Paris/July 17

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Latest revision as of 17:48, 7 October 2007

PCRs

The assembly PCRs did not work... we'll try them again !
The Lox66-DapAColi PCRs worked very well in all conditions

=> Gel purification

PCR : Lox71-FtsZ

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL
55°C	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	1 Lox71-FtsZ-F	10µM 2.5µL	YES
2m30'	Oligo R 10µM	2 FtsZ-R	10µM 2.5µL	Image (click to enlarge)
Number cycles	Water	34µL
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	toothpick in glycerol stock of MG1655		1-2

PCR : Lox66-DapA-Subtilis

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL
55°C	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	8 Lox66-DapASubtilis-F	10µM 2.5µL	YES
2m30'	Oligo R 10µM	9 DapASubtilis R	10µM 2.5µL	Image (click to enlarge)
Number cycles	Water	34µL
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	0.5µL of Subtilis DNA from stock (DB tube in fridge)		3-4

PCR : Assembly PCR DGAT

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL
55°C	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM		10µM 2.5µL	NO
2m30'	Oligo R 10µM		10µM 2.5µL	Image (click to enlarge)
Number cycles	Water	34µL
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	1µL of PCR product DGAT 1 1µL of PCR product DGAT 2		7-8

PCR : Assembly PCR FtsZ-FtsA

PCR Settings	Buffer (5x)	5x 10µL		Expected size
Annealing (°C)	MgCl₂ 10µM	10µM 0µL
55°C	dNTP 10µM	10µM 1µL		Success
Time Elongation	Oligo F 10µM	3	10µM 2.5µL	NO
2m30'	Oligo R 10µM	2	10µM 2.5µL	Image (click to enlarge)
Number cycles	Water	34µL
30	Polymerase	Phusion 0.5µL		Band (0=ladder)
	DNA	1µL of PCR product FtsZ 1 1µL of PCR product FtsZ 2		5-6

I make a gel to check PCR products. To put on gel :

50µL of PCR product
5µL of loading buffer.

As the precedent one didn't work, we decided to change our way to do it. We made the PCR reactions within two main steps :

the 2 PCR products are used as a matrix and added to the PCR mix without the primers ! - then 15 typical cycles
We add the primers to the PCR mix and run the total for 30 cycles.

We tested the PCR construction. It didn't give anything !

DGAT = a smear + 1 band at 100kb (primers)
FtsZ = nothing at all...

Let's do it again tomorow i order to find out where the problem could be from.

DAP solution contamination test

We spread on LB plates DAP solution :

50µL of H₂0 (control)
50µL of aliquot DAP 50mM (in use)
50µL of DAP 1mM
50µL of DAP 0.2 mM
50µL of DAP 50mM

+ filtration of the DAP stock
See Results.

Transduction of MG1655 with P1 stock made on w121

As it still didn't work, we try for now with culture of MG1655 in exponential growth rate (ExpGR)
OD=0.6

Preparation of samples:
- Control (900µL LB MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 5µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 50µL Phage + 900µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
- 500µL Phage + 500µL LB (MgSO4 30mM; CaCl2 15mM) + 100µL MG1655 Culture ExpGR
20mn @ 37°C = adsorption
Centrifugation 1mn @ 10000rpm
Resuspension in LB + Na Citrate 20mM + DAP 300µM
1H growth @ 37°C
Plate on LB + Na Citrate + Erythromycin + DAP

Transformations of Biobricks : results

Transformation of biobricks : we obtained hundreds of clones for each biobrick. Transformation in DH5alpha subcloning efficiency
Spread on LB-Amp

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)

Transduction of w121 strain

Titration of the stock of phage from 12.7.7.
See results.

Digestions

We did as follow :

25µL of plasmid miniprep
15,5µL H20
5µL of NE2 Buffer 10X (Biolabs)
0,5µL of BSA 100X
2µL of Enzyme1 (Biolabs)
2µL of Enzyme2 (Biolabs)

We have 2 clones for each plasmid, we digested both as follow :

pJ23100 with EcoR1/Spe1 and Spe1/Pst1
pJ23107 with EcoR1/Spe1 and Spe1/Pst1
I0500 with Ecor1/Pst1
B0015 with EcoR1/Pst1
Lox66 DapA E.coli (PCR product) with EcoR1/Pst1 and Xba1/Pst1

After 3 hours of digestion, running on a 1% agarose gel.
We cut the bands and put them all at -20°C for next morning.

