"
Team:Lyon-INSA-ENS/Realisation/Week7
From 2011.igem.org
Week 7
From Monday the 25th of July to Friday the 29th of July 2011
Monday
Strain construction
Miniprep of some other parts from the transformed bacteria. Digestion and electrophoresis to control the presence of the right insert.
PCR and mutagenesis of rcn, csgBA, csgEFG
Miniprep of bacteria with mutated part CsgAB. Digestion to check restriction profile (removal of intern PstI site).
Tuesday
Adherence Tests
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.
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.
Strain construction
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)
PCR and mutagenesis of rcn, csgBA, csgEFG
Send of plasmid with the right profile for part CsgAB to sequencing. Extraction of new MC4100 strain's DNA. PCR with fresh DNA and Taq Phusion.
Wednesday
Strain construction
Midiprep and nanodrop of the following ligated or newly obtained plasmids :
NiCoT : 112,2 ng/µL
2M-2L-Tet : 109.6 ng/µL
2M-2L-Kan : 128 ng/µL
18A-OmpR234 : 59.5 ng/µL
rcn-Curli : 116 ng/µL
2M-24E : 92.3 ng/µL
Fermentas digestion : 2M-2L-Tet (X+P), 2M-2L-Kan (X+P), rcn-curli(E+P), 2M-24E(X+P), Curli(S+P)
PCR and mutagenesis of rcn, csgBA, csgEFG
PCR did not work.
Thursday
Strain construction
Standard ligation of :
-Prcn into Cn backbone
-rcn-curli into Cn backbone
-Pcurli into 2M-2L-Kan
-Pcurli into 2M-2L-Tet
TSS transformation into NM522.
PCR and mutagenesis of rcn, csgBA, csgEFG
PCR with genomic DNA of MC4100 with Taq DNA Polymerase and Phusion
Friday
PCR and mutagenesis of rcn, csgBA, csgEFG
PCR did not work. New try with different concentrations of primers (half, normal and twice) with Taq DNA polymerase and Phusion.
