Team:UCLouvain/OurProject/Auxotrophs Overview

Auxotroph approach details

Our aim is to create a rapid UV-response system, so people can be rapidly warned of an UV-exposure. For this reason, we decided to work with a photocaged amino acid. One of our PIs, Professor Patrice Soumillon, had already worked with those compounds during his doctoral thesis and knew how efficiently they work. We used the ortho-nitrobenzyl tyrosine (ONB-Tyr) for its relatively low price to compare with others photocaged amino acids. The cage, the ONB molecule, is cleaved when exposed to UV rays, and let the tyrosine free for protein synthesis (Fig 1).

We placed the ONB-Tyr inside a liquid cell culture. Our cell culture is a tyrosine auxotroph (TyrA-) E. coli culture. Those cells are containing a plasmid with a red fluorescent protein (RFP) gene. For that, we used the biobrick BBa_K577882, developed by the Boston University and Wellensly College for iGEM 2011 (Fig 2). It is composed of an pBad promoter, a RBS, a RFP, and a terminator. The RPF originally comes from a coral, Discosoma striata and gives a red color.

We used a pBad promoter to induce the RPF expression when the biomass is sufficient to produce a good signal.

We worked with a liquid M9 minimal medium, adding ONB-Tyr. As our chassis is Tyr auxotroph, it can’t produce the RFP, expected when exposed to UV rays, as the uncaged tyrosine is free for protein synthesis.

Finally, when the badge is exposed to UV rays, the tyrosine is uncaged, making it free for the RFP synthesis, and the solution turns red!

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