Team:CU-Boulder

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Latest revision as of 01:37, 4 October 2012

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Welcome to the CU-iGEM 2012 wiki! This is the first time the University of Colorado has competed in the International Genetically Engineered Machines competition, and we are excited to show you the science coming out of Boulder, Colorado! We are a team of five undergraduates advised by our two graduate mentors, and a smattering of post-docs. This year is also the grand opening of the brand new Biofrontiers building for bioscience, and biotechnology research. We have been fortunate enough to have lab space in the new building.

Quorum STOPPING

Quorum sensing is a system of bacterial communication that coordinates gene expression based on population density. Quorum sensing dictates many functions within bacteria such as when pathogenic gene products are released, biofilm formation, and food rot. Inhibiting quorum sensing became the goal of CU-Boulder’s 2012 iGEM team. This led us to find or create many interesting BioBrick parts such as Aiia, a protein that degrades specific quorums, Sdia, a transcription factor that is more sensitive to AHLs than the standard LuxR, and NucB, a nuclease that degrades preexisting biofilm.
Instead of using a pathogenic bacteria, our team utilized the Lux gene brick from Vibrio fischeri. This gram-negative bacteria uses quorum sensing to produce bioluminescence, while living in the gut of bobtail squid (shown above). By using V. fischeri, our team eliminated the risk of working with pathogenic bacteria and bioluminescence proved to be easily quantifiable using plate reader experiments. As none of the presubmitted Lux bricks worked for our team, we isolated each of the 5 essential genes (LuxA,B,C,D,and E) and well an extremely important bioluminescent gene, LuxG, from a V. fischeri strain to create our model system. For more detailed information on our project visit our Project page.

The video below is an animated overview of quorum sensing.

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+<br>
+<center><h2>Quorum STOPPING</h2></center>
+<br>
+<center>
+<img src="https://static.igem.org/mediawiki/2012/d/d1/Media_5a7257dc5595cb7be3df7954509f3e90.jpeg" width=290 height=193 />
+</center>
- <table width="100%" cellpadding="0" cellspacing="10" border="0">
+<br>
-    <tr>
+&nbsp;&nbsp;&nbsp;&nbsp;Quorum sensing is a system of bacterial communication that coordinates gene expression based on population density. Quorum sensing dictates many functions within bacteria such as when pathogenic gene products are released, biofilm formation, and food rot. Inhibiting quorum sensing became the goal of CU-Boulder’s 2012 iGEM team. This led us to find or create many interesting BioBrick parts such as Aiia, a protein that degrades specific quorums, Sdia, a transcription factor that is more sensitive to AHLs than the standard LuxR, and NucB, a nuclease that degrades preexisting biofilm. <br>
-        <td width="50%" valign="top">
+&nbsp;&nbsp;&nbsp;&nbsp;Instead of using a pathogenic bacteria, our team utilized the Lux gene brick from Vibrio fischeri. This gram-negative bacteria uses quorum sensing to produce bioluminescence, while living in the gut of bobtail squid (shown above). By using V. fischeri, our team eliminated the risk of working with pathogenic bacteria and bioluminescence proved to be easily quantifiable using plate reader experiments. As none of the presubmitted Lux bricks worked for our team, we isolated each of the 5 essential genes (LuxA,B,C,D,and E) and well an extremely important bioluminescent gene, LuxG, from a V. fischeri strain to create our model system. For more detailed information on our project visit our <a href="https://2012.igem.org/Team:CU-Boulder/Project">Project</a> page.
+<br><br>
-            <div align=center> <b> Bacterial Nightlight </b> </div> <br>
+The video below is an animated overview of quorum sensing.
+<br><br>
-            <div align=center> <img src="https://static.igem.org/mediawiki/2012/9/9b/Nemo.jpg"> </div>
+<center><object width="420" height="315"><param name="movie" value="http://www.youtube.com/v/YJWKWYQfSi0?version=3&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="http://www.youtube.com/v/YJWKWYQfSi0?version=3&amp;hl=en_US" type="application/x-shockwave-flash" width="420" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object></center>
-            <p>Certain organisms use bioluminescence for communication, mating, or intimidating predators.
-            Our project aims to harness the bioluminescence genes, known as the Lux operon, from Aliivibrio fischeri,
-            to make a genetically engineered E. Coli machine. The six genes of the 9-kilobase Lux operon, LuxCDABE and G,
-            are regulated by LuxI and LuxR and produce blue-green light through the following reaction: </p>
-           <p style="text-align: center;">
-                        <b> FMNH2+O2+R-CHO → FMN + R-COOH + H2O + Light </b></p>
-           <p>Instead of simply transforming an E.Coli with the Lux operon under a single promoter, we have isolated
-           each of the six genes independently and placed them under different promoters to optimize the system
-           and produce the maximum amount of light. The entire system is regulated by a light-repressed promoter
-           so the bacteria only luminesce during nighttime. In conclusion, we have created the first ever bacteria night light!
-           Possible real world applications for this are endless and include illuminating street signs, marking hiking trails, and
-           even giving light to impoverished areas of the world that do not have access to electricity.</p>
-        </td>
-        <td width="50%" valign="top">
-         <div align=center> <b> No More Biofilm! </b> </div> <br>
-            <div align=center> <img src="https://static.igem.org/mediawiki/2012/9/92/Mr-clean.jpg" width="306" height="208"> </div>
-            <p>Our project harnesses the use of quorum sensing to prevent bacteria from activating pathogenic factors triggered by AHLs. We developed a model system with Allivibrio fisheri. A. fisheri luminesces when activated by AHLs, which is a much safer alternative to pathogenic bacteria. We grew up our E. coli with Vibrio fisheri, and showed that the recombinant E. coli could detect V. fisheri’s AHLs, and inhibit V. fisheri from activating its quorum response genes by not producing light.The detection system then secretes factors that reduce the concentration of AHLs in the environment, thereby inhibiting the bacteria that use AHLs from communicating with each other. This is useful in preventing pathology from bacteria. When a threshold level of AHLs is reached in pathogenic bacteria, the signals activate transcription of pathogenic factors such as biofilm formation and host invasion signals (Raffa et al, 2005). We propose that disrupting the AHLs will inhibit these bacteria from creating biofilms or activating their pathogenic genes. In order to detect AHLs, we took advantage of the Salmonella enterica serovar typhimurium AHL receptor SdiA, a LuxR homolog. The SdiA transcription factor has been shown to be more sensitive to a greater diversity of AHLs than LuxR (Smith et al, 2008). Therefore it will be activated by AHLs present before the AHLs can activate genes in the surrounding bacteria. We also synthesized LuxR mutants that were more sensitive than LuxR at detecting the AHL 3OC6HSL, so they would be activated before LuxR gets activated. We planned to couple the new LuxR homologs to the AHLase Aiia, to demonstrate how we could inhibit activation of pathological factors. We also designed a construct with the new part, NucB, a nuclease that has been shown to digest extracellular DNA.  </p>
-        </td>
-    </tr>
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