Team:ETH Zurich/expresults/integrases

From 2014.igem.org

Integrases

The design of our XOR gates was based on integrase logic (ref bonnet). This means, depending on the input molecules integrases can be expressed, subsequently switch a terminator sequence previously blocking gene expression, and then the output gene can be transcribed. The details of this approach are explained here (ref recombinase gates).

In order to characterize the integrase system described above, we first combined the pLux promoter with one of our integrase genes followed directly by mCherry to have a direct measure of expression. Also, this system includes an XOR buffer gate per default blocking transcription of sfGFP. Upon integrase activity and switching the gate into ON-state, the terminator should have been removed and sfGFP should have been expressed. We designed three different constructs for characterization of the recombinases and their cross-activity. However, the measurement of fluorescent proteins, with both a plate reader and a FACS (ref), did not indicate GFP expression even though mCherry was clearly detectable upon induction in platereader experiments.

PUT GRAPHS HERE

As our constructs did not show the expected functionality, we decided to directly use the plasmids described by Bonnet et al. which where obtained from addgene (ref, ref). However, we were using a TOP10 strain not expressing TetR by default (as compared to DH5alphaZ1) and as a result our strain had to be co-transformed with an additional plasmid encoding TetR. Also, we used defined M9 medium with 0.4% glycerol and 1%CAA (ref) instead of proprietary defined medium (Teknova Hi-Def Azure medium). As of today, this set-up did not allow us to get the integrase XOR gate running. Anyhow, we are not giving up on this and are proceeding with debugging our construct further and hope to find a solution until the Giant Jamboree in Boston.