Team:CU-Boulder/Transform

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• Refinement •

Summary

The first step to improve our visualization for our photocages would be to change from the eGFP from last year which required a similar excitation laser to the wavelength which should shift cis- AzoPhe to its trans- isomer. We decided it would be best to use a red fluorescent protein. However, as our visualization depends very much on puncta of fluorescence, we wanted to be very sure that the RFP was not forming dimers or tetramers, as the wild type RFP is known to do. Even the engineered monomeric RFPs such as mCherry have been seen to sometimes dimerize, and so we elected to use the highly engineered non-dimerizing Fusion Red protein (https://www.ncbi.nlm.nih.gov/pubmed/23149748).
We cloned the Fusion Red vector into BioBrick standards using routine PCR, digest, and ligation, in the process adding the EutC 1-19 amino acid tag for compartment localization. Initial visualization showed that Fusion Red is expressed at very high levels within the cell, preventing us from distinguishing the compartment-localized protein from the background. To eliminate background, we decided to add a LVA protein degradation tag, reasoning that the protease to which LVA targets proteins should not be able to reach the compartment internal Fusion Red.

This new protein construct functioned as well as we had hoped, demonstrating only bright red fluorescence inside of EutS compartments. With this new protein, our visualization system was now greatly improved over that of the CU Boulder 2016 iGEM team, and we could decisively improve the characterization of last year’s compartments.

Better still, we were able to provide this system to our collaborating iGEM team in Bielefeld, who was able to replicate the same results in their lab. This was particularly triumphant for our team, as until now our plasmids have only been shown to work in our lab under our specific conditions. Validation in another lab on the other side of the world means that future iGEM teams should be able to use EutS compartments for their own projects without too much headache!

Figure 1: EutC 1-19 tagged Fusion Red without LVA tag on pSB1C3, co-transformed with EutS on pSB4A5.

Figure 2-3: EutC 1-19 tagged Fusion Red with new LVA tag in EutS Compartments, made by co- transforming both EutS on pSB4A5 and Fusion Red on 1C3.