Team:Paris/August 26

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

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

Electrophoresis settings

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

Results of the electrophoresis
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



 * 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)



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'

=Cloning of EnvZ*=

Ligation

 * 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=


 * Protocol stocks
 * Protocol miniprep

=Construction of pFlhB - mRFP Tripart (LVA+)=

Aim : Construction of  "pFlhB-RBS-mRFP-dbl ter" (pFlhB-I732078) 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
Protocol Digestion

Gel Verification
Protocol

=Screening of the cloning of pFlgA-GFP Generator=

Minipreps and glycerol stock
=Construction for Synchronisation=