Team:Lyon-INSA-ENS/Realisation/Week3

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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/Week3Fr">Version Fran&ccedil;aise</a>
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     <h1 style="color: white;"> Week 3 </h1>     
     <h1 style="color: white;"> Week 3 </h1>     
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     <p style="text-align : center"> <small> From Monday the 20th of June to Friday the 24th of June 2011 </small> </p>
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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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   <h6 style="text-align :left"> Monday </h6>
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   <h6 style="text-align :left"> Monday </h6> <HR>
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<p style = "line-height : 1.5em">
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<FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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One transformed colony seemed correct, the others were probably satellite colonies without the plasmid.<br/>
+
 
-
Start of 5mL liquid culture of the main colony. <br/>
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Another <b>PCR</b> is launched to collect more DNA from MC4100 (Failure).<br/>
-
Plating of the satellite colonies to check the presence of the plasmid <br/>
+
Alcoholic precipitation of PCR product from the previous week.<br/>
-
    </p>
+
 
 +
Culture of the wrong strains for the transformation...(Failure : the strain already had a plasmid)<br/>
 +
</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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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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<b>Culture</b> of NM522 cells for later transformations and Curli for extraction. <br/><br/><br/>
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</p>
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<p style = "line-height : 1.5em"><FONT COLOR="green"> <b>PCR and mutagenesis of rcn, csgBA, csgEFG</b> </FONT> <br><br>
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 +
<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.
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<h6 style="text-align :left"> Wednesday </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>
 +
 
 +
<b>CaCl2 chemical transformation</b> ( V=10mL, 2µL DNA ) of some iGEM kit distribution DNA : <br/><br/>
 +
 
 +
<U>Plate 1 : </U><br/>
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18A (constitutive promoter, Amp )<br/>
 +
2M ( strong RBS, Amp ) <br/>
 +
5L ( weak RBS, Amp )<br/>
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22M ( RBS+YFP, Amp)<br/><br/>
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<U>Plate 2 : </U><br/>
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24E (YFP, Amp + Kan ) <br/>
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2L (GFP, Amp ) <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 white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.<br/><br/>
 +
 
 +
<b>Transformation</b> in NM522 of part CsgAB.
 +
    </p>
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  <h6 style="text-align :left"> Tuesday </h6>
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<h6 style="text-align :left"> Thursday </h6> <HR>
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4 more new clones have grown : start of 5mL liquid cultures on these clones.
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    </p>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
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    <br/> <br/>  
+
<b>Miniprep</b> using the QuickPure kit, digestion and electrophoresis of the Curli plasmid.<br/><br/>
-
<h6 style="text-align :left"> Wednesday </h6>
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Start of liquid cultures of two individual clones for each of the 6 iGEM plasmids <br/>
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<p style=" line-height : 1.5em">
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<b>Miniprep</b> using the QuickPure kit of the 18A, 2M, 22M, 24E and 2L plasmids.<br/><br/><br/>
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Miniprep on the four clones. Verification of plasmid by digestion (EcoRI, HindIII)
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    </p>
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</p>
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<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 CsgEFG and RcnR. <b>Digestion</b> of plasmid with EcoRI to check part insertions. <br/>
 +
    </p>
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    <br/>
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    <p style = "line-height : 1.5em">
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The local newspaper "VIVA" interviewed us. They asked about our project, our team and about iGEM's organization.
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<h6 style="text-align :left"> Thursday </h6>
 
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PCR from genomic DNA from E.coli strain MC4100 for three genes : RcnR (cobalt receptor), CsgAB (curli operon, part 1) and CsgEFG (curli operon, part 2)
 
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<h6 style="text-align :left"> Friday </h6>
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<h6 style="text-align :left"> Friday </h6> <HR>
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<p style = "line-height : 1.5em">
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
 +
 +
<b>Miniprep</b> using the QuickPure kit of 5L plasmid. <br/><br/>
 +
 +
<b>Ozyme digestion</b> with :<br/>
 +
S + P : 18A, 2M, 5L<br/>
 +
X + P : 22M, 2L, 24E<br/><br/>
 +
 +
<b>Electrophoresis</b> of the digested plasmids versus the non digested.<br/>
 +
 +
-> All the strains had the correct plasmid : they were plated on LB + amp medium.<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>Miniprep</b> of clones with Csg AB. <b>Digestion</b> of plasmid with EcoRI. Send to GATC for <b>sequencing</b>.
 +
 +
 +
  </p>
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 +
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    <br/> <br/>
     <br/> <br/>
     <br/> <br/>
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    <p>
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              <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/Week4"/><font color="grey"><b>Next Week</b></font></a>
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Latest revision as of 23:34, 21 September 2011







Week 3


From Monday the 27th of June to Friday the 1st of July 2011







Monday

PCR and mutagenesis of rcn, csgBA, csgEFG

Another PCR is launched to collect more DNA from MC4100 (Failure).
Alcoholic precipitation of PCR product from the previous week.
Culture of the wrong strains for the transformation...(Failure : the strain already had a plasmid)



Tuesday

Strain construction

Culture of NM522 cells for later transformations and Curli for extraction.


PCR and mutagenesis of rcn, csgBA, csgEFG

Ligation of PCR products in pGem-T easy vector. Transformation in NM522 strain of the ligation product. Selection on ampicilline, X-Gal, IPTG plate. Only CsgAB was not transformed this day.





Wednesday

Strain construction

CaCl2 chemical transformation ( V=10mL, 2µL DNA ) of some iGEM kit distribution DNA :

Plate 1 :
18A (constitutive promoter, Amp )
2M ( strong RBS, Amp )
5L ( weak RBS, Amp )
22M ( RBS+YFP, Amp)

Plate 2 :
24E (YFP, Amp + Kan )
2L (GFP, Amp )


PCR and mutagenesis of rcn, csgBA, csgEFG

Selection of white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.

Transformation in NM522 of part CsgAB.





Thursday

Strain construction

Miniprep using the QuickPure kit, digestion and electrophoresis of the Curli plasmid.

Start of liquid cultures of two individual clones for each of the 6 iGEM plasmids
Miniprep using the QuickPure kit of the 18A, 2M, 22M, 24E and 2L plasmids.


PCR and mutagenesis of rcn, csgBA, csgEFG

Miniprep of clones with CsgEFG and RcnR. Digestion of plasmid with EcoRI to check part insertions.


The local newspaper "VIVA" interviewed us. They asked about our project, our team and about iGEM's organization.





Friday

Strain construction

Miniprep using the QuickPure kit of 5L plasmid.

Ozyme digestion with :
S + P : 18A, 2M, 5L
X + P : 22M, 2L, 24E

Electrophoresis of the digested plasmids versus the non digested.
-> All the strains had the correct plasmid : they were plated on LB + amp medium.


PCR and mutagenesis of rcn, csgBA, csgEFG

Miniprep of clones with Csg AB. Digestion of plasmid with EcoRI. Send to GATC for sequencing.







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ENS assystem Biomérieux INSA INSA
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