http://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&feed=atom&action=historyTeam:CU-Boulder/Project - Revision history2024-03-29T06:44:36ZRevision history for this page on the wikiMediaWiki 1.16.0http://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=266935&oldid=prevJacksoag at 03:26, 4 October 20122012-10-04T03:26:15Z<p></p>
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</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=266888&oldid=prevJacksoag at 03:24, 4 October 20122012-10-04T03:24:53Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br><br></div></td></tr>
</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=266848&oldid=prevJacksoag at 03:23, 4 October 20122012-10-04T03:23:46Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br><br></div></td></tr>
</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=266751&oldid=prevJacksoag at 03:21, 4 October 20122012-10-04T03:21:16Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br><br></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br><br></div></td></tr>
</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=259390&oldid=prevJacksoag at 19:36, 3 October 20122012-10-03T19:36:39Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><p style="text-align: center;">Applications</p></h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><p style="text-align: center;">Applications</p></h1></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <ins class="diffchange diffchange-inline"><br></ins><br></div></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>While our project has relevance and application today, if it was continued to be worked on, there is much more that could be accomplished. Many plants have defense mechanisms against biofilm, secreting proteins that inhibit the formation of biofilm similar to our project. If in the future we could express our construct in plants, we could produce healthier plants less prone to disease and with a longer shelf life, while at the same time limiting the amount of pesticide needed to keep them from spoiling. The plants would effectively be defending themselves from pathogenic bacterial invasion and farmers would be selling a more healthy product. <br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>While our project has relevance and application today, if it was continued to be worked on, there is much more that could be accomplished. Many plants have defense mechanisms against biofilm, secreting proteins that inhibit the formation of biofilm similar to our project. If in the future we could express our construct in plants, we could produce healthier plants less prone to disease and with a longer shelf life, while at the same time limiting the amount of pesticide needed to keep them from spoiling. The plants would effectively be defending themselves from pathogenic bacterial invasion and farmers would be selling a more healthy product. <ins class="diffchange diffchange-inline"><br></ins><br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Another future application for our project is developing the AHL recognition complex. Our complex recognizes a broad range of AHLs because we wanted our construct to react to a broad range of bacteria. If we could “tune” our receptors to recognize species-specific AHLs, we could determine bacterial environmental composition. If we could take a broad range of reporting mechanisms all with different color outputs and have them activate when the species-specific AHL is present, then we would know exactly which bacterial species is present. By having a cocktail of these constructs with specific reporters linked to specific AHL receptors, we could efficiently visualize a microbial environment at low cost. This could be a useful tool diagnostically where today physicians learn so much about the body from bacterial fecal population; a bacterial population so crucial to humans that fecal transplants are undergone to re-culture humans with poor bacterial environments. By adding this cocktail to fecal matter, it would be possible to image and observe the type of fluorescence in the feces in order to make a diagnosis. This would be especially useful to physicians in the third world who lack modern medical equipment. This goes much further than humans as well; farmers could also cheaply survey bacterial composition of their soil with this construct and assess the fertility of the land. AHLs could prove to become very important in years to come and have a real effect on our interaction with the microbial world. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Another future application for our project is developing the AHL recognition complex. Our complex recognizes a broad range of AHLs because we wanted our construct to react to a broad range of bacteria. If we could “tune” our receptors to recognize species-specific AHLs, we could determine bacterial environmental composition. If we could take a broad range of reporting mechanisms all with different color outputs and have them activate when the species-specific AHL is present, then we would know exactly which bacterial species is present. By having a cocktail of these constructs with specific reporters linked to specific AHL receptors, we could efficiently visualize a microbial environment at low cost. This could be a useful tool diagnostically where today physicians learn so much about the body from bacterial fecal population; a bacterial population so crucial to humans that fecal transplants are undergone to re-culture humans with poor bacterial environments. By adding this cocktail to fecal matter, it would be possible to image and observe the type of fluorescence in the feces in order to make a diagnosis. This would be especially useful to physicians in the third world who lack modern medical equipment. This goes much further than humans as well; farmers could also cheaply survey bacterial composition of their soil with this construct and assess the fertility of the land. AHLs could prove to become very important in years to come and have a real effect on our interaction with the microbial world. </div></td></tr>
