Team:Lyon-INSA-ENS/Realisation/Week3

From 2011.igem.org

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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/>
<b>Culture</b> of NM522 cells for later transformations and Curli for extraction. <br/><br/>
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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/>
<b>CaCl2 chemical transformation</b> ( V=10mL, 2µL DNA ) of some iGEM kit distribution DNA : <br/><br/>
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2L (GFP, Amp ) <br/><br/>
2L (GFP, Amp ) <br/><br/>
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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>Selection</b> of white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.<br/><br/>
<b>Selection</b> of white colonies on the plate for part RcnR and CsgEFG. Liquid culture for miniprep.<br/><br/>
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<FONT COLOR="red"> <b>Strain construction</b> </FONT> <br><br>
<b>Miniprep</b> using the QuickPure kit, digestion and electrophoresis of the Curli plasmid.<br/><br/>
<b>Miniprep</b> using the QuickPure kit, digestion and electrophoresis of the Curli plasmid.<br/><br/>

Revision as of 15:04, 13 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


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.









ENS assystem Biomérieux INSA INSA