As described above, the hybrid promoter functions the opposite of how we eventually want it to; namely, we want nitrogenase to be highly repressed in light conditions (where a lot of oxygen will be present to poison nitrogenase), and highly expressed in the dark. Therefore we need to create a genetic switch that functions essentially as a NOT gate, where in high light conditions, our product (currently EYFP), will be highly repressed.
Different switches based on RNA have been well documented in E. coli; we have several options. First we are testing the repression mechanism using a basic pTet/TetR, repression mechanism. By expressing TetR, the system driven by the hybrid promoter should show strong repression in light, and strong expression in dark.

Our next step would have been to swap out the pTet/TetR repression with a system of CRISPR interference to block transcription initiation or elongation based on binding to the template or non-template strand of a chosen sequence [9]. In our case, we would tune the sgRNA to bind to the -35 box of our hybrid promoter in order to block transcription. The reason we want to use CRISPRi is because it has been shown to be functional in both prokaryotes and eukaryotes, although slightly more complex, this becomes a much more powerful tool once people transition to eukaryotic environments. Both of these genetic switches should be able to highly repress our fluorescent protein when induced with light.

Meanwhile, once the other half of our iGem team gets nif cluster working in E. coli, we should be able to swap out the fluorescent protein for the nif cluster in our plasmid to regulate the nif cluster in E. Coli with light. In dark conditions, the switch will be “off”, namely there should be fluorescence; and in the light, the system should be repressed. We expect to see higher levels of repression with our hybrid promoter and greater dynamic fold change.

Figure above: Original Light induction experiment once all the cloning was over. No fluorescence seen.

After all our cloning mishaps we finally were able to test our light induction system. However, nothing worked as expected. The data represented above is the fluorescence normalized to the absorbance, or OD of the strains we grew. We essentially had the same fluorescence as our negative control, and even when we induced with 250ng/mL of ATC we saw no fluorescence. This led us to run a few more experiments to troubleshoot the plasmids.

Our original positive control was under control of constitutive promoter, J23100, and was the only thing we got to fluoresce. We were concerned with competing translation start sites so we cloned a plasmid that modified the RBS to allow for EYFP to fold properly, but fluorescence only got weaker. We then decided to swap out the pTet promoter and lo- and behold got fluorescence. Our next step was to swap out the reporter mechanism of our experimental plasmids.

Figure above: Final Light experimental data