Team:Paris/August 20

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

Spreading the clones in order to obtain single colonies
The plates obtained from the speading of yesterday can't be used because there are not single colonies. We have to try again, but with a stronger dilution of the bacteria or with a smaller volume of spreading.
 * Resuspension of some bacteria from the glycerol stock into 1 mL of LB+antibiotic
 * Dilution 10 and dilution 100
 * Spreading of 100 µL of each dilution on a LB plate containing the right antibiotic
 * Overnight incubation (37°C)

=Miniprep and stock glycerol of stable strains with biobricks 2008=


 * Protocol stocks
 * Protocol Miniprep

=Construction for FIFO=

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

Measurement of concentration of minipreps
standard protocol

Digestion
Protocol Digestion

Gel Extraction
Protocol

=> Following a mistake, the right E0430(D166) and E0432(D167) digestion, have not been purified. So we need to repeat the same digestion experiment tomorrow morning.

=Construction of: promotor-rbs-LasR-dbl ter=

PCR Screening
Protocol

Minipreps
=Promoter characterization plasmids=

Ligation
Our ligations from yesterday didn't work. The positive control for transformation worked.

Digestion
We had a problem with a gel extraction so we have to make again the digestions from yesterday, Other digestions made:

Protocol Digestion

D179 D180 D181 D182 D183 D184 D185

=Sequencing= *We succeed for FlgA promoter *FlgB and flhB don't match with our expected sequences. We decided to cut our Miniprep product with other enzymes to check our sequence.
 * We received results of our sequencing from COCHIN

Digestion
Protocol
 * D176 : pFlgB digested with ApoI
 * D177 : pFlgB digested with NruI
 * D178 : pFlhB digested with BstAPI

Screening
Gel 1‎ Gel 2

Conclusion => the sequence of pFlgB and pFlhB are not good we will tried to isolate pFlhB again.