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</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=259384&oldid=prevJacksoag at 19:36, 3 October 20122012-10-03T19:36:09Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><p style="text-align: center;">Applications</p></h1></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h1><p style="text-align: center;">Applications</p></h1></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Biofilms are all around us, from our bathrooms to our gums and teeth. By utilizing our construct - which intercepts the bacterial signal preventing the quorum threshold from being met - the majority of biofilms would be prevented from forming. This could be utilized in a variety of different applications such as applying our construct to areas that may not receive regular cleaning. For instance applying our construct throughout a shower curtain would reduce biofilm activity and inhibit bacterial flourishing in the shower. Theoretically, stationary bodies of water prone to generating intense bacterial mats on their surface could be preemptively stopped by adding our construct. Biofilms also form in the plants we eat, usually resulting in disease of the plant and spoilage of food. Our construct could be a marker for consumers applied to encased produce (such as bananas). If our reporter mechanism began to glow intensely, it would be a good indicator that the food was beginning to turn bad. <br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">While our project has relevance and application today, if it was continued to be worked on, there is much more that could be accomplished. Many plants have defense mechanisms against biofilm, secreting proteins that inhibit the formation of biofilm similar to our project. If in the future we could express our construct in plants, we could produce healthier plants less prone to disease and with a longer shelf life, while at the same time limiting the amount of pesticide needed to keep them from spoiling. The plants would effectively be defending themselves from pathogenic bacterial invasion and farmers would be selling a more healthy product. <br></ins></div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">Another future application for our project is developing the AHL recognition complex. Our complex recognizes a broad range of AHLs because we wanted our construct to react to a broad range of bacteria. If we could “tune” our receptors to recognize species-specific AHLs, we could determine bacterial environmental composition. If we could take a broad range of reporting mechanisms all with different color outputs and have them activate when the species-specific AHL is present, then we would know exactly which bacterial species is present. By having a cocktail of these constructs with specific reporters linked to specific AHL receptors, we could efficiently visualize a microbial environment at low cost. This could be a useful tool diagnostically where today physicians learn so much about the body from bacterial fecal population; a bacterial population so crucial to humans that fecal transplants are undergone to re-culture humans with poor bacterial environments. By adding this cocktail to fecal matter, it would be possible to image and observe the type of fluorescence in the feces in order to make a diagnosis. This would be especially useful to physicians in the third world who lack modern medical equipment. This goes much further than humans as well; farmers could also cheaply survey bacterial composition of their soil with this construct and assess the fertility of the land. AHLs could prove to become very important in years to come and have a real effect on our interaction with the microbial world. </ins></div></td></tr>
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</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=259298&oldid=prevJacksoag at 19:24, 3 October 20122012-10-03T19:24:47Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. '''Test the SdiA pSrgE cassette’s inhibition effects with VF acting as a pathogen analogue''':</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>3. '''Test the SdiA pSrgE cassette’s inhibition effects with VF acting as a pathogen analogue''':</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Co-culture the SdiA/pSrgE/YFP/Aiia cells with V. fisheri and use the plate reader to show that co-cultures will keep V. fisheri from emitting light at given ODs. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Co-culture the SdiA/pSrgE/YFP/Aiia cells with V. fisheri and use the plate reader to show that co-cultures will keep V. fisheri from emitting light at given ODs. </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;"></ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. '''Isolate and submit NucB''': Isolate NucB and standardize it to biobrick format.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>4. '''Isolate and submit NucB''': Isolate NucB and standardize it to biobrick format.</div></td></tr>
