />
<br />
<br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<b>Digestion</b> of R1, R2, C1, N1, N2 minipreps by E and electrophoresis : <br/><br/><br />
R2 : 2kb, not good <br/><br />
R1 : 2.8kb, validated.<br/><br />
N2 : 2kb, 5kb , not good<br/><br />
N1 : 8kb , not good<br/><br />
C1 : 3kb, 2.5kb , not good<br/><br/><br />
<br />
Plating on LB+Amp of two clones of each transformation (08/25): MC4100/piG6 and MC4100/piG2.<br/><br/><br />
<br />
<b>Miniprep</b> of two clones of : S19/p115, S19/p116, S19/p127, S19/p157, NM522/p115, NM522/p116, NM522/p127, NM522/p157, NM522/p10 to control then the strains we are using for fluorescence tests.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"><b>Plasmid Collection</b></FONT> <br/><br/><br />
<b>Midiprep</b> of: p10, p115, p127, p116 and piG30 (transformed in NM522) for storage in plasmid collection after control.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Flocculation Test </b></FONT> <br/><br/><br />
Start of a <b>flocculation</b> test in two media LB/2 and M63G:<br/><br />
-5mL of medium<br/><br />
-50µL of Amp<br/><br />
-One clone of the transformation plates (08/25): MC4100/piG2 and MC4100/piG6 (negative control)<br/><br />
-Co 10µM for MC4100/piG2<br/><br />
These liquid cultures are let over the weekend at 30°C and low stirring (70).<br/><br/><br/><br />
</p><br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Results of Fluorescence Tests </b></FONT> <br/><br/><br />
Measuring the fluorescence and the OD600 of all the liquid cultures started on Thursday.<br/><br/><br/><br />
</p><br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Results of Adherence Test - pRcn-csgBAEFG Characterization </b></FONT> <br/><br/><br />
Revealing of the 24 well plates started on Wednesday.<br/><br />
Measuring OD600 and Crystal Violet coloration.<br/><br/><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence Test </b></FONT> <br/><br/><br />
Start of <b>24 well plates </b>: <br />
<br/><br />
(1) in M63G : NM522 (negative control),PHL818 (positive control),NM522/piG3 (without cobalt and with 0.1mM cobalt),NM522/p10 (without cobalt and with 0.1mM cobalt),S19(without cobalt,with 0.05 mM cobalt and with 0.1 mM cobalt) and NM522/p56 (without cobalt, with 0.05 mM cobalt and with 0.1 cobalt).<br />
<br/><br />
(2) in LB/2 : NM522 (negative control),PHL818 (positive control),NM522/piG3 (without cobalt and with 0.1mM cobalt),NM522/p10 (without cobalt and with 0.1mM cobalt),S19(without cobalt,with 0.1 mM cobalt and with 0.15 mM cobalt) and NM522/p56 (without cobalt, with 0.1 mM cobalt and with 0.15 cobalt).<br />
<br/><br />
<br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b> Others </b></FONT> <br/><br/><br />
Start of 2X5 mL cultures of NMYO ( 5mL LB, 50µL saturated NMYO, 50µL Kan)<br/><br/><br />
Because no NiCoT plasmid was correct, the transformation was abandonned.<br/><br/><br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
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<br/> <br/><br />
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<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week9"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week11"/><font color="grey"><b>Next Week</b></font></a><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week9
Team:Lyon-INSA-ENS/Realisation/Week9
2011-09-21T23:35:53Z
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<div id="language";><br />
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</div><br />
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<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 9 </h1> <br />
</div><br />
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<br/><br />
<p style="text-align : center"> <small> From Monday the 15th of August to Friday the 19th of August 2011 </small> </p><br />
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<h6 style="text-align :left"> Monday </h6> <HR><br />
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<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<br />
<b>Transformation and plating</b> of NM522 with : pIG3 (3µL), pIG16. (3µL)<br/> <br />
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</p> <br />
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<h6 style="text-align :left"> Tuesday </h6> <HR><br />
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<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Plating of individual or non individual clones from the previous NM522-pIG16 and NM522-pIG3 cultures on LB+amp medium.<br/> Incubation at 37°C overnight.<br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/><br />
Start of 5mL cultures for adherence tests from the collection : <br/><br />
LB : NM522 <br/><br />
LB/2 : NM522, S3, S4, S15, S19 <br/><br />
M63 : NM522, S15, S3, S19, S4, S18 <br />
</p><br />
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</p><br />
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<h6 style="text-align :left"> Wednesday </h6> <HR><br />
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<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
Start of 3x5mL cultures of NM522 in LB medium from 50µL of a saturated NM522 culture for a later transformation.<br/><br />
Start of 5mL cultures of the individual clones plated on the previous day. <br/><br/><br />
<br />
<b>Transformation</b> and <b>plating</b> of NM522 on LB+Amp with : pIG6 (3µL), pIG7 (3µL), pIG24 (2µL), p56 (2µL), p10 (2µL), pIG16+p157 (3µL each), pIG16+p115(3µL each), pIG16+p127(3µL each), pIG16+p116 ( 3µL and 5µL respectively ), pIG25 (1µL). <br/><br/><br />
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<b>Transformation</b> and <b>plating</b> of S19 on LB+Amp with : p157(3µL), p115(3µL), 3 replica each.<br/><br/><br />
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<b>Extraction</b> of pIG16 from S19 with the QIAGen miniprep protocol. <br/><br/><br/><br />
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</p><br />
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<div style="border:1px solid black;float:left;margin-left:3%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/d/de/14.06_plaque.jpg" width="180px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em;margin-left:30%;"><br />
<FONT COLOR="purple"> <b>Adherence Test</b> </FONT> <br/><br/><br />
Start of <b>24 well plate</b> adherence test in M63G medium with PHL818<br/>( positive control ), NM522( negative control ), S18 + Amp + CoCl2<br/>( in increasing concentration ) <br/> <br/><br/><br/><br />
<br />
</p><br />
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<p style=" line-height : 1.5em"><br />
<FONT COLOR="orange"> <b>Others</b> </FONT> <br/><br/><br />
Test of antibiotics : start of 5mL cultures ( 5mL LB, 50µL antibiotic, 10µL saturated NM522) of NM522 with antibiotics to test if the antibiotic is still good for Kan, Tet, Cm and Amp<br/><br />
The bacteria with Amp grow : a new solution is made ( 5x1mL mother solutions at 100mg/mL from solid Amp, and 2x10mL solutions at 100µg/mL by dilution of 10µL of mother solution into 10mL sterile water ).<br />
</p><br />
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<br/><br/><br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br><br />
Launched an overnight PCR using the synthetised rcn-CsgBAEFG part as the template, and primers used during the first try to obtain the csg construct : 3' primer for csgBA and 5' primer for csgEFG<br><br />
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</p> <br />
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</p> <br />
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<h6 style="text-align :left"> Thursday </h6> <HR><br />
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<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
