Team:Paris/August 26

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=Extraction of EnvZ* and OmpR* from ''E. coli'' genome=
=Extraction of EnvZ* and OmpR* from ''E. coli'' genome=
==Name of the PCR==
==Name of the PCR==
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==Protocol==
==Protocol==
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|style="background: #cbff7B"|  ~ 1000 bp
|style="background: #cbff7B"|  ~ 1000 bp
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=Cloning of EnvZ*=
=Cloning of EnvZ*=
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*transformation of TOP10 competent cells with 5 µL of ligation product
*transformation of TOP10 competent cells with 5 µL of ligation product
*spreading on LB plates + ampicilline and incubation overnight at 37°C
*spreading on LB plates + ampicilline and incubation overnight at 37°C
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='''Miniprep and stock glycerolof New Biobrick'''=
='''Miniprep and stock glycerolof New Biobrick'''=
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|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_regulatory.png]]<br>gfp generator - pTet  
|[[Image:Icon_rbs.png]][[Image:Icon_reporter.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_terminator.png]][[Image:Part_icon_regulatory.png]]<br>gfp generator - pTet  
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='''Construction of pFlhB - mRFP Tripart (LVA+)'''=
='''Construction of pFlhB - mRFP Tripart (LVA+)'''=
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='''Screening of the cloning of pFlgA-GFP Generator'''=
='''Screening of the cloning of pFlgA-GFP Generator'''=
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FlgA-GFP generator
FlgA-GFP generator
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='''Construction for Synchronisation'''=
='''Construction for Synchronisation'''=

Latest revision as of 17:59, 11 September 2008

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Contents

Extraction of EnvZ* and OmpR* from E. coli genome

Name of the PCR

Name What's in ? Template DNA Oligo F Oligo R
PCR 147 EnvZ* S 120 O 126 O 127
PCR 148 OmpR* S 119 O 138 O 139
T 147 nothing no template O 126 O 127
T 148 nothing no template O 138 O 139

Protocol

Protocol

  • 10 µL Phusion HF Buffer 5X
  • 2.5 µL Oligo F 10 mM
  • 2.5 µL Oligo R 10 mM
  • 1 µL dNTP
  • 1 µL Template DNA
  • 33 µL H2O

PCR Programme

  • 98°C 30s Initial denaturation
  • CYCLE 30X
  • 98°C 10s Denaturation
  • 60°C 30s Annealing
  • 72°C 45s Elongation
  • END OF CYCLE
  • 72°C 5min Terminal Elongation

Resuts

Results of the cloning of EnvZ* and OmpR*

Electrophoresis settings

  • Gel 1% agar
  • 10µL Ladder 1kb
  • 10µL Ladder 100bp
  • 4µL DNA + 2µL Loading Blue

Results of the electrophoresis

Name Gene Well Expected size Measured size
PCR 147 EnvZ* 2 1412 bp 1400 bp
T 147 no sample 3
PCR 148 OmpR* 4 779 bp 800 bp
T 148 no sample 5 
Conclusion : All the PCR worked perfectly well !

Cleaning of the PCR products

Standard protocol 

The cleaned PCR products are stored in the freezer overnight.


PCR Promoters and Genes FlhDC/FliA

PCR Promoters FlhDC and Gene

  • PCR 137 = pFlhDC (O111-F / O113-R)
  • PCR 141 = Gene FlhDC (O134-F / O131-R)
  • PCR 125 = pFlgB (O102-F / O103-R)
  • PCR 126 = pFlhB (O108-F / O109-R)

Program: Gradient
Denaturation :
98°C-5'
Cycling 1 (3X) :
98°C-10"
Gradient 61°C +/-10°C - 30
 72°C-30"
Cycling 2 (25X) :
98°C-10"
72°C-30"
Elongation :
72°C-5'

Vf=20µL
H20=13,4µL
Buffer5X=4µL
dNTP=0,4µL
O1=1µL
O2=1µL
Phusion=0,2µL

PCR Promoters FliA

(49-60)PCR promoter FliA & Promoter+Gene
  • pFliA (rbs) (O145-F / O144-R)
  • pFliA (O145-F / O146-R)
  • pFliA +Gene FliA (O145-F / O151-R)

Program: promoter
Denaturation :
98°C-5'
Cycling 1 (3X) :
98°C-10"
55°C - 30
 72°C-30"
Cycling 2 (30X) :
98°C-10"
65°C-30" 72°C-30"
Elongation :
72°C-5'

Vf=50µL
H20=33,5µL
Buffer5X=10µL
dNTP=1µL
O1=2,5µL
O2=2,5µL
Phusion=0,5µL

PCR mutagenesis FliA

  • PCRFliA1 (O143-F / O152-R)
  • PCR 145 = PCRFliA1' (O148-F / O152-R)
  • PCR 146 = PCRFliA2 (O153-F / O150-R)
  • PCRFliA3 (O148-F / O150-R)
(1-24)PCRFliA1
(25-48)PCRFliA2
(61-72)PCRFliA1'
(73-84)PCRFliA3


Program: PCRFliA1
Denaturation :
98°C-5'
Cycling 1 (30X) :
98°C-10"
Gradient 66°C +/-6°C - 25
 72°C-20"
Elongation :
72°C-5'

Program: PCRFliA1'
Denaturation :
98°C-5'
Cycling 1 (30X) :
98°C-10"
72°C-20"
Elongation :
72°C-5'

Program: PCRFliA2
Denaturation :
98°C-5'
Cycling 1 (3X) :
98°C-10"
Gradient 61°C +/-10°C - 25
 72°C-20"
Cycling 2 (30X) :
98°C-10"
72°C-30"
Elongation :
72°C-5'

