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	|n_oligoR= 13 DPst1-DGAT-R		|n_oligoR= 13 DPst1-DGAT-R
	|v_oligoR= 2.5µL		|v_oligoR= 2.5µL
-	|water= 34µl	+	|water= 33µl
	|pol= Phusion 0.5µL		|pol= Phusion 0.5µL
-	|DNA= Miniprep pKS::DGAT	+	|DNA= Miniprep pKS::DGAT (1µL = 138ng)
		+	|Size=
		+	|Success=
		+	|Image=
		+	|Band=
		+	|}}
		+
		+
		+	{{Paris_PCR_0| Title = DGAT2
		+	|Annealing= 50
		+	|Elongation= 2m00'
		+	|Cycles= 35
		+	|Buffer=  5x 10µL
		+	|MgCl2= 10µM 0µL
		+	|dNTP= 10µM 1µL
		+	|n_oligoF= 12 DPst1-DGAT
		+	|v_oligoF= 2.5µL
		+	|n_oligoR= 27 Rev-DGAT1
		+	|v_oligoR= 2.5µL
		+	|water= 33µl
		+	|pol= Phusion 0.5µL
		+	|DNA= Miniprep pKS::DGAT (1µL = 138ng)
	|Size=		|Size=
	|Success=		|Success=

---

Revision as of 10:15, 31 July 2007

Contents 1 w121 growth 2 Transduction of DapA deletion in MG1655 3 Migration of digestion products 4 MiniPrep 5 Digestion reactions 6 PCRs 7 E.Coli pKS::DGAT

w121 growth

w121 culture launched on 22/07/07 did not grow ON: I had forgot to add DAP to LB-Erythro growth medium.

at 12h30, the following culture was launched:

• w121 in 2ml LB-Erythro-DAP

We were unable to perform growth kinetics analysis of w121 in different DAP-supplemented media (because the machine is occupied for the night

Transduction of DapA deletion in MG1655

• The transduction seems to have worked: isolation of clones on LB and LB+DAP for verification.

Migration of digestion products

MiniPrep

• BBa_J23107

Digestion reactions

Each of the digestion reactions that follow were performed as follows:

• 20µL DNA solution (miniprep or PCR product)
• 5µL Buffer 10x
• 0.5µL BSA 100x
• 2µL of each of the 2 enzymes indicated
• H20 qsp 50µL

NEB3 buffer used for XbaI/PstI double digestion NEBEcoRI buffer used for both EcoRI/SpeI & EcoRI/PstI double digestion reactions

Digestion Products
Number	Product Name	Matrix Name	Enzyme 1	Enzyme 2	size	Description
D22	pSB1A2 open vector	BBa_J61047 (MP9.1 & MP9.2)	EcoI	PstI		Open pSB1A2 vector for BioBrick EcoRI/PstI insertio
D23	AraC/pBad promoter fw-insert	BBa_I0500 (MP4.1 & MP4.2)	EcoI	SpeI		AraC/pBad promoter ready for use as a forward insert
D24	eCFP bw-insert	BBa_E0422 (MP6.1 & MP6.2)	XbaI	PstI		eCFP (RBS-OFR_LVA-T) ready for use as a backward insert
D25	PoPs to GFP converter	BBa_E0421 (MP7.1 & MP7.2)	XbaI	PstI		PoPs to GFP converter ready for use as a backward insert
D26	gfp-tripart	BBa_E0840 (MP8.1 & MP8.2)	XbaI	PstI		gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert
D27	lox71-ftsZ bw-insert	lox71-ftsZ PCR product P7	XbaI	PstI		lox71-ftsZ(unmutated) ready for use as a backward insert

PCRs

PCR : Lox71-FtsA-FtsZ
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			2058
60		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3 Lox71-FtsA-F	2.5µL
3m00'		Oligo R 10µM	2 FtsZ-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Toothpick in MG16655 Glycerol

PCR : Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			2058
70°C (5x) without oligos + 60°C (35x)		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	3 Lox71-FtsA-F	2.5µL
3m00'		Oligo R 10µM	2 FtsZ-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
40x		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	8.5µl Lox71-FtsA-FtsZ-1 (~50ng) + 17.5µl FtsZ-2 (~50ng)

PCR : B0030-DapAColi
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			952
60°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	o20 RBS-DapAColi	2.5µL
3m00'		Oligo R 10µM	o7 DapAColi-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
35x		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Toothpick in MG16655 Glycerol

PCR : B0030-DapASubtilis
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL			946
60°C		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	o20 RBS-DapASubtilis	2.5µL
3m00'		Oligo R 10µM	o9 DapASubtilis-R	2.5µL		Image (click to enlarge)
Number cycles		Water	34µl			[[Image:|30px]]
35x		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Toothpick in MG16655 Glycerol

PCR : DGAT1
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
50		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	27 ForDGAT1	2.5µL
2m00'		Oligo R 10µM	13 DPst1-DGAT-R	2.5µL		Image (click to enlarge)
Number cycles		Water	33µl			[[Image:|30px]]
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Miniprep pKS::DGAT (1µL = 138ng)

PCR : DGAT2
PCR Settings		Buffer (5x)	5x 10µL			Expected size
Annealing (°C)		MgCl2 10µM	10µM 0µL
50		dNTP 10µM	10µM 1µL			Success
Time Elongation		Oligo F 10µM	12 DPst1-DGAT	2.5µL
2m00'		Oligo R 10µM	27 Rev-DGAT1	2.5µL		Image (click to enlarge)
Number cycles		Water	33µl			[[Image:|30px]]
35		Polymerase	Phusion 0.5µL			Band (0=ladder)
		DNA	Miniprep pKS::DGAT (1µL = 138ng)

E.Coli pKS::DGAT

We look under microscopy 5 days after incubation of E.coli transformed by pKS::DGAT and the control E.coli transformed by part B0015 on different LB medium (See July 18).

• Observation:

We can observe E.coli single cells (100X).

• Interpretation:

We can observe lipid inclusion into E.coli transformed by pKS::DGAT with IPTG induction. With or without adding oleate we do not observe significant differences. We could think that only dead cells are fluorescent (DGAT could kill the cells) but with cell death marker (green) we can see that cell death is not increased with DGAT.