The transformation gave too many bacteria : plating of all the previous transformed strains on a new Petri dish in order to isolate individual clones.<br/><br/><br />
<br />
<b>Digestion</b> of pIG16 (E+P) and electrophoresis : the digestion had failed or the extracted plasmid was not pIG16.<br/><br />
<b>Digestion</b> of pIG16 by E,X,S,P,E+P,X+S : E,X and S show a linearization of the plasmid, P shows nothing, E+P shows a linearization and X+S extracts the part -> P was inactive. <br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Adherence Test Preparation</b> </FONT> <br/><br/><br />
<br />
Start of 5mL cultures for 24 well plates :<br/><br />
M63 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm)<br/><br />
LB/2 : PHL818, NM522/pUC18 (Amp), NM522/pSB1C3(Cm), S17 (Cm), S18(Amp), NM522<br/><br />
LB : NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116, NM522<br/><br/><br />
<br />
<br/><br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br><br />
Revealing of the PCR : it is positive, but the negative control is positive too. PCR re-done.<br><br />
<br />
</p> <br />
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<br />
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<br />
</p> <br />
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<br />
<h6 style="text-align :left"> Friday </h6><HR><br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Transformations and controls for future tests</b> </FONT> <br/><br/><br />
<b>Plating</b> of NM522/pIG16/p157, NM522/pIG16/p115, NM522/pIG16/p127, NM522/pIG16/p116 (who have a double resistance) on their second resistance ( Kan ).<br/><br />
<b>Extraction</b> (QIAgen) and <b>digestion</b>(Fermentas) of these plasmids by E : only one plasmid was in the strains -> the transformations need to be restarted.<br/><br/><br />
<br />
<br />
The NM522/pIG24 (Cm) strain grows on Amp plates and not Cm. Moreover, pIG24 is a linear plasmid and should not have transformed : plating of NM522, NM522/pIG24 on LB+Amp and start of 5mL cultures ( LB+Amp ) of the same strains to test Amp efficiency or contamination of NM522 with an AmpR strain.<br/><br/><br/><br />
<br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"> <b>Adherence and Flocculation Test</b> </FONT> <br/><br/><br />
<br />
Start of <b>24 well plates</b> :<br/><br />
(1) M63 : PHL818 ( positive control ), NM522 and NM522/pUC18( negative controls ), S18 + Co ( in increasing concentration)-> to characterize rcn-csgBAEFG <br/><br />
(2) M63 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak<br/><br />
(3) LB/2 : PHL818, NM522, NM522/pUC18 (with and without cobalt), S17 and S18 ( with cobalt in increasing concentrations )-> to test the leak<br/><br />
<br />
<br><br />
Start of tubes for flocculation : the same as plates (2) and (3).<br><br />
<br />
<br/><br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#1E8B22><b>Re-synthetising csg-BAEFG with standard iGEM restriction sites</b></FONT> <br><br><br />
Revealing PCR results, they show a ~2800 bp DNA band, in accordance with the expected size.<br><br />
<br />
</p> <br />
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</p> <br />
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<br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week8"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week10"/><font color="grey"><b>Next Week</b></font></a><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week8
Team:Lyon-INSA-ENS/Realisation/Week8
2011-09-21T23:35:41Z
<p>Suxiaohui: </p>
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</div><br />
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<div class="contenugrand2";><br />
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<br/> <br/><br />
<br/> <br />
<br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 8 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 1st of August to Friday the 5th of August 2011 </small> </p><br />
<br />
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<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
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<br/><br />
<br />
<br />
<br />
<p style=" line-height : 1.5em; "><br />
<FONT COLOR="purple"><b> Adherence and Flocculation Tests</b></FONT> <br><br><br />
<b>24 well plate</b> with LB/2 or M63 medium to test bacterial adherence in response to cobalt :<br><br />
positive control with an adherent strain PHL818 ( MG1655 malT::Tn10 adr101=OmpR234 ), negative control with a non adherent strain NM522, and our synthesized part Rcn-CsgBAEFG (Amp) with increasing concentrations of cobalt ( in CoCl2 form).<br><br />
</p><br />
<br />
<p style=" line-height : 1.5em;"><br />
<br><br />
<b>Flocculation test</b> for the same strains with LB/2 or M63G medium.<br><br />
<br><br/><br />
<br />
</p><br />
<p style=" line-height : 1.5em;"><FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> of the transformed colonies with the following plasmids : Prcn / Pcurli-GFP / Pcurli - Operon curli.<br><br />
-> none of the isolated clones had the Prcn or Pcurli-GFP plasmid, start of liquid culture of new clones.<br><br />
-> two clones had the Pcurli-curli operon part : storage in the collection <br><br />
<br />
</p> <br />
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<br><br><br />
<br />
<h6 style="text-align :left"> Tuesday </h6><HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence and Flocculation Tests</b></FONT> <br><br><br />
<br />
In LB/2, the flocculation tests show flocculation for the adherent strain (PHL818), none for the negative control. Our strain shows a small flocculation that increases with the concentration in cobalt.<br><br />
No flocculation in M63 medium for our strain : LB/2 will be used only for the next tests. <br><br />
<br><br />
New <b>flocculation tests</b> with a wider range of cobalt concentration in LB or LB/2 and using the Prcn-CsgBAEFG (Cm) synthesized part <br><br />
</p><br />
<br />
<div style="border:1px solid black;float:right;margin-right:5%; width : 210px"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f5/02.08_24_well_plate.jpg" width="210px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em; width : 400px"><br />
<br><br />
Revealing of the 24 well plate from the previous day, using 2 different methods : methyl violet (to visualise the formation of the biofilm at the surface of the well ) or direct OD600 measure.<br><br />
</p><br />
<br><br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> of the clones started the previous day <br><br />
-> two clones have the Prcn part and one Pcurli-GFP.<br> <br />
<br><br />
<b>Storage</b> of PHL818, PHL1273, Rcn-CsgBAEFG (Cm) = S3 = S17, Rcn-CsgBAEFG (Amp) = S4 = S18<br><br />
</p> <br />
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<br />
<br />
<h6 style="text-align :left"> Wednesday </h6><HR><br />
<br />
<br><br />
<br />
<div style="border:1px solid black;float:right;margin-right:5%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/03.08_24_well_plate.jpg" width="180px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em ; width : 440px"><br />
<FONT COLOR="purple"><b> Adherence and Flocculation Tests</b></FONT> <br><br><br />
Measurement of the OD600 of the LB/2 plate, which gives an adherence percentage for each strain.<br />
<br/> <br/><br />
Revealing of the M63 medium 24 well plate using the methyl violet ( 1 well) or OD600 method ( 3 wells ).<br />
<br> <br/> <br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<br><br />
The flocculation tests from the previous day give better results in LB/2 compared to LB , but they're not significative. We'll use LB/2 for future tests.<br><br/><br />
<br />
