Team:Paris/August 15

=Construction of OmpR*+RBS and EnvZ*+RBS= I did some digestions (today), ligations (tommorow) and screening (the day after tommorow). I tried to build : RBS (B0034) + OmpR* and RBS (B0034) + EnvZ*

Protocol

 * X µL of Template DNA
 * Buffer (n°2) 10X : 3µL
 * BSA 100X : 0.3µL
 * Pure water qsp 30 µL
 * 1 µL of each enzyme


 * Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes). Then 10°C overnight.

=Creation of a registry of pFliL, pFlhDC, and FlhDC=

Transformation of the ligations we did yesterday
We transformed L 143, L 144 and the negative controls T1 and T2, using Invitrogen's TOP10 chemically competent cells standard protocol.

Protocol
We followed the standard protocol of amplification in Two steps. PCR program used : PHUSION2

Results
Settings Gel 1%
 * Ladder 1 kb 10 µL
 * 4µL Template DNA + 2µL Loading Blue
 * 1 % Agar

We managed to amplify flhDC !

Settings Gel 2%
 * Ladder 100 bp 10 µL
 * 4µL Template DNA + 2µL Loading Blue
 * 2 % Agar

We managed to amplify the promoter of flhDC

Digestions
After having succeded in amplifying the promoter and the gene of flhDC, we decided to clone it into a plasmid. The first step is the digestion.

Protocol

 * X µL of Template DNA
 * Buffer (n°2) 10X : 3µL
 * BSA 100X : 0.3µL
 * Pure water qsp 30 µL
 * 1 µL of each enzyme

Then 10°C overnight.
 * Incubate during about 3h at 37°C, then 20 minutes at 65°C (to inactivate the enzymes).

=Construction of pLas-TetR-GFP tripart & rbs-LasR-dble ter=

Measurement of concentration of minipreps
standard protocol

Digestion
Protocol Digestion

Ligation
Protocol Ligation

=PCR screening of cloning of E0240, flhD, flhC and pflhDC+flhDC=

Analysis of yesterday PCR screening
Electrophoresis
 * 1% agarose gel
 * 10 µL loaded

Gel 1 Gel 2 Gel 3

=Starting the construction of the Promoter characterization plasmid=

Measurement of concentration of minipreps
standard protocol

Digestion
Protocol Digestion