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Team:Lyon-INSA-ENS/Realisation/Week6
From 2011.igem.org
Week 6
From Monday the 18th of July to Friday the 22nd of July 2011
Monday
Strain construction
Concerned parts : RBS-GFP-LVA (Cn),BBa_J23119 ( 18A, plate 1 )(Amp), Curli (Cn), ompR234 (Cn)
Start of 5mL liquid cultures from a Petri dish culture ( 5mL sterile LB, 50µL antibiotic ,bacteria). Incubation at 37°C
Later, start of 150mL liquid cultures from the previous 5mL cultures ( 150 mL sterile LB ,300 µL from the grown 5mL bacterial culture, 1.5 mL antibiotic ( Amp or Cn ) )
Incubation at 37°C overnight.
10X concentration and plating of 100 µL of transformed bacteria from the previous week : 2M+24E ( amp ), Curli + GFP ( Kan ), Kan + 2L ( Kan ), positive control ( Puc18 plasmid ), negative control ( water )
Incubation at 37°C
Tuesday
Strain construction
Midiprep to extract DNA from the previous liquid cultures.
Nanodrop quantification :
OmpR234 : 59.5 ng/µL
GFP : 109.5 ng/µL
18A : 73.7 ng/µL
Curli : 82.6 ng/µL
The 260/280 ratio is lower than 2 in all cases.
PCR and mutagenesis of rcn, csgBA, csgEFG
A critical error in part CsgAB and a less important error in part CsgEFG were detected by sequencing. Restart mutagenesis on another plasmid with part CsgAB without the fatal error. Restart construction of part csgEFG from the beginning. Another clone of CsgEFG is sent to sequencing .Part RcnR is correct.
Wednesday
Strain construction
Digestion of several biobricks : RBS, GFP, YFP, Curli, OmpR234.
Electrophoresis to control the digestions.
PCR and mutagenesis of rcn, csgBA, csgEFG
Mutagenesis on the other clone of part CsgAB worked. Digestion with DpnI to eliminate parental plasmids. On the order hand, PCR with Pfu polymerase on MC4100 to amplify part CsgEFG did not work. Retry with extracted DNA of MC4100.
Thursday
A french radio, RCF, interviewed us.
Strain construction
Standard or 3A ligation of the previously cut biobricks. TSS transformation into NM522.
PCR and mutagenesis of rcn, csgBA, csgEFG
Transformation of NM522 with the mutated plasmid of part csgAB.
Friday
Strain construction
Start of 5mL liquid cultures of selected individual colonies containing the biobricks.
Extraction of the plasmids to test the presence of the insert.
PCR and mutagenesis of rcn, csgBA, csgEFG
Liquid culture of transformed colonies with csgAB for miniprep