<div style="border:1px solid black;float:left;margin-left:5%; width : 448px"><br />
<img src="https://static.igem.org/mediawiki/2011/4/46/03.08_LB_grand_test_floc.jpg" /> <br />
</div><br />
<br />
<p style=" line-height : 1.5em; width : 200px ; margin-left : 70%"><br />
<br><br/><br/><br />
The flocculation tests from the previous day give better results in LB/2 compared to LB , but they're not significative. We'll use LB/2 for future tests.<br />
<br/><br/><br/><br/> <br/><br/><br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="blue"><b>Plasmid Collection</b></FONT> <br><br><br />
<b>Nanodrop</b> quantification of some plasmids in the collection.<br><br><br><br />
<br />
</p> <br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#12B1AF><b>Cobalt accumulating strain reconstruction</b></FONT> <br><br><br />
MC4100 E.coli put in LB medium, with 10mM MgSO4 and 10mM CaCl2<br><br />
<br />
</p> <br />
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<br />
<h6 style="text-align :left"> Thursday </h6><HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Tests</b></FONT> <br><br><br />
Start of new <b>24 well plates</b>, to study:<br />
</p><br />
<ul style=" line-height : 1.5em"><br />
<li> the effect of cobalt (in increasing concentration), </il><br />
<li> the difference between Amp or Cm strains, -> it works better in Amp </il><br />
<li> the effect of the presence of antibiotic, -> it works better with Antibiotic</il><br />
<li> the effect of prior treatment of the plate with EDTA, -> it is better when plate is rinse</li><br />
<li> the effect of EDTA (in increasing concentration) on the strain. -> no useful </il><br />
</ul><br />
<br><br />
<p style=" line-height : 1.5em"><br />
Characterization of OmpR234 with a <b>24 well plate</b> and a <b>flocculation test</b>.<br><br />
<br><br />
</p><br />
<br />
<div style="float:left;margin-left:5%; width : 170px"><br />
<img src="https://static.igem.org/mediawiki/2011/8/89/05.08_test_CFA.jpg" width="170px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em; margin-left: 35%"><br />
<br/> <br/><br />
<b>Test in CFA medium</b> to show the formation of biofilms by colouring them red using the following strains( 10µL each ) :<br><br />
PHL818 (positive control),NM522 (negative control), and test bacteria containing 18A promoter ( alone without the coding part ),our synthesized parts (Amp or Cm)<br><br />
-> not very conclusive, a little circle of light around the bacteria.<br />
<br><br/> <br/><br/><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction : Prcn-GFP(LVA)</b> </FONT> <br><br><br />
<br />
<b>Digestion</b> of the plasmid with Prcn (S+P) = linearization -> error: the part goes out from the plasmid <br><br />
-> use of other enzyme tubes, with the same results, same with a simple digestion -> our enzyme tubes are contaminated<br><br />
digestion of the plasmid with strong RRB-GFP (X+P) -> OK<br><br />
<br><br/><br />
<br />
</p><br />
<p style=" line-height : 1.5em"><FONT COLOR="blue"><b>Strain Collection</b></FONT> <br><br><br />
<b>Storage</b> of MC4100 (Str) = S7, NM522 = S11, NMYO = S13 and NM522/NicoT (Amp) = S14<br />
</p><br/><br/><br/><br />
<br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR=#12B1AF><b>Cobalt accumulating strain reconstruction</b></FONT> <br><br><br />
Created a dilution range for the P1 phage we've been given (10-2 to 10-10)<br><br />
Phage adsorbption onto our target strain <br/><br />
Overnight culture, 37°C <br/><br />
<br />
</p> <br />
<br />
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<br />
<h6 style="text-align :left"> Friday</h6><HR><br />
<br><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Tests</b></FONT> <br><br/><br />
Repeat of the <b>flocculation test</b> for the characterization of OmpR234.<br><br />
<br><br />
Repeat of the <b>CFA medium test</b>.<br><br />
<br><br />
Revealing of the four 24 well plates from thursday.<br />
</p> <br />
<br />
<br />
<br/><br/><br/><p style=" line-height : 1.5em"><br />
<FONT COLOR=#12B1AF><b>Cobalt accumulating strain reconstruction</b></FONT> <br><br><br />
Revealing of the lysis plates : results are negative, it seems the phage are inactive. Re-construction post-poned indefinitely.<br/><br />
<br />
</p> <br />
<br />
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<br/> <br/><br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week7"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week9"/><font color="grey"><b>Next Week</b></font></a><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week7
Team:Lyon-INSA-ENS/Realisation/Week7
2011-09-21T23:35:28Z
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<br />
<div class="contenugrand2";><br />
<br />
<br/> <br/><br />
<br/><br />
<br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 7 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 25th of July to Friday the 29th of July 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> of some other parts from the transformed bacteria. <b>Digestion</b> and <b>electrophoresis</b> to control the presence of the right insert.<br/> <br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> of bacteria with mutated part CsgAB. <b>Digestion</b> to check restriction profile (removal of intern PstI site).<br />
</p> <br />
<br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/> <br/> <br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="purple"><b> Adherence Tests</b></FONT> <br><br><br />
Start of a compared culture of a strain transformed with 18A ( constitutive promoter ) only and 18A+OmpR234 ( part which is supposed to induce the formation of a biofilm ) in LB medium, 37°C to check the qualitative behaviour of the OmpR234 part.<br/> <br />
OmpR234 part seems to make the bacteria adherent, the experiment was restarted with a different protocol ( LB diluted by 2 in water, 30°C ) to enhance the adherence.<br/> <br/><br />
</p><br />
<br/><br />
<br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 200px"><br />
<img src="https://static.igem.org/mediawiki/2011/7/74/Boite-petri.jpg" width="200px"/> <br />
</div><br />
<br />
<br />
<p style=" line-height : 1.5em; margin-left : 35%"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
Start of 5mL liquid cultures for minipreps : 18A-OmpR234, 2M-2L Tet, 2M-2L-Kan, rcn-curli, 2M-24E, NiCoT ( a transporter for cobalt that makes the bacteria import cobalt from the medium) <br/> <br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
Send of plasmid with the right profile for part CsgAB to <b>sequencing</b>. <b>Extraction</b> of new MC4100 strain's DNA. <b>PCR</b> with fresh DNA and Taq Phusion.<br />
</p> <br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Midiprep</b> and <b>nanodrop</b> of the following ligated or newly obtained plasmids :<br/><br />
NiCoT : 112,2 ng/µL <br/><br />
2M-2L-Tet : 109.6 ng/µL <br/><br />
2M-2L-Kan : 128 ng/µL <br/><br />
18A-OmpR234 : 59.5 ng/µL <br/><br />
rcn-Curli : 116 ng/µL <br/><br />
2M-24E : 92.3 ng/µL <br/><br/><br />
<br />
<b>Fermentas digestion</b> : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P) <br />
<br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
<b>PCR</b> did not work.<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Standard <b>ligation</b> of :<br/><br />
-Prcn into Cn backbone<br/><br />
-rcn-curli into Cn backbone<br/><br />
-Pcurli into 2M-2L-Kan <br/><br />
-Pcurli into 2M-2L-Tet <br/><br/><br />
<br />
<b>TSS transformation</b> into NM522.<br />
<br />
</p> <br />
<br />
<br/> <br/><br/><br />
<br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
PCR with genomic DNA of MC4100 with Taq DNA Polymerase and Phusion<br />
</p> <br />
<br />
<br/> <br/> <br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Friday </h6> <HR><br />
