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

Measurement of the concentration of D187 purified

Protocol (it's same that for Miniprep)

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

Cloning of EnvZ* in pSB1A2

Transformation results

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

Cloning of OmpR*

Digestion

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

Name of the digestions

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.

Ligation

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

EcorI/PstI digestion of mutated 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 !