Team:Paris/August 17

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

Analysis of the transformation we did yesterday
{|border="1" style="text-align: center"

Number of colonies and flurorescence

 * Ligation name
 * Description
 * Antibio
 * Number Colonies observed
 * Fluorescence
 * Comments
 * colspan="6" |Ligations
 * L150
 * D105(BV) - D146(BI) pLas - rbs-TetR-GFP Tripart
 * Amp
 * 3
 * No
 * style="background: #ff6d73"|Problem
 * L151
 * D147(FI) - D125(FV) rbs-LasR - Double terminator
 * Kan
 * 21
 * No
 * style="background: #cbff7B"|OK
 * colspan="6" |Controls
 * C1
 * D105(BV)
 * Amp
 * 150
 * NO
 * style="background: #cbff7B"|OK
 * C2
 * D125(FV)
 * Kan
 * 2
 * No
 * style="background: #cbff7B"|OK
 * Positive Control
 * pUC19
 * Amp
 * 416 (efficiency 4.10^7)
 * No
 * style="background: #cbff7B"|OK
 * }
 * Positive Control
 * pUC19
 * Amp
 * 416 (efficiency 4.10^7)
 * No
 * style="background: #cbff7B"|OK
 * }

PCR Screening
Protocol

Conclusion : L150 doesn't success, measured size match with the promoter size only, we will check the size of L101 by screening and if it doesn't match with the good size (1827pb) we will try again the ligation : rbs-TetR (S03879) with GFP tripart (E0840). L151 is ok we have to put a strong promoter before the part rbs-lasR-double terminator :GREAT !

Minipreps & Stocks

 * Cultured in 7,5ml of LB with a thoothpick of a colony.
 * Culture O/N at 37°C of :

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

Evaluation of the number of colonies
Remarks: L143 and L144 did not work once again. Maybe there is a problem with the digestion, because the primers  used present only two nucleotides after the restriction sites. what we should do is cutting pfliL with xbaI and SpeI. As the vector will autoligate, we should use a phosphatase to prevent this phenomenon. For L147 and its control (T4), we do not observe any red fluorescence. It means that the digestion work well, because if it was not digested, the strong promoter J23100 should express mRFP! We will repeat all the ligation anyway.

=Construction of OmpR*+RBS and EnvZ*+RBS: transformation=