Team:Paris/August 21

= Cloning of FlhB promoter=

Protocol
Resuspension of 1 colony E.coli K12 strain MG 1655 in 100µl of water.
 * Preparation of the template :

1µl of dNTP 10µl Buffer Phusion 5X 2.5µl O 108 2.5µl O 109 1µl Template 1µl Phusion 33µL pure water
 * Preparation of PCR mix :

Result


=Construction for FIFO=

Aim : Construction of pFlgA - YFP tripart (+/- LVA)  "pFlgA-RBS-YFP-dbl ter" (pFlgA-E0430/E0432)

Digestion
Protocol Digestion

Gel Extraction
Protocol

Measurement of the concentration of D166 & D167 purified
Protocol (it's same that for Miniprep)

Ligation
Protocol

=Screening of the cloning of E0240 and FlhDC+promotor=

We obtained colonies isolated with the dilution 1/100; 13 clones were analysed by PCR

PCR screening
reaction mixture (25 µL) PCR screening programm
 * 12,5 µL Quick load PCR Mixture 2X
 * 0,5 µL O18
 * 0,5 µL O19
 * 11,5 µL water
 * elongation time: 1 min 30
 * primers: O18 and O19
 * positive control: S158 (pSB3K3)
 * negative control: no template

Electrophoresis



 * 1% agarose gel
 * 10 µL loaded

Results: *The clone of E0240 (S159.1) always have several bands amplified by PCR. It might contain different plasmids. *The clone of FlhDC+promotor (S161.1) don't have the correct size band. It also doesn't have the insert in the plasmid.

=Construction for synchronization=

Ligations
Protocol

=Promoter characterization plasmids=

Ligations from digestions from 20th
Top 10 cells were used

Protocol