<br />
<br/> <br/> <br />
<br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
PCR did not work. New try with different concentrations of primers (half, normal and twice) with Taq DNA polymerase and Phusion. </p> <br />
<br />
<br/> <br/> <br />
<br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week6"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week8"/><font color="grey"><b>Next Week</b></font></a><br />
<br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week6
Team:Lyon-INSA-ENS/Realisation/Week6
2011-09-21T23:35:18Z
<p>Suxiaohui: </p>
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<br />
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<br />
<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
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<br />
<body><br />
<br />
<div id="language";><br />
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</div><br />
<br />
<div class="contenugrand2";><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 6 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 18th of July to Friday the 22nd of July 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR> <br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn) <br />
<br/><br/><br />
Start of 5mL liquid cultures from a Petri dish culture ( 5mL sterile LB, 50µL antibiotic ,bacteria). Incubation at 37°C<br />
<br/><br/><br />
Later, start of 150mL liquid cultures from the previous 5mL cultures ( 150 mL sterile LB ,300 µL from the grown 5mL bacterial culture, 1.5 mL antibiotic ( Amp or Cn ) )<br />
Incubation at 37°C overnight. <br />
<br/><br/><br/><br />
10X concentration and plating of 100 µL of transformed bacteria from the previous week : 2M+24E ( amp ), Curli + GFP ( Kan ), Kan + 2L ( Kan ), positive control ( Puc18 plasmid ), negative control ( water ) <br/><br />
Incubation at 37°C <br/><br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/> <br />
<br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Midiprep</b> to extract DNA from the previous liquid cultures.<br />
<br/><br />
Nanodrop quantification :<br />
<br/><br />
OmpR234 : 59.5 ng/µL<br />
<br/><br />
GFP : 109.5 ng/µL<br />
<br/><br />
18A : 73.7 ng/µL<br />
<br/><br />
Curli : 82.6 ng/µL<br />
<br/><br />
The 260/280 ratio is lower than 2 in all cases.<br/> <br/> <br/><br />
<br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
A critical error in part CsgAB and a less important error in part CsgEFG were detected by sequencing. Restart mutagenesis on another plasmid with part CsgAB without the fatal error. Restart construction of part csgEFG from the beginning. Another clone of CsgEFG is sent to sequencing .Part RcnR is correct. <br />
</p> <br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Digestion</b> of several biobricks : RBS, GFP, YFP, Curli, OmpR234.<br />
Electrophoresis to control the digestions.<br/> <br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
Mutagenesis on the other clone of part CsgAB worked. Digestion with DpnI to eliminate parental plasmids. On the order hand, PCR with Pfu polymerase on MC4100 to amplify part CsgEFG did not work. Retry with extracted DNA of MC4100.<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
A french radio, <B>RCF</B>, interviewed us.<br><br><br />
</p><br />
<br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Standard or 3A<b> ligation </b>of the previously cut biobricks. <b>TSS transformation</b> into NM522.<br/><br/><br/><br />
<br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
Transformation of NM522 with the mutated plasmid of part csgAB.<br/><br/><br />
</p><br />
<br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Friday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Start of 5mL liquid cultures of selected individual colonies containing the biobricks.<br />
<b>Extraction</b> of the plasmids to test the presence of the insert. <br/> <br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
Liquid culture of transformed colonies with csgAB for miniprep<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week5"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week7"/><font color="grey"><b>Next Week</b></font></a><br />
<br/><br />
</p><br />
<br/> <br/><br />
<br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week5
Team:Lyon-INSA-ENS/Realisation/Week5
2011-09-21T23:35:03Z
<p>Suxiaohui: </p>
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<img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week5Fr">Version Fran&ccedil;aise</a><br />
</div><br />
<br />
<div class="contenugrand2";><br />
<br />
<br/> <br/><br />
<br/> <br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 5 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 11th of July to Friday the 15th of July 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
Visit of Centraco : a french nuclear waste treatment plant <br/><br/><br/><br />
<br />
</p><br />
<p style=" line-height : 1.5em"><FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Start of 5mL cultures of 18A (Amp), 24E (Amp) and Curli(Cn). Incubation at 37°C <br/><br />
Start of 50 mL cultures ( 50mL sterile LB, 500µL antibiotic, 100µL from the 5mL cultures ). Incubation at 37°C overnight.<br />
</p><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/> <br />
<br />
<div style="border:1px solid black;float:left;margin-left:3%; width : 180px"><br />
<img src="https://static.igem.org/mediawiki/2011/f/f9/Electrophorese.jpg" width="180px"/> <br />
</div><br />
<br />
<p style=" line-height : 1.5em; margin-left : 30%; length: 300px"><br />
<br/> <br/><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> of the ligations from the previous week to check the insert : 5L+2L, 5L+24E, 2M+2L, 2M+24E, 2L-Tet, 24E-Tet<br/><br />
4 clones are chosen for each ligation. <br/><br />
<b>Digestion</b> and <b>electrophoresis</b> : no clone seemed to contain the insert. Decision to verify the digestion, ligation and transformation protocols. <br/><br/><br />
</p><br />
<br />
<br/><br />
<br />
<p style=" line-height : 1.5em"><br />
<br />
<b>Midiprep</b> of 18A, 24E and curli parts.<br/><br />
Nanodrop analysis : <br/><br />
-18A : 16.7 ng/µL <br/><br />
-Curli : 40.5 ng/µL <br/><br />
-24E : 190.8 ng/µL <br/><br/><br/><br />
<br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
The sequencing results were not enough good due to too high concentration of mutated plasmid. Resent of dillute sample.<br/> <br/> <br />
</p> <br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
Another french newspaper <B>"Le progrès"</B> interviewed us.<br><br><br/><br />
<br />
</p><br />
<p style=" line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Start of 5mL cultures of 22M, 2M, 2L, 5L, OmpR234 and RBS-GFP-LVA ( synthesized part with an unstable GFP )<br/><br/><br />
<br />
Start of 50mL cultures for the same parts.<br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> of the clones with the mutated plasmid to get more plasmid for the next step. <br/><br/><br />
<br />
</p> <br />
<br />
<br/> <br/><br />
<br/> <br/> <br />
<br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
French national day but... we worked.<br/><br/><br/><br />
<br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
</p> <br />
<br />
<p style=" line-height : 1.5em"><br />
<b>Midiprep</b> of 22M, 2M, 2L, 5L, OmpR234, RBS-GFP-LVA.<br />
</p> <br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week4"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week6"/><font color="grey"><b>Next Week</b></font></a><br />
<br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week4
Team:Lyon-INSA-ENS/Realisation/Week4
2011-09-21T23:34:52Z
<p>Suxiaohui: </p>
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<br />
<br />
