Paris/July 23

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 +
[[Paris/July 22|yesterday]] -- [[Paris/July 24|tomorrow]] <br>
== w121 growth ==
== 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:  
at 12h30, the following culture was launched:  
*w121 in 2ml LB-Erythro-DAP
*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 ==
== Transduction of DapA deletion in MG1655 ==
Line 13: Line 20:
* <bbpart>BBa_J23107</bbpart>
* <bbpart>BBa_J23107</bbpart>
-
== Anealing of lox66 and lox71 ==
 
-
== Ligations and transformations==
+
== 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
 +
 
 +
 
 +
{|
 +
|- style="background: #ccccff;"
 +
! colspan="5" style="background: #ccccff;" | Digestion Products
 +
|- style="background: #ccccff; text-align: center;"
 +
|width=5%| Number
 +
|width=20%| Product Name
 +
|width=30%| Matrix Name
 +
|width=5%| Enzyme 1
 +
|width=5%| Enzyme 2
 +
|width=5%| size
 +
|width=35%| Description
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D22
 +
|pSB1A2 open vector
 +
|<bbpart>BBa_J61047</bbpart> (MP9.1 & MP9.2)
 +
|EcoI
 +
|PstI
 +
|
 +
|Open pSB1A2 vector for BioBrick EcoRI/PstI insertio
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D23
 +
|AraC/pBad promoter fw-insert
 +
|<bbpart>BBa_I0500</bbpart> (MP4.1 & MP4.2)
 +
|EcoI
 +
|SpeI
 +
|
 +
|AraC/pBad promoter ready for use as a forward insert
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D24
 +
|eCFP bw-insert
 +
|<bbpart>BBa_E0422</bbpart> (MP6.1 & MP6.2)
 +
|XbaI
 +
|PstI
 +
|
 +
|eCFP (RBS-OFR_LVA-T) ready for use as a backward insert
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D25
 +
|PoPs to GFP converter
 +
|<bbpart>BBa_E0421</bbpart> (MP7.1 & MP7.2)
 +
|XbaI
 +
|PstI
 +
|
 +
|PoPs to GFP converter ready for use as a backward insert
 +
|
 +
|
 +
|- style="background: #cccccc;" 
 +
| style="background: #ccffcc;" |D26
 +
|gfp-tripart
 +
|<bbpart>BBa_E0840</bbpart> (MP8.1 & MP8.2)
 +
|XbaI
 +
|PstI
 +
|
 +
|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 ==
 +
 
 +
{{Paris_PCR_0| Title = Lox71-FtsA-FtsZ
 +
|Annealing= 60
 +
|Elongation= 3m00'
 +
|Cycles= 35
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 3 Lox71-FtsA-F
 +
|v_oligoF= 2.5µL
 +
|n_oligoR= 2 FtsZ-R
 +
|v_oligoR= 2.5µL
 +
|water= 34µl
 +
|pol= Phusion 0.5µL
 +
|DNA= Toothpick in MG16655 Glycerol
 +
|Size= 2058
 +
|Success=
 +
|Image=
 +
|Band=
 +
|}}
 +
 
 +
 
 +
{{Paris_PCR_0| Title = Assembly PCR Lox71-FtsA-FtsZ-1 + FtsZ-2
 +
|Annealing= 70°C (5x) without oligos + 60°C (35x)
 +
|Elongation= 3m00'
 +
|Cycles= 40x
 +
|Buffer=  5x 10µL
 +
|MgCl2= 10µM 0µL
 +
|dNTP= 10µM 1µL
 +
|n_oligoF= 3 Lox71-FtsA-F
 +
|v_oligoF= 2.5µL
 +
|n_oligoR= 2 FtsZ-R
 +
|v_oligoR= 2.5µL
 +
|water= 34µl
 +
|pol= Phusion 0.5µL
 +
|DNA= 8.5µl Lox71-FtsA-FtsZ-1 (~50ng) + 17.5µl FtsZ-2 (~50ng)
 +
|Size= 2058
 +
|Success=
 +
|Image=
 +
|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]]

Latest revision as of 17:49, 7 October 2007

yesterday -- tomorrow

Contents

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


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 DGAT107232007.jpg
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 DGAT107232007.jpg
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).

Coli dgat 07232007.jpg


  • 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.

Coli dgat death 07232007.jpg
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