Exp1: test for flipping and cross-talk

The first experiment is design to test if integrases and excisionases pairs can flip a target sequence in both directions in a controlled manner. Then, we will test if these integrases are specific by putting them in presence of non-target sites. If this first set of experiment is successfull, we will then perform a kinetic analysis of flipping, by varying time of induction and concentrations of recombinases.

In a first time we will characterize 3 Integrases/Excisionases pairs.

=Experimental design=

Steady state experiment: Does it flip ?
E.coli for simplicity
 * organism

A target "flipper" plasmid bearing a T7 promoter flanked by specific attP and attB or attL and AttR recombination sites in antiparallel orientation will be cotranformed in e. coli cells with a plasmid expressing the corresponding integrase, or the integrase and excisionase pair. Each target sequence will be flanked on each side by a gene encoding a different fluorescent protein, enabling the monitoring of promoter orientation.
 * Brief description



Cells will be allowed to grow and to reach steady stte of the reaction and the pourcentage of flipping cells will be determined. In a second time, integrases and excisionases will be tested for their ability to act on non-cognate sites (cross talk).


 * Constructs

First, we work with standards parts. We need to choose which Biobrick format we want to use (see here).

Standard chosen is Freiburg: allow protein fusion, compatible with BBa

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