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<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
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</div><br />
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</div><br />
<br />
<br />
<div class="contenugrand2";><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 4 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 4th of July to Friday the 8th of July 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Additional <b>minipreps</b> using the QuickPure protocol of the same 6 parts ( 5 replica each ) <br/><br />
<b>Digestion</b> as previously<br/><br/><br />
<br />
<b>Ligation</b> to obtain : RBS ( strong and weak ) + GFP, RBS ( strong and weak ) + YFP. This corresponds respectively to 2M+2L, 5L+2L, 2M+24E, 5L+24E assemblies.<br/><br/><br />
<br />
Start of a 5mL culture of NM522 cells.<br/><br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).<br/><br />
<br />
<b>Transformation</b> of 5µL ( S series ) or 10 µL ( D series ) from the previous ligations into NM522 (V=15mL) using a CaCl2 chemical transformation. Positive control was done with 1µL Puc18, negative with 5µL water. <br/> <br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
Sequencing results obtained. Mutagenesis of the plasmids with the least errors by quick change method to remove unwanted restriction sites (EcoRI or PstI).<br/><br />
</p><br />
<br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br />
<br/><br />
<br />
<div style="border:1px solid black;float:right;margin-right:6%;"><br />
<div style="border : 3px solid green;"><br />
<div style="border : 1px solid black;"><br />
<img src="https://static.igem.org/mediawiki/2011/d/d3/Notebook-tournage.jpg" width="120px" /><br />
</div><br />
</div><br />
</div><br />
<br />
<p style = "line-height : 1.5em;width : 530px"><br />
We shoot our "Cobalt Buster's Clip" which introduces, in a funny way, our team : students, instructors and advisors. We dedicate the all afternoon to this shooting.<br><br><br/><br />
</p> <br />
<br />
<p style = "line-height : 1.5em; width : 530px"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
Selection of individual transformed colonies and start of solid and liquid culture.<br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<b>Digestion</b> of PCR/mutagenesis product with DpnI to eliminate parental plasmid. <b>Transformation</b> of NM522 strains with plasmid. Culture and selection on ampicilline plates.<br />
</p><br />
<br/><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br/><br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> using the QuickPure protocol of the previous liquid cultures.<br/><br/><br />
<br />
<b>Ozyme digestion</b> of the plasmids by X+P.<br/><br/><br />
<br />
<b>Electrophoresis</b> of the digested and non digested plasmids : we have observed either no DNA, no insert, or a plasmid with a correct size insert.<br/><br />
<br />
However, due to the small size of the RBS, a flaw in our protocol ( choice of antibiotic resistances ) doesn't allow us to detect a difference between 24E or 2L plasmids compared to their ligated equivalents. Thus we can't conclude that the 800 bp inserts correspond to the ligated parts.<br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<b>Selection</b> of clones on plate and liquid culture for miniprep. <b>Digestion</b> of purified PCR product in Tango buffer to have a better efficiency. <b>Transformation</b> of NM522 with purified plasmids.<br />
<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Friday </h6> <HR><br />
<p style = "line-height : 1.5em;width : 560px"><br />
<br/><br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<b>Miniprep</b> of clones with mutated plasmids. Restriction to check the removal of unwanted restriction sites (EcoRI or PstI). Send of plasmids with the expected profile to sequencing.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week3"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week5"/><font color="grey"><b>Next Week</b></font></a><br />
<br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week3
Team:Lyon-INSA-ENS/Realisation/Week3
2011-09-21T23:34:42Z
<p>Suxiaohui: </p>
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<div>{{Lyon-INSA-ENS/header}}<br />
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<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
<a href="https://2011.igem.org/Main_Page" ><br />
<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<body><br />
<br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Week3Fr">Version Fran&ccedil;aise</a><br />
</div><br />
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<div class="contenugrand2";><br />
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<br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 3 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 27th of June to Friday the 1st of July 2011 </small> </p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br/><br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
Another <b>PCR</b> is launched to collect more DNA from MC4100 (Failure).<br/><br />
Alcoholic precipitation of PCR product from the previous week.<br/><br />
<br />
Culture of the wrong strains for the transformation...(Failure : the strain already had a plasmid)<br/><br />
</p><br />
<br/> <br><br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br/><br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Culture</b> of NM522 cells for later transformations and Curli for extraction. <br/><br/><br/><br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
<b>Ligation</b> of PCR products in pGem-T easy vector. <b>Transformation</b> in NM522 strain of the ligation product. Selection on ampicilline, X-Gal, IPTG plate. Only CsgAB was not transformed this day.<br />
</p><br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br/><br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>CaCl2 chemical transformation</b> ( V=10mL, 2µL DNA ) of some iGEM kit distribution DNA : <br/><br/><br />
<br />
<U>Plate 1 : </U><br/><br />
18A (constitutive promoter, Amp )<br/><br />
2M ( strong RBS, Amp ) <br/><br />
5L ( weak RBS, Amp )<br/><br />
22M ( RBS+YFP, Amp)<br/><br/><br />
<br />
<U>Plate 2 : </U><br/><br />
24E (YFP, Amp + Kan ) <br/><br />
2L (GFP, Amp ) <br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<b>Selection</b> of white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.<br/><br/><br />
<br />
<b>Transformation</b> in NM522 of part CsgAB.<br />
</p><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br/><br />
<p style = "line-height : 1.5em"><br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<b>Miniprep</b> using the QuickPure kit, digestion and electrophoresis of the Curli plasmid.<br/><br/><br />
<br />
Start of liquid cultures of two individual clones for each of the 6 iGEM plasmids <br/><br />
<b>Miniprep</b> using the QuickPure kit of the 18A, 2M, 22M, 24E and 2L plasmids.<br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<b>Miniprep</b> of clones with CsgEFG and RcnR. <b>Digestion</b> of plasmid with EcoRI to check part insertions. <br/><br />
</p><br />
<br />
<br/><br />
<br />
<p style = "line-height : 1.5em"><br />
The local newspaper "VIVA" interviewed us. They asked about our project, our team and about iGEM's organization.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<h6 style="text-align :left"> Friday </h6> <HR><br />
<br />
<br/><br />
<br />
<p style = "line-height : 1.5em"><br />
<br />
<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br><br />
<br />
<b>Miniprep</b> using the QuickPure kit of 5L plasmid. <br/><br/><br />
<br />
<b>Ozyme digestion</b> with :<br/><br />
S + P : 18A, 2M, 5L<br/><br />
X + P : 22M, 2L, 24E<br/><br/><br />
