Paris/July 23

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Latest revision as of 17:49, 7 October 2007

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 mutagenesis DGAT

DGAT has Pst1 restriction site. We want to mutagenize it to do a biobrick.

PCR : DGAT1

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		1087
50	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	27 ForDGAT1	2.5µL	YES
2m00'	Oligo R 10µM	13 DPst1-DGAT-R	2.5µL	Image (click to enlarge)
Number cycles	Water	33µl
35	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	Miniprep pKS::DGAT (1µL = 138ng)		1

PCR : DGAT2

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL		396
50	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	12 DPst1-DGAT	2.5µL	YES
2m00'	Oligo R 10µM	27 Rev-DGAT1	2.5µL	Image (click to enlarge)
Number cycles	Water	33µl
35	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	Miniprep pKS::DGAT (1µL = 138ng)		2

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.

@@ Line 1: / Line 1: @@
+[[Paris/July 22|yesterday]] -- [[Paris/July 24|tomorrow]] <br>
 == w121 growth ==
@@ Line 67: / Line 68: @@
 |- style="background: #cccccc;"
 | style="background: #ccffcc;" |D24
-|lox71-ftsZ bw-insert
-|lox71-ftsZ PCR product P7
-|XbaI
-|PstI
-|
-|lox71-ftsZ(unmutated) ready for use as a backward insert
-|
-|
-|- style="background: #cccccc;"
-| style="background: #ccffcc;" |D25
 |eCFP bw-insert
 |<bbpart>BBa_E0422</bbpart> (MP6.1 & MP6.2)
@@ Line 86: / Line 77: @@
 |
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |D26
+| style="background: #ccffcc;" |D25
 |PoPs to GFP converter
 |<bbpart>BBa_E0421</bbpart> (MP7.1 & MP7.2)
@@ Line 96: / Line 87: @@
 |
 |- style="background: #cccccc;"
-| style="background: #ccffcc;" |D27
+| style="background: #ccffcc;" |D26
 |gfp-tripart
 |<bbpart>BBa_E0840</bbpart> (MP8.1 & MP8.2)
@@ Line 103: / Line 94: @@
 |
 |gfp-tri part (strongRBS-ORF-T) ready for use as a backward insert
+|
+|
+|- style="background: #cccccc;"
+| style="background: #ccffcc;" |D27
+|lox71-ftsZ bw-insert
+|lox71-ftsZ PCR product P7
+|XbaI
+|PstI
+|
+|lox71-ftsZ(unmutated) ready for use as a backward insert
 |
 |
 |}
 == PCRs ==
@@ Line 127: / Line 129: @@
 |Band=
 |}}
 {{Paris_PCR_0| Title = Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2
@@ Line 147: / Line 150: @@
 |Band=
 |}}
+{{Paris_PCR_0| Title = B0030-DapAColi
+|Annealing= 60°C
+|Elongation= 3m00'
+|Cycles= 35x
+|Buffer=  5x 10µL
+|MgCl2= 10µM 0µL
+|dNTP= 10µM 1µL
+|n_oligoF= o20 RBS-DapAColi
+|v_oligoF= 2.5µL
+|n_oligoR= o7 DapAColi-R
+|v_oligoR= 2.5µL
+|water= 34µl
+|pol= Phusion 0.5µL
+|DNA= Toothpick in MG16655 Glycerol
+|Size= 952
+|Success=
+|Image=
+|Band=
+|}}
+{{Paris_PCR_0| Title = B0030-DapASubtilis
+|Annealing= 60°C
+|Elongation= 3m00'
+|Cycles= 35x
+|Buffer=  5x 10µL
+|MgCl2= 10µM 0µL
+|dNTP= 10µM 1µL
+|n_oligoF= o20 RBS-DapASubtilis
+|v_oligoF= 2.5µL
+|n_oligoR= o9 DapASubtilis-R
+|v_oligoR= 2.5µL
+|water= 34µl
+|pol= Phusion 0.5µL
+|DNA= Toothpick in MG16655 Glycerol
+|Size= 946
+|Success=
+|Image=
+|Band=
+|}}
+== PCR mutagenesis DGAT ==
+DGAT has Pst1 restriction site. We want to mutagenize it to do a biobrick.
+{{Paris_PCR_0| Title = DGAT1
+|Annealing= 50
+|Elongation= 2m00'
+|Cycles= 35
+|Buffer=  5x 10µL
+|MgCl2= 10µM 0µL
+|dNTP= 10µM 1µL
+|n_oligoF= 27 ForDGAT1
+|v_oligoF= 2.5µL
+|n_oligoR= 13 DPst1-DGAT-R
+|v_oligoR= 2.5µL
+|water= 33µl
+|pol= Phusion 0.5µL
+|DNA= Miniprep pKS::DGAT (1µL = 138ng)
+|Size= 1087
+|Success= YES
+|Image= DGAT107232007.jpg
+|Band= 1
+|}}
+<br>
+{{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= 396
+|Success= YES
+|Image= DGAT107232007.jpg
+|Band= 2
+|}}
+<br>
+== 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 [[Paris/July_18#E.coli_pKS::DGAT|July 18]]).
+* Observation:
+We can observe E.coli single cells (100X).
+[[Image: coli_dgat_07232007.jpg|center|900px]]
+* 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.
+[[Image: coli_dgat_death_07232007.jpg|center]]

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