Team:Paris/August 27

=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.

Measurement of the concentration of D187 purified
Protocol (it's same that for Miniprep)

=> the experiments of Gel Extraction have failed, so we need to repeat the step of digestion.

Digestion
Protocol Digestion

Gel Verification
Protocol

=Cloning of EnvZ* in pSB1A2=

PCR screening

 * screening programm
 * elongation time: 2 min
 * number of cycle: 24
 * total volume reaction: 25 µL
 * primers used: O18 and O19
 * positive control: S158 (pSB3K3)
 * negative control: no template

Electrophoresis


No correct clone The 8 other clones were also screened. PCR elongation time: 2 min 30 Electrophoresis Results: All the clones analysed were not correct.

=Cloning of OmpR*=

Determination of the concentration of DNA
We used the biophotometer
 * 5 µL of template DNA or 5 µL of EB buffer for th blank
 * 55 µL of pure water

Protocol of digestion

 * D 188 : 3 µL of PCR 148
 * D 189 : 3 µL of MP 101.2
 * 3µL Buffer (n°2) 10X
 * 0.3µL BSA 100X
 * 22.7 µL of pure Water
 * 1 µL of each enzyme


 * Incubate during about 2h30 at 37°C
 * 20 minutes at 65°C

Cleaning of the digestion products
Standard protocol.

Determination of the concentration of DNA
We used the biophotometer
 * 10 µL of template DNA or 10 µL of EB buffer for th blank
 * 50 µL of pure water

Protocol of ligation L171

 * 2 µL Ligase Buffer 10X
 * 1.5 µL D 189 (vector)
 * 5 µL D 188 (insert)
 * 11.5 µL pure Water (qsp 20 µL)
 * 1 µL T4 DNA ligase at 400 000 U/mL concentration
 * O/N at 16°C

=Checking mutagenesis FliA=

For this, i digested mutated FliA and non-mutated FliA with EcoRI and PstI and put in migration the digestion products running on gels. Results : No digestion for the mutated sequence --> successful mission !