Construction for Synchronization

Transformation of the ligations we did yesterday

Construction of pFlgA - GFP Generator

Aim : Construction of "pFlgA-RBS-GFP-dbl ter" (pFlgA-E0240)

Results of the transformation we did the day before yesterday

=> Need to screen to know which clones we can use for the  of  pFlgA promotor characterization.

Cloning of EnvZ*

The sequencing of EnvZ* previously cloned, revealed a loss of about 300 bp. EnvZ* contains indeed an EcoRI restriction site within its sequence. So we can't use this enzyme during the cloning.

Digestion

Reaction mixture

• 4 µL of PCR129 (or 2 µL of MP108)
• 3 µL of 10X buffer n°2
• 0,3 µL of 100X BSA
• 1 µL of XbaI
• 1 µL of PstI
• 20,7 µL (or 22,7 µL) of water

Incubation at 37°C during 2H25, and then ~20 min at 65°C

Electrophoresis

Gel after excision

1% agarose gel

• EnvZ*: 3 µL of digestion products + 1 µL of loading blue + 2 µL of water
• pSB1A2: 30 µL of digestion products + 6 µL of loading blue

Purification

• EnvZ*: digestion product purified directly by Qiagen kit
• pSB1A2: excision of the band (2 kb) from the gel and purification by the Qiaquick Gel Extraction kit

elution in 30 µL of buffer EB
then kept at - 20°C

Promoter characterization plasmids

Transformation results: ligations from August 21th

Top 10 cells were used

Protocol

Screening of the cloning of pFlgA-YFP Tripart (LVA+/-)

Electrophoresis

Minipreps and glycerol stock