Difference between revisions of "Team:UCLouvain/Approach1"
| Line 23: | Line 23: | ||
<img | <img | ||
| − | src="https://static.igem.org/mediawiki/2017/8/86/UCLOUVAIN_-_uncaging_of_ONB-Tyr.png" class="in_text_img" style="width : 10 px;"> | + | src="https://static.igem.org/mediawiki/2017/8/86/UCLOUVAIN_-_uncaging_of_ONB-Tyr.png" class="in_text_img" style="width: 10 px;"> |
<p>We placed the ONB-Tyr inside a liquid cell culture. Our cell culture is a tyrosine auxotroph (TyrA-) E. coli culture. Those cells contain a plasmid with a red fluorescent protein (RFP) gene. We used the RFP gene from the BBa_K577882 Biobrick, developed by Boston University and Wellensly College for iGEM 2011 (Fig 2). It consists of a pBad promoter, a RBS, a RFP, and a terminator. The RFP originally comes from a coral, Discosoma striata and gives a red color. </p> | <p>We placed the ONB-Tyr inside a liquid cell culture. Our cell culture is a tyrosine auxotroph (TyrA-) E. coli culture. Those cells contain a plasmid with a red fluorescent protein (RFP) gene. We used the RFP gene from the BBa_K577882 Biobrick, developed by Boston University and Wellensly College for iGEM 2011 (Fig 2). It consists of a pBad promoter, a RBS, a RFP, and a terminator. The RFP originally comes from a coral, Discosoma striata and gives a red color. </p> | ||
Revision as of 23:51, 30 October 2017
Our aim is to create a rapid UV-response system, so people can be rapidly warned of an UV-exposure. 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 cage, the ONB molecule, is cleaved when exposed to UV rays, and releases the tyrosine 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 contain a plasmid with a red fluorescent protein (RFP) gene. We used the RFP gene from the BBa_K577882 Biobrick, developed by Boston University and Wellensly College for iGEM 2011 (Fig 2). It consists of a pBad promoter, a RBS, a RFP, and a terminator. The RFP originally comes from a coral, Discosoma striata and gives a red color.
We used a pBad promoter to induce the RFP 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, except 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 available for RFP synthesis, and the solution turns red!