Program: PCRFliA3
Denaturation :
98°C-30'
Cycling 1 (3X) :
98°C-10"
72°C-30"
Cycling 2 (5X) :
98°C-10"
98°C->72°C low decreasing 1.0°C/s
72°C-30"
Break - Add Oligo O148/O150
Cycling 3 (20X) :
98°C-10"
72°C-30"
Elongation :
72°C-5'

Name Promotor Well Expected size Measured size
PCRFliA1 Upstream part of FliA 2-27 197 bp ~ 150 bp
PCRFliA1' Upstream part of FliA 61-72 210 bp ~ 180 bp
PCRFliA2 Downstream part of FliA 28-53 670 bp ~ 650 bp
PCRFliA3 Mutated FliA 73-84 800 bp ~ 800 bp
PCR pFliA+rbs 54-56 325 bp ~ 350 bp
PCR PFliA 57-58 310 bp ~ 320 bp
PCR pFliA+Gene FliA 59-60 1100 bp ~ 1000 bp


Cloning of EnvZ*

Concentration measurement by Biophotometer

Digestion n° Name [DNA] in µg/mL A260/A280
D159 EnvZ* 10 µg/mL 1.35
D116 pSB1A2 17 µg/mL 1.02

Ligation

control insert / vector mass ratio
1,8 / 1 2,4 / 1 3,1 / 1
D159 EnvZ* (µL) 0 6 8 8
D116 pSB1A2 (µL) 2 2 2 1,5
10X T4 ligase (µL) 2 2 2 2
T4 DNA ligase (µL) 1 1 1 1
water (µL) 15 9 7 7,5
  • 5 hours at room temperature
  • transformation of TOP10 competent cells with 5 µL of ligation product
  • spreading on LB plates + ampicilline and incubation overnight at 37°C


Miniprep and stock glycerolof New Biobrick

Miniprep Strain Antiobiotic Name Description
MP101.1 S109.1 Amp J23101 Part icon regulatory.png
constitutive promotor
MP101.2 S109.2
MP101.1 S109.1 Amp J23101 Part icon regulatory.png
constitutive promotor
MP101.2 S109.2
MP104.1 S111.1 Amp R0040 Part icon regulatory.png
ptetR repressible
MP104.2 S111.2
MP104.1 S111.1 Amp R0040 Part icon regulatory.png
ptetR repressible
MP104.2 S111.2
MP105.1 S112.1 Amp S03154 Icon rbs.pngIcon reporter.png
RBS-lasI
MP105.2 S112.2
MP105.1 S112.1 Amp S03154 Icon rbs.pngIcon reporter.png
RBS-lasI
MP105.2 S112.2
MP143 S157 Amp E0240 (in pSB1A2) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS GFP dbl term
MP1151 S150 Amp L122
D107 (BV) - D130 (BI) 		Icon rbs.pngIcon reporter.pngIcon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS-lasI - ECFP
MP157 S156 Kan L138 (E0240 in pSB3K3) Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
gfp generator
MP158.1 S160.1 Amp L141 Part icon regulatory.png
pFlhD
MP158.2 S160.2
MP158.3 S160.3
MP171 S171 Amp L167
D181 (BV) - D184 (BI) 		Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.pngPart icon regulatory.png
gfp generator - pTet


Construction of pFlhB - mRFP Tripart (LVA+)

Aim : Construction of "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078) Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
We can only do the construction with mRFP Tripart (LVA+) because the stable strain with the Biobricks I732011 (mRFP Tripart LVA-) don't to growth.

Digestion

Digestion

Protocol Digestion

Name Template DNA Description Vol MP (µl) Vol H2O (µl) Enzymes
D187 MP168.1 RBS - mRFP - term (FV) 9.00 15.7 EcoRI and XbaI

Gel Verification

Protocol

Gel Verification of D187 digestion
Well 1 2 3 4 5 6
Sample 100 pb ladder MP168.1 no sample D187 no sample
Expected size (pb) 2 955 2 940
Measured size (pb) 2 900 2 900


Screening of the cloning of pFlgA-GFP Generator

Electrophoresis

Screening of L164
well n° 1 2 3 4 5 6 7 8 9
sample 1kb ladder MP172.6 MP143.1 L161.1 L161.2 L161.3 L161.4 L161.5 L161.6
expected size (pb) 973 1176 1 426
measured size 900 1 100 1 300 1 300 1 300 1 300 1 300 1 300

Minipreps and glycerol stock

Miniprep Glycerol Stock Ligation Antibotic Name
MP175.1 S177.1 L164.1 Amp Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

FlgA-GFP generator

MP175.2 S177.2 L164.2 Amp Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

FlgA-GFP generator

MP175.4 S177.4 L164.4 Amp Part icon regulatory.pngPart icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png

FlgA-GFP generator


Construction for Synchronisation

Results of the transformation we did yesterday

Ligation name Description Antibio Number Colonies observed Fluorescence Comments
Ligations
L159 D109(FI) - D125(FV)
rbs-lasI - double terminator 		Kan - - to do again
L162 D107(BV) - D163(BI)
rbs-lasI - gfp generator 		Amp - - to do again
L162 D110(BV) - D163(BI)
rbs-TetR - gfp generator 		Amp - - to do again
Controls
TL159 D125(FV) Kan - - to do again
TL162 D107(BV) Amp - - to do again
TL163 D110(BV) AMp - - to do again
Positive Control pUC19 Amp No OK
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