Exponential culture of E.coli pKS::DGAT and microscopy

Protocol

- dilution 1/200 of the liquid culture overnight of E.coli pKS::DGAT and E.coli control (see July 16).

- incubation 2hours 37°C

- add:

oleate : 0/0.5mM/2mM
IPTG : 0/0.5mM

- incubate 1 hour 37°C

- add Nile Red (0.5µg/mL medium)

- microscopy pictures

Result:

No differences between the different culture condition (+-IPTG / +-oleate / E.coli transformed or not).

We decide to test after overnight culture: no result.

@@ Line 1: / Line 1: @@
+[[Paris/July 16|yesterday]] -- [[Paris/July 18|tomorrow]] <br>
 == PCRs ==
@@ Line 5: / Line 6: @@
 => Gel purification
-{{Paris_PCR_0| Title = FtsZ-E.Coli
+{{Paris_PCR_0| Title = Lox71-FtsZ
 |Name= Ftsz-E.Coli
 |Annealing= 55°C
@@ Line 22: / Line 23: @@
 |Size=
 |Success= YES
-|Image=
+|Image=Paris_17_07-GEL03.jpg
-|Band=
+|Band=1-2
 |}}
-{{Paris_PCR_0| Title = DapA-Subtilis
+{{Paris_PCR_0| Title = Lox66-DapA-Subtilis
 |Name= DapA-Subtilis
 |Annealing= 55°C
@@ Line 43: / Line 44: @@
 |Size=
 |Success=YES
-|Image=
+|Image=Paris_17_07-GEL03.jpg
-|Band=
+|Band=3-4
 |}}
@@ Line 64: / Line 65: @@
 |Size=
 |Success=NO
-|Image=
+|Image=Paris_17_07-GEL03.jpg
-|Band=
+|Band=7-8
 |}}
@@ Line 85: / Line 86: @@
 |Size=
 |Success=NO
-|Image=
+|Image=Paris_17_07-GEL03.jpg
-|Band=
+|Band=5-6
 |}}
@@ Line 143: / Line 144: @@
 Titration of the stock of phage from 12.7.7.
 <br>
+See [[Paris/July_18#Transduction of w121 strain|results]].
 == Digestions ==
 We did as follow : <br>
-µL of plasmid miniprep<br>
+*25µL of plasmid miniprep<br>
-,5µL H20<br>
+*15,5µL H20<br>
-µL of NE2 Buffer 10X (Biolabs)<br>
+*5µL of NE2 Buffer 10X (Biolabs)<br>
-,5µL of BSA 100X<br>
+*0,5µL of BSA 100X<br>
-µL of Enzyme1 (Biolabs)<br>
+*2µL of Enzyme1 (Biolabs)<br>
-µL of Enzyme2 (Biolabs)<br>
+*2µL of Enzyme2 (Biolabs)<br>
 <br>
 We have 2 clones for each plasmid, we digested both as follow :
@@ Line 162: / Line 165: @@
 <br>
 We cut the bands and put them all at -20°C for next morning.
+== Exponential culture of E.coli pKS::DGAT and microscopy ==
+* Protocol
+- dilution 1/200 of the liquid culture overnight of E.coli pKS::DGAT and E.coli control (see [[Paris/July_16#Plasmid_pKs::DGAT_expression_in_E._Coli|July 16]]).<br>
+- incubation 2hours 37°C <br>
+- add:
+# oleate : 0/0.5mM/2mM
+# IPTG : 0/0.5mM <br>
+- incubate 1 hour 37°C <br>
+- add Nile Red (0.5µg/mL medium) <br>
+- microscopy pictures <br>
+* Result:
+No differences between the different culture condition (+-IPTG / +-oleate / E.coli transformed or not).
+We decide to test after overnight culture: no result.

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