<br />
<b>Electrophoresis</b> of the digested plasmids versus the non digested.<br/><br />
<br />
-> All the strains had the correct plasmid : they were plated on LB + amp medium.<br/><br/><br/><br />
<br />
</p><br />
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<b>Miniprep</b> of clones with Csg AB. <b>Digestion</b> of plasmid with EcoRI. Send to GATC for <b>sequencing</b>.<br />
<br />
<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week3"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week4"/><font color="grey"><b>Next Week</b></font></a><br />
<br/><br />
</p><br />
<br/> <br/><br />
<br />
<br />
</div><br />
</body><br />
</html><br />
<br />
<br />
{{Lyon-INSA-ENS/footer}}</div>
Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week2
Team:Lyon-INSA-ENS/Realisation/Week2
2011-09-21T23:34:32Z
<p>Suxiaohui: </p>
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<a href="https://2011.igem.org/Main_Page" ><br />
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</a><br />
</div><br />
<br />
<body><br />
<br />
<div id="language";><br />
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</div><br />
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<div class="contenugrand2";><br />
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<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 2 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Monday the 20th of June to Friday the 24th of June 2011 </small> </p><br />
<br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Monday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="orange"> <b> Extraction of NiCoT </b></FONT> <br><br><br />
<br />
One transformed colony (NicoT in NM522) seemed correct, the others were probably satellite colonies without the plasmid.<br/><br />
Start of 5mL liquid culture of the main colony. <br/><br />
Plating of the satellite colonies to check the presence of the plasmid <br/><br />
</p><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Tuesday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="orange"> <b> Extraction of NiCoT </b></FONT> <br><br><br />
<br />
4 more new clones have grown : start of 5mL liquid cultures on these clones.<br />
</p><br />
<br/> <br/> <br />
<br/> <br/> <br />
<br />
<h6 style="text-align :left"> Wednesday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="orange"> <b> Extraction of NiCoT </b></FONT> <br><br><br />
<br />
Miniprep on the four clones. Verification of plasmid by <b>digestion</b> (EcoRI, HindIII)<br />
</p><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br><br />
<br />
<b>PCR</b> from genomic DNA from E.coli strain MC4100 to amplify three genes : RcnR (cobalt receptor), CsgAB (curli operon, part 1) and CsgEFG (curli operon, part 2)<br />
</p> <br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week2"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week3"/><font color="grey"><b>Next Week</b></font></a><br />
<br/><br />
</p><br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Notebook/Week1
Team:Lyon-INSA-ENS/Realisation/Notebook/Week1
2011-09-21T23:34:22Z
<p>Suxiaohui: </p>
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<div style="float : left; margin-top : -10px; margin-left : -200px"><br />
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<img src="https://static.igem.org/mediawiki/2011/6/67/Team_INSA-Lyon_IGEM_Home.png" title="iGEM's main page" /><br />
</a><br />
</div><br />
<br />
<br />
<body><br />
<br />
<div id="language";><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Drapeau_francais.jpg"; width=20px; /> <a href="/Team:Lyon-INSA-ENS/Realisation/Notebook/Week1Fr">Version Fran&ccedil;aise</a><br />
</div><br />
<br />
<div class="contenugrand2";><br />
<br />
<br/> <br/><br />
<br/> <br />
<br />
<div class="cadre" ; style="background-color:green;margin-left : 8%" ><br />
<br/><br />
<h1 style="color: white;"> Week 1 </h1> <br />
</div><br />
<br />
<br/><br />
<p style="text-align : center"> <small> From Thursday the 16th of June to Friday the 17th of June 2011 </small> </p><br />
<br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<br />
<br />
<h6 style="text-align :left"> Thursday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="orange"> <b> Extraction of NiCoT </b></FONT> <br><br><br />
<br />
Culture of NM 522 cells for later transformations<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<h6 style="text-align :left"> Friday </h6> <HR><br />
<br/><br />
<p style=" line-height : 1.5em"><br />
<br />
<FONT COLOR="orange"> <b> Extraction of NiCoT </b></FONT> <br><br><br />
<br />
<b>Extraction</b> of plasmid with NicoT gene from filter paper. <b>Transformation</b> of the extracted DNA in NM522 strain using TSS. Culture and selection of transformants on ampicilline plate during the week end.<br />
</p><br />
<br />
<br/> <br/><br />
<br/> <br/><br />
<br/> <br/><br />
<br />
<p><br />
<a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Notebook/Week1"/><font color="grey"><b>Previous Week</b></font></a><br />
<a style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week2"/><font color="grey"><b>Next Week</b></font></a><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Project/Achievement
Team:Lyon-INSA-ENS/Project/Achievement
2011-09-21T23:33:33Z
<p>Suxiaohui: </p>
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<p id="top"> <font color="green" size="6"><br />
Achievements<br><HR><br />
<br/><br />
</font><br />
</p><br />
<br />
<br><br> <br />
<p style="line-height:1.5em;text-indent:0%;"><br />
<ul style="list-style-image:url('https://static.igem.org/mediawiki/2011/4/4d/Chek.JPG'); margin-left:50px;"><br />
<br />
<li>We lived a <b>great human experience</b>, met new friends and <b>had fun</b>!!</li><br><br><br />
<li>We acquired <b>new knowledge and competencies</b> dealing with scientific and logistic issues.</li><br><br><br />
<li>We submitted <b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Project/Parts">2 documented parts</a></b> to the registry and showed that they work as expected.</li><br><br><br />
<li>We characterized a <b><a href="http://partsregistry.org/Part:BBa_K342003">pre-existing part</a></b>.</li><br><br><br />
<li>We <b><a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Sponsors/Collaboration">collaborated</a></b> with another iGEM team : TU Delft.</li><br><br><br />
<li><p style="line-height:1.5em;text-indent:0%;"><br />
We <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Communication/Main">presented our project and Synthetic Biology</a> in many ways : in different <b>websites</b>,<br/> in <b>newspapers</b>, on the <b>radio</b>, on <b>TV</b>, … and even <b>in music</b> ; in order to inform a public <br/>as varied as possible.<br />
</p></li><br><br><br />
<li>We <b>get our project close to reality</b> meeting experts to consider <b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Project/Industrialization">industrialization</a></b>.</li><br/><br/><br />
<li>We explored <b><a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Safety/Intro"> safety implications </a></b> of the project as a whole.</li><br/><br />
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Suxiaohui
http://2011.igem.org/Team:Lyon-INSA-ENS/Project/Industrialization
Team:Lyon-INSA-ENS/Project/Industrialization
2011-09-21T23:33:15Z
<p>Suxiaohui: </p>
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Industrialization <br><HR><br />
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<ul style="list-style-type:circle;margin-left:10%;"> <br />
<li> <a href="#brainstorming"> <font color="green"> <b> Team Brainstorming </b> </font> </a> </li><br />
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<li> <a href="#biofilter"> <font color="green"> <b> Why a "Cobalt Buster" biofilter in nuclear power plants ? </b> </font> </a> </li> <br />
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<li> <a href="#circuit"> <font color="green"> <b> Why not in the primary circuit ? </b> </font> </a> </li><br />
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<li> <a href="#where"> <font color="green"> <b> Where to use "Cobalt Buster" ?</b> </font> </a> </li> <br />
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<li> <a href="#prospects"> <font color="green"> <b> Other prospects for the "Cobalt Buster" project ?</b> </font> </a> </li> <br />
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<li> <a href="#further"> <font color="green"> <b> To go further : Economic analysis of the electronuclear pattern</b> </font> </a> </li> <br />
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Team brainstorming <br><HR><br />
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<big>After several months of reflection and review of the scientific literature, the "Cobalt Buster" biofilter was born as a filter dedicated to the primary water circuit of nuclear power plants ! </big></p><br />
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Why a "Cobalt Buster" biofilter in nuclear power plants ?<br><HR><br />
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<big>1- </big>It is known that a <b> pulse of radioactive Cobalt emission occurs in the primary circuit </b> of water, during the maintenance of nuclear power plants when they open the reactor core. This pulse <b>damages the ion exchange resins </b>used to filter the water and reduce its radioactive level. </p><br/><br/><br/><br />
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<big>2- </big> A major concern of the nuclear industry is <b>the reduction of waste volume </b> and a previous modeling estimated that the "Cobalt Buster" strain is very efficient (Appl Microbio Biotechnol 2009 81:571- 578):</p><br/><br />
<p style="line-height:1.5em ; text-align : center"> <b>4 kg of modified bacteria = 8000 kg of ion exchange resins </b></p><br/><br />
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<big>3- </big> <b>Drastic reduction of the costs of waste processing and conditioning is also a major issue </b>for nuclear industry. The production of biofilters is less expensive and the biofilter may prevent damage caused to the resins. It could significantly reduce costs of rehabilitation of primary circuit wastewater.</p><br/><br/><br />
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<big>4- </big> Maintenance phases generate a shortfall of millions of euros and <b>reduction of the duration of maintenance phases</b> represent a major issue.</p><br/><br/><br />
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<big>5- </big> Storage stations of moderately radioactive waste <b>are rapidly full. Our bacteria could help in downgrading the high volumes of high radioactive waste to low radioactive waste</b>, which are stocked in other stations, usually more spacious. Radioactive cobalt would be concentrated in smaller volumes thanks to the increase of the efficiency of filtration.</p><br/><br/><br />
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Why not in the primary circuit ? (Experts advice)<br><HR><br />
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<b>To include our project into a realistic approach</b> and close to concerns of today and tomorrow's engineers, <b>we notably have discussed the realization of our biofilter with M. Brette (Assistant professor in INSA Lyon, doctor in economic sciences) and our partners (Assystem, EDF)</b>. We also have a realistic idea of the technical difficulties thanks to the visits we made in nuclear installations (central of Tricastin, area of Centraco) and discussions we had with a Chemist of the nuclear power plant of Bugey.</p><br/><br />
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To get an industrial application technically realistic, our biofilter has to be designed as a modular innovation, what means that this solution can be applied without any major modification of the structure of the power plant or of the treatment center (plumbing, circuits of the different components, ...). Thus, design of the cartridge of the biofilter is fundamental to be able to be used into the circuit of nuclear effluents, then be treated as radioactive waste.</b><br />
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<b style="line-height:1.5em">From those discussions we know that our <b>project is plausible and could interest industrialists</b> but some changes have to be made regarding the uses of our biofilters.</b></p><br/><br/><br/><br/> <br />
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<big>1- </big> Cobalt released during the opening of the nuclear reactor may represent <b>150 TeraBecquerel (TBq) of radioactivity</b> (500 m3 of contaminated water with a radioactive Cobalt estimated level of 300 gigaBq / m3).</p> <br />
<p style="line-height:1.5em"> If the Cobalt biofilter is used as shown above, <b>dose rate for only 1 of the 150 TBq will represent 0,4 sievert per hour (Sv/h) whereas the authorized rate is up to 20 mSv per year</b>. </p><br/><br/><br />
<p style="line-height:1.5em ; text-align : center"> Calculation of dose rate:<br><br />
<b>Dose Rate = 0.54 * C * E * P / d²</b></p><br/><br />
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with <br><br />
C = the activity Curie <br><br />
E = energy radiation in MeV <br><br />
P = the percentage of emission <br><br />
d = distance from the radiation source <br><br/><br />
To treat 1TBq of Co60 with d = 1m (1TBq = 30 Curie)<br><br />
EP = 2.5 </p><br/><br />
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we can estimate Dose rate <br><br />
DR = (0.54 * 30 * 2.5) / 1² <br><br />
DR = 40 rad / h <br><br />
DR = 0.4 Gy / h <br><br />
DR = 0.4 Sv / h </p><br/><br/><br />
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<p style="line-height:1.5em ; margin-left : 35%"> This exposure rate supposed that if we want to use our "Cobalt Buster" biofilter, human operators will have to be protected from this high level of radiation, which means that a <b>concrete wall of at least one meter has to be built</b> for each parallel biofilter, and all <b>manipulations have to be automated</b>. This would involve a major modification of the structure of nuclear power plants.</p><br/><br />
<p style="line-height:1.5em;margin-left : 35%">These changes involve too important costs as in France, <b style="line-height:1.5em">a modification in one power plant must be also done in the 58 other power plants of the nuclear fleet, for standardization and safety reasons.</b></p><br/><br />
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<big>2- </big> We also have to consider that during the conventional operation, <b>pressure in the primary circuit can reach up to 155 bars, and temperature up to 327°C (621°F)</b>. As the maximum rate of temperature decrease is estimated at 28°C/hr (82°F/hr)and acceptable temperature for our biofilter is between 20°C to 45 °C, <b>it implies waiting 4 to 5 hours after the opening of the nuclear reactor</b>, before starting the cobalt decontamination.</p> <br />
<p style="line-height:1.5em">It could be too long because <b>stopping the reactor costs one million euros a day </b>and the maintenance time has to be as short as possible.</p> <br />
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<big>3- </big> In the primary circuit, cobalt is in form of ions and particles. <br />
<b style="line-height:1.5em"> Cobalt particles may represent the majority of cobalt and the initial bioremediation strain is design to capture ions of cobalt </b>.</p><br/><br />
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At this stage of the project we could not assess the ability of the biofilter to capture cobalt particles. However, the final "Cobalt Buster" strain will produce amyloid fibers (curli) that could allow it to fix cobalt particles on its surface. </p><br />
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Where to use "Cobalt Buster" ? (Experts advice) <br><HR><br />
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<big>1- </big> According to the experts, the "Cobalt Buster" biofilter could be used in the treatment of other effluents, such as those of dismantling stations or stations of treatments of liquid effluents (STEL).</p><br/><br/><br/><br />
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In these stations the radioactivity is lower, but the problems related to the cobalt still exist, and <b>temperature and pressure are compatible with the survival of our biofilter </b> (atmospheric pressure and ambient temperature).</p><br/><br/><br />
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<b style="line-height:1.5em">Moreover, our biofilter may be adapted on an existing filter and a collaboration subjected to non-disclosure agreement is being discussed with our partner ASSYSTEM.</b></p><br />
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<big>2- </big> The filter could also be used in a <b>bubbling type system to treat contaminated air </b>during the decommissioning of power plants.</p><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br/><br />
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Other prospects for the "Cobalt Buster" project ? <br><HR><br />
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As shown by the number of teams interested in strain adherence (<a href="https://2011.igem.org/Team:TU-Delft">TU-Delft 2011</a>), metal absorption (<a href="https://2010.igem.org/Team:Peking">Peking 2010</a>), or radioactivity (<a href="https://2011.igem.org/Team:Osaka">Osaka 2011</a>; <a href="https://2011.igem.org/Team:NYC_Software">NYC_Software 2011</a>), <b>collaborations and common brainstorming may enhance future prospects.</b></p><br />
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<b>We initiated a collaboration with <a href="https://2011.igem.org/Team:TU-Delft">TU-Delft</a> team in order to compare the efficiency of our two approaches</b> and determine if the overproduction of curli is the best strategy to optimize the adhesion of <i>E. coli</i> strains.</p><br />
<p style="line-height:1.5em"> <br />
<b>Collaborations with Osaka and NYC teams should be considered to optimize the radioactive cobalt bioremediation</b> of our "Cobalt Buster" strain.</p><br />
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<b>To go further</b>, outside the context of electronuclear pattern, <b>we considered a wider use of the biofilter.</b> The system could involve a <b>large variety of bioremediation strains</b>, each optimized for the capture of one metal and why not other pollutants (hydrocarbons, antibiotics...). These strains <b>could be associated in a complex biofilm to create a broad-spectrum biofilter</b>, as shown in the following video !<br />
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<iframe frameborder="0" width="480" height="360" src="http://www.dailymotion.com/embed/video/xl7q47"></iframe><br /><a href="http://www.dailymotion.com/video/xl7q47_schema-prospects_tech" target="_blank">sch&eacute;ma prospects</a> <i>par <a href="http://www.dailymotion.com/iGEM_Lyon_2011" target="_blank">iGEM_Lyon_2011</a></i><br />
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<p id="further"> <br><font color="green" size="4"><br />
<big> To go Further :</big> Economic analysis of the electronuclear pattern <br><HR><br />
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<big><b>The electronuclear pattern</b></big></p><br/><br />
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Because of the implied technologies, ways and the potential risks, nuclear and electronuclear pattern are strategic fields. There are four sectors :<br />
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<b>The Upstream aims to supply centrals in nuclear fuel</b>. It groups together several links : mines (mining exploration and natural uranium extraction), chemistry (purification and conversion of uranium to uranium hexafluorure), enrichment (augmentation of isotope U235 content from 0,7% to 3-5%).<br />
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<b>The Construction put together conception, studies and engineering</b> for each project of central, fabrication of components, installation and starting of centrals.<br />
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<b>The operators of Exploitation are cautious about the well functioning of centrals daily</b>, and calibrate the power of reactor according to the needs of electric network. Maintenance includes activities necessary for upkeep, modernization and extension of the lifetime of nuclear centrals. The outages are important points of this activity: indeed reactors are stopped sometimes during several weeks to refill in fuel and large-scale maintenance operations.<br />
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<b>The Downstream</b> of the pattern is divided in two different activities: <b>used fuel</b> (recycling in MOX for a reuse), and </b>life-ending of nuclear installations</b> (dismantling, redevelopment of areas).<br />
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<b>Because of the huge financial and technological means</b> needed for the development of a business in the electronuclear field, <b>threat of new competitors (new businesses incoming into the market) is low</b>. Consequently, just a few large groups share the four lines of business, even at the world scale (for example AREVA, EDF, GE Energy or Mitsubishi). Suppliers, operators and customers inside the pattern are interconnected, and are quite often subsidiaries of these multinational companies. Competition is very strong because contracts are rare and huge.<br />
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<b>Thus,sources of pressure on this field are numerous and varied</b>. Political influence on the electronuclear field is quite important: in France, nuclear holds a paramount place, but <b>politics can at any moment decide to favor other ways of electricity production, as in Germany (solar, wind turbines, etc.)</b>. Therefore role of public power in different countries and of international organizations (for example AIEA, ANDRA) is crucial, because those are the ones which will decide, fix rules and directives. The application of agreements against the global warming by public power (Kyoto, Copenhagen) can also impact directly and positively on the electronuclear field, this one giving out no CO2, to produce electricity. <b>However, there are currently two main problems: the becoming of nuclear wastes</b> (for now, stocking of wastes of low, medium and high activity), and <b>the risk of an accident whose consequences would be disastrous for environment and population</b>. Voted laws restrict this field to allow a permanent control, avoid accidents and protect populations. Indeed, from a social outlook, <b>the apprehension facing this strength and the different accidents which occurred is still present</b> <a href="https://2011.igem.org/Team:Lyon-INSA-ENS/Safety/Intro"> (Safety)</a>. Nevertheless, because of the increase of the price of fossil energies (petroleum, gas) and thanks to the researches on new generations of more efficient reactors, the electronuclear field keeps its competitiveness on the energy market.<br />
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