Difference between revisions of "Team:UCLouvain/OurProject/Auxotrophs Overview"

Our aim is to create a rapid UV-response system, so people can be rapidly warned of potentially harmful exposure to sunlight. For this reason, we’ve decided to work with a photocaged amino acid. One of our PIs, Professor Patrice Soumillon, has already worked with those compounds during his doctoral thesis and knows how efficiently they work. We’ve used the ortho-nitrobenzyl tyrosine (ONB-Tyr) for its relatively low price compared with other photocaged amino acids. The oNB cage is cleaved when exposed to UVs (312nm), and releases the tyrosine for protein synthesis (Fig 1).

We placed the ONB-Tyr inside a liquid cell culture. We studied a tyrosine auxotroph (TyrA-) E. coli strain. Those cells contain a plasmid including a gen encoding for a red fluorescent protein (RFP). This plasmid was furnished in the iGEM DNA distribution kit (BBa_K577882), developed by Boston University and Wellensly College in 2011 (Fig 2). The RFP originally comes from a coral, Discosoma striata and gives a red color.

We used a pBad promoter to induce the RPF expression with arabinose when the biomass is sufficient to produce a visible indicator.

We worked with a liquid M9 minimal medium, adding ONB-Tyr. As our chassis is Tyrosine auxotroph, it can’t produce the RFP, except when exposed to UV light making the uncaged tyrosine is avalaible for protein synthesis.

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