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

Revision as of 11:33, 1 November 2017

iGEM UCLouvain Team iGEM UCLouvain Team

Auxotrophic Approach

BactaSun Overview

Auxotrophic approach details

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).

*Figure 1 : the photo-uncaging of the ONB-Tyr (https://static.igem.org/mediawiki/2016/2/24/T--Aachen--onbtyrosincleavagereaction.png-modified)*

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.

*Figure 2 : the composition of the BBa_K577882 biobrick (http://partsregistry.org/Part:BBa_K577882))*

*Figure 3 : description of the biobricks contained in BBa_K577882 biobrick)*

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!

... Auxotrophic Approach Results

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-					<p>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 <i>ortho</i>-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).</p>
+					<p>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 <i>ortho</i>-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).</p>
 <figure>
 					<img src="https://static.igem.org/mediawiki/2017/a/a3/UCLouvain_Overview2.png" class="in_text_img" style="width: 500px;">
 <figcaption> <center> <font size = "2"><i> Figure 1 : the photo-uncaging of the ONB-Tyr (https://static.igem.org/mediawiki/2016/2/24/T--Aachen--onbtyrosincleavagereaction.png-modified)</i></font></figcaption>
 </figure>
+<br>
-					<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, <i>Discosoma striata</i> and gives a red color.</p>
+					<p>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, <i>Discosoma striata</i> and gives a red color.</p>
-					<p>We used a pBad promoter to induce the RPF expression when the biomass is sufficient to produce a good signal.</p>
+					<p>We used a pBad promoter to induce the RPF expression with arabinose when the biomass is sufficient to produce a visible indicator.</p>
 			<figure>
 					<img src="https://static.igem.org/mediawiki/2017/b/b1/UCLouvain_App1_2.png" class="in_text_img" style="width: 500px;">
@@ Line 37: / Line 38: @@
 </figure>
-					<p>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 avalaible for protein synthesis.</p>
+					<p>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.</p>
-					<p>Finally, when the badge is exposed to UV rays, the tyrosine is uncaged, making it avalaible for the RFP synthesis, and the solution turns red!</p>
+					<p>Finally, when the badge is exposed to sunlight, the tyrosine is uncaged, making it avalaible for the RFP synthesis, and the solution turns red!</p>
+<img src="https://static.igem.org/mediawiki/2017/0/0e/UCLOUVAIN_-APPRAOCH1_VISUAL.jpeg" class="in_text_img" style="width: 500px;">
                                          <a href="https://2017.igem.org/Team:UCLouvain/OurProject/Approach1/Results" class="btn blue" style="float:right;"><span>... Auxotrophic Approach Results </span></a></div>
 				</div>