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 7 Transformations of Biobricks

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 (0.5 and 2mM), that should in theory increase TG synthesis.

Results : we don't see the inducible 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.

We can also wait to see accumulation of TG. Indeed in Acinetobacter, the process is pretty long (2-4 days). It could be the same for E.coli transformed by pKS::DGAT.

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

The Lox66-DapAColi PCR did not work... so we try a gradient of annealing temperatures

PCR : Lox66-DapAColi
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
50-65°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	6 Lox66-DapAColi-F	10µM 2.5µL		YES
2m00'		Oligo R 10µM	7 DapAColi-R	10µM 2.5µL		Image (click to enlarge)
Number cycles		Water	34µL
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	toothpick in glycerol stock of MG1655			1 to 6

Transformations of Biobricks

As in the protocol given in the registry. Transformation of biobricks :

• BBa_B0030 pSB1A2 : RBS (well 3G plate 1)
• BBa_E0422 pSB1A2 : ECFP (RBS+LVA+Term) (well 11G plate 1)
• BBa_E0241 pSB1A2 : PoPs to GFP converter (well 15c plate 2)
• BBa_E0840 gfp tri-part; strong rbs (well 16E plate 1)
• BBa_J61047 pSB1A2 : Cre ORF (well 8P plate 4)

Transformation in DH5alpha subcloning efficiency
Spread on LB-Amp