Part:BBa_K1132037:Experience

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In vitro characterization of BBa_K1132034

The basic idea is to perform the switch with a cell lysis containing the recombinase and the plasmid containing the gate to be switched. After incubation, transformation of the plasmid containing the gate allows the quantification of switched versus non switched plasmids. Experiments have been performed as described in the protocol. http://2013.igem.org/Team:INSA_Toulouse/contenu/lab_practice/notebook/protocols/charac_recomb

Results

In the absence of recombinase, the colonies are white. As a result, non-specific recombinases cannot switch the AND gate.

We tested the AND gate with FimE. After incubation with FimE, the colonies are still white (this is normal!), therefore visual inspection of the switch was not possible. The only possibility was to sequence independent clones due to lack of time we did not have the results (see genotype section). We analyzed five independent colonies by sequencing, but none was switched. Several hypotheses can be made. First, the presence of the PhiC31 switching sequences may inhibit.

The AND was also tested with the Siuti’s construction parts containing PhiC31, but not switched has been obtained. Results might be due to low expression levels of PhiC31 that would lead to partial switch in in vitro conditions. A second explanation would be that the site we have designed is less efficient when the FimE recombinase has not yet performed the primary switch. More experiments are needed to verify PhiC31’s efficiency.

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