Team:ETH Zurich/expresults/integrases

From 2014.igem.org

Integrases

The design of our XOR gates was based on integrase logic^[9]. 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. This approach is explained here.

In order to characterize the integrase system described above, we first combined the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] with one of our integrase genes bxb1, followed directly by a red fluorescent protein (RFP, mCherry) to make the expression accessible. Also, this system includes an XOR buffer gate per default blocking transcription of sfGFP. Upon BXB1 activity and switching the gate into ON-state, the terminator should have been removed and sfGFP should have been expressed. We intially 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 flow cytometer, did not indicate sfGFP expression due to recombinase activity. Nevertheless, RFP was clearly detectable upon induction in plate reader experiments suggesting that the induction itself worked (see fig.4 and fig.5).

Fig. 4 Expression of red fluorescent protein (RFP) and green fluorescent protein (GFP) 250 min after induction with 3OC6-HSL. RFP is under the control of the [http://parts.igem.org/Part:BBa_R0062 pLux promoter (BBa_R0062)] together with the recombinase BXB1. Upon expression of BXB1 a buffer gate should have been swtiched to ON-state producing GFP. Data points are mean values of triplicate measurements ± standard deviation.

Fig. 5 . Data points are mean values of triplicate measurements ± standard deviation.

As our constructs did not show the expected functionality, we decided to directly use the plasmids described by Bonnet et al.^[9] which where obtained from addgene ([http://www.addgene.org/44456 Dual-recombinase-controller], [http://www.addgene.org/44453/ XOR gate-V2.0]). However, we were using a [http://www.openwetware.org/wiki/E._coli_genotypes#TOP10_.28Invitrogen.29 TOP10] strain not expressing TetR by default (as compared to [http://www.expressys.com/main_strains.html 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 instead of proprietary defined medium ([http://teknova-blog.com/hi-def-azure-media Teknova Hi-Def Azure medium]). As of today, this set-up did not allow us to get the integrase XOR gate running. 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.