Team:Paris/August 26

From 2008.igem.org

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|pFliA+rbs
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|325 bp
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|style="background: #cbff7B"| ~ 350 bp
|style="background: #cbff7B"| ~ 350 bp

Revision as of 17:39, 27 August 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 nothing 3 nothing nothing
PCR 148 OmpR* 4 779 bp 800 bp
T 148 nothing 5 nothing nothing 
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

  • pFlhDC (O111-F / O113-R)
  • Gene FlhDC (O134-F / O131-R)
  • pFlgB (O102-F / O103-R)
  • 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

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) - PCRFliA1' (O148-F / O152-R)
  • PCRFliA2 (O153-F / O150-R)
  • PCRFliA3 (O148-F / O150-R)
PCRFliA1
PCRFliA2
PCRFliA1'
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 50-60 210 bp ~ 180 bp
PCRFliA2 Downstream part of FliA 28-53 670 bp ~ 650 bp
PCRFliA3 Mutated FliA 62-71 800 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

Name Digestion n° [DNA] in µg/mL A260/A280
EnvZ* D159 10 µg/mL 1.35
pSB1A2 D116 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 Name Description
MP1.1 S1.1 Icon rbs.png
RBS
MP1.2 S1.2
MP1.1 S1.1 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP+ LVA+ term
MP1.2 S1.2
MP1.1 S1.1 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP+ LVA- term
MP1.2 S1.2
MP1.1 S1.1 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ YFP LVA+ term
MP1.2 S1.2
MP1.1 S1.1 Icon rbs.pngIcon reporter.pngPart icon terminator.pngPart icon terminator.png
RBS+ ECFP LVA- term
MP1.2 S1.2

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

File:KR000.JPG
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)
Measured size (pb)


Screening of the cloning of pFlgA-GFP Generator

Electrophoresis

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) 876 1 126
measured size
File:KR000.jpg
Screening of L164

Minipreps and glycerol stock

Miniprep Glycerol Stock Ligation Name
MP1 S1 L164 FlgA-rbs-GFP-dbl ter
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