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</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=259292&oldid=prevJacksoag at 19:24, 3 October 20122012-10-03T19:24:07Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also identified and obtained a novel biofilm degradation enzyme, NucB. NucB is a deoxyribonuclease synthesized by <i>Bacillus subtilis</i>, a sporulating gram-positive bacteria. When <i>B. subtilis</i> goes through sporulation, it divides asymmetrically and generates two compartments of unequal size and different developmental fates. The smaller compartment eventually develops into a mature spore, while the larger compartment serves to protect and nurture the developing spore (Eichenberger et al., 2003). NucB is part of a large operon that responds to regulator molecules activated in the large compartment during sporulation. Its task is to degrade nucleic acids present in surrounding biofilm in order for the spore to escape and travel to a new location. DNA in biofilms is responsible for increased viscoelastic and adhesive properties. Since our goal is to prevent biofilm formation, we wanted to express NucB as an additional method of prevention. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>We also identified and obtained a novel biofilm degradation enzyme, NucB. NucB is a deoxyribonuclease synthesized by <i>Bacillus subtilis</i>, a sporulating gram-positive bacteria. When <i>B. subtilis</i> goes through sporulation, it divides asymmetrically and generates two compartments of unequal size and different developmental fates. The smaller compartment eventually develops into a mature spore, while the larger compartment serves to protect and nurture the developing spore (Eichenberger et al., 2003). NucB is part of a large operon that responds to regulator molecules activated in the large compartment during sporulation. Its task is to degrade nucleic acids present in surrounding biofilm in order for the spore to escape and travel to a new location. DNA in biofilms is responsible for increased viscoelastic and adhesive properties. Since our goal is to prevent biofilm formation, we wanted to express NucB as an additional method of prevention. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="experiments2"></a></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><a name="experiments2"></a></div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3><p style="text-align: center;">Project 2: Experiments</p></h3></div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div><h3><p style="text-align: center;">Project 2: Experiments</p></h3></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>1. '''Test whether the SdiA pSrgE cassette is more sensitive to extracellular AHLs than the LuxR LuxpR cassette<del class="diffchange diffchange-inline">.</del>'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>1. '''Test whether the SdiA pSrgE cassette is more sensitive to extracellular AHLs than the LuxR LuxpR cassette'''<ins class="diffchange diffchange-inline">:</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This will be done by placing an RFP behind the cassette, and using the plate reader to determine the fluorescence at a given concentration of RFP.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>This will be done by placing an RFP behind the cassette, and using the plate reader to determine the fluorescence at a given concentration of RFP.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>2. '''Use the SdiA pSrgE cassette’s sensitivity to inhibit the LuxR LuxpR system'''<del class="diffchange diffchange-inline">. </del></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>2. '''Use the SdiA pSrgE cassette’s sensitivity to inhibit the LuxR LuxpR system'''<ins class="diffchange diffchange-inline">: </ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Attach an AHLase and YFP to the more sensitive SdiA/pSrgE cassette. Co-culture the AHLase/YFP construct with a LuxR/LuxpR-RFP bacteria. The culture should turn fluoresce yellow, and not turn red.</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Attach an AHLase and YFP to the more sensitive SdiA/pSrgE cassette. Co-culture the AHLase/YFP construct with a LuxR/LuxpR-RFP bacteria. The culture should turn fluoresce yellow, and not turn red.</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>3. '''Test the SdiA pSrgE cassette’s inhibition effects with <del class="diffchange diffchange-inline">Vibrio fisheri </del>acting as a pathogen analogue<del class="diffchange diffchange-inline">.</del>'''</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>3. '''Test the SdiA pSrgE cassette’s inhibition effects with <ins class="diffchange diffchange-inline">VF </ins>acting as a pathogen analogue'''<ins class="diffchange diffchange-inline">:</ins></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Co-culture the SdiA/pSrgE/YFP/Aiia cells with V. fisheri and use the plate reader to show that co-cultures will keep V. fisheri from emitting light at given ODs. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Co-culture the SdiA/pSrgE/YFP/Aiