Team:Lyon-INSA-ENS/Realisation/Week4

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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/Week4Fr">Version Fran&ccedil;aise</a>
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     <h1 style="color: white;"> Week 4 </h1>     
     <h1 style="color: white;"> Week 4 </h1>     
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<br/>
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     <p style="text-align : center"> <small> From Monday the 27th of June to Friday the 1st of July 2011 </small> </p>
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     <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/>
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   <h6 style="text-align :left"> Monday </h6> <HR>
   <h6 style="text-align :left"> Monday </h6> <HR>
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     <br/>
     <br/>
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<p style = "line-height : 1.5em">
 
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Another PCR is launched to collect more DNA (Failure).<br/>
 
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Culture of the wrong strains for the transformation...(Failure : the strain already had a plasmid)<br/>
 
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Alcoholic precipitation of PCR product from the previous week.<br/>
 
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</p>
 
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    <br/> <br>
 
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  <h6 style="text-align :left"> Tuesday </h6> <HR>
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    <p style = "line-height : 1.5em">
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  <br/>
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<p style = "line-height : 1.5em">
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Culture of NM522 cells for later transformations and Curli for extraction. <br/><br/>
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Ligation of PCR products in pGem-T easy vector to make them standard parts. Transformation in NM522 strain of the ligation product. Selection on ampicilline, X-Gal, IPTG plate. Only CsgAB was not transformed this day.
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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    </p>
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    <br/> <br/>
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    <br/> <br/>  
+
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Additional <b>minipreps</b> using the QuickPure protocol of the same 6 parts ( 5 replica each ) <br/>
 +
<b>Digestion</b> as previously<br/><br/>
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<h6 style="text-align :left"> Wednesday </h6> <HR>
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<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/>
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    <br/>
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    <p style = "line-height : 1.5em">
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Miniprep, digestion and electrophoresis of the Curli plasmid.<br/><br/>
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Selection of white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.<br/><br/>
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Start of a 5mL culture of NM522 cells.<br/>
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    </p>
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Transformation in NM522 of part CsgAB.
 
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    </p>
 
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
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  <h6 style="text-align :left"> Tuesday </h6> <HR>
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    <br/>
 +
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    <p style = "line-height : 1.5em">
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
 +
 +
Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).<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/>
 +
 +
</p>
 +
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <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/>
 +
  </p>
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 +
    <br/> <br/>   
 +
    <br/> <br/> 
   
   
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<h6 style="text-align :left"> Thursday </h6> <HR>
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<h6 style="text-align :left"> Wednesday </h6> <HR>
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     <br/>
     <br/>
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      <p style = "line-height : 1.5em">
 
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CaCl2 chemical transformation of some iGEM kit distribution DNA : <br/><br/>
 
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<U>Plate 1 : </U><br/>
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    <div style="border:1px solid black;float:right;margin-right:6%;">
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18A (constitutive promoter, Amp )<br/>
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        <div style="border : 3px solid green;">
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2M ( strong RBS, Amp ) <br/>
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          <div style="border : 1px solid black;">
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5L ( weak RBS, Amp )<br/>
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              <img src="https://static.igem.org/mediawiki/2011/d/d3/Notebook-tournage.jpg" width="120px" />
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22M ( RBS+YFP, Amp)<br/><br/>
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          </div>
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        </div>
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    </div>
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<U>Plate 2 : </U><br/>
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  <p style = "line-height : 1.5em;width : 530px">
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24E (YFP, Amp + Kan ) <br/>
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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/>
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2L (GFP, Amp ) <br/><br/>
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  </p>
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Miniprep of clones with CsgEFG and RcnR. Digestion of plasmid with EcoRI to check part insertions. <br/>
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     <p style = "line-height : 1.5em; width : 530px">
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     </p>
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    <br/>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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    <p style = "line-height : 1.5em">
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Selection of individual transformed colonies and start of solid and liquid culture.<br/><br/><br/>
-
The local newspaper "VIVA" interviewed us. They ask about our project, our team and about iGEM's organization.
+
 
-
    </p>
+
</p>
 +
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <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.
 +
  </p>
 +
    <br/>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
     <br/> <br/>
-
 
+
<h6 style="text-align :left"> Thursday </h6> <HR>
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<h6 style="text-align :left"> Friday </h6> <HR>
+
-
 
+
     <br/>
     <br/>
 +
    <p style = "line-height : 1.5em">
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<p style = "line-height : 1.5em">
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
-
Miniprep using the QuickPure kit of the previous 6 iGEM parts. <br/><br/>
+
-
Digestion with :<br/>
+
<b>Miniprep</b> using the QuickPure protocol of the previous liquid cultures.<br/><br/>
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S + P : 18A, 2M, 5L<br/>
+
-
X + P : 22M, 2L, 24E<br/><br/>
+
-
Electrophoresis of the digested plasmids versus the non digested.<br/>
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<b>Ozyme digestion</b> of the plasmids by X+P.<br/><br/>
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All the strains had the correct plasmid : they were plated on LB + amp medium.<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/>
-
Miniprep of clones with Csg AB. Digestion of plasmid with EcoRI. Send to GATC for sequencing.
+
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/>
 +
</p>
 +
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <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.
   </p>
   </p>
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<h6 style="text-align :left"> Friday </h6> <HR>
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<p style = "line-height : 1.5em;width : 560px">
 +
<br/>
 +
</p>
 +
<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <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.
 +
</p>
 +
 +
    <br/> <br/>
 +
    <br/> <br/>
 +
    <br/> <br/>
 +
    <p>
 +
              <a  href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week3"/><font color="grey"><b>Previous Week</b></font></a>
 +
              <a  style = "float : right"; href="https://2011.igem.org/Team:Lyon-INSA-ENS/Realisation/Week5"/><font color="grey"><b>Next Week</b></font></a>
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              <br/>
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    <br/> <br/>
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Latest revision as of 23:34, 21 September 2011







Week 4


From Monday the 4th of July to Friday the 8th of July 2011







Monday

Strain construction

Additional minipreps using the QuickPure protocol of the same 6 parts ( 5 replica each )
Digestion as previously

Ligation 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.

Start of a 5mL culture of NM522 cells.





Tuesday

Strain construction

Start of a 50mL culture of NM522 from the overnight preculture ( 50mL sterile LB, 250µL of preculture ).
Transformation 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.


PCR and mutagenesis of rcn, csgBA, csgEFG

Sequencing results obtained. Mutagenesis of the plasmids with the least errors by quick change method to remove unwanted restriction sites (EcoRI or PstI).





Wednesday

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.


Strain construction

Selection of individual transformed colonies and start of solid and liquid culture.


PCR and mutagenesis of rcn, csgBA, csgEFG

Digestion of PCR/mutagenesis product with DpnI to eliminate parental plasmid. Transformation of NM522 strains with plasmid. Culture and selection on ampicilline plates.






Thursday

Strain construction

Miniprep using the QuickPure protocol of the previous liquid cultures.

Ozyme digestion of the plasmids by X+P.

Electrophoresis of the digested and non digested plasmids : we have observed either no DNA, no insert, or a plasmid with a correct size insert.
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.


PCR and mutagenesis of rcn, csgBA, csgEFG

Selection of clones on plate and liquid culture for miniprep. Digestion of purified PCR product in Tango buffer to have a better efficiency. Transformation of NM522 with purified plasmids.





Friday


PCR and mutagenesis of rcn, csgBA, csgEFG

Miniprep 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.







Previous Week Next Week






ENS assystem Biomérieux INSA INSA
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