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Team:Lyon-INSA-ENS/Realisation/Week3
From 2011.igem.org
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.
