From 2007.igem.org

Contents 1 Plasmid pKs::DGAT expression in E. Coli 2 Growth kinetics of w121 strain 3 Transduction of MG1655 with P1 stock made on w121 4 MiniPreps 5 PCR purification 6 PCRs

Plasmid pKs::DGAT expression in E. Coli

We tried to see TG with NR died on E. Coli cells transfected with pKs::DGAT, an IPTG-inducible promoter. We tried different growth media containing more or less oleate, that should in theory increase TG synthesis.

Results : we don't see the inductible effect of IPTG. We can think that :

• Either the fluorescence without IPTG is due to a leak of the promoter
• Either DGAT is not induce in presence of IPTG, and the fluorescence we see is only a background.

Growth kinetics of w121 strain

Results of the previous day : we lost everything because of a crash of the computer :( Sorry Eimad !

Transduction of MG1655 with P1 stock made on w121

• Control (1mL LB MgSO4 30mM; CaCl2 15mM)
• 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
• 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON
• 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ON

=>For the n^th time, it is not working : we have only contaminants.

MiniPreps

• I0500 clones 1, 2
• pJ23107 clones 1, 2

PCR purification

• Lox71-FtsZ1
• FtsZ2
• DGAT1
• DGAT2

PCRs

The Lox66-DapAColi PCR did not work... We'll try again with different annealing temperature

Assembly PCRs: Lox71-FtsA-FtsZ-1 + FtsZ-2 DGAT-1 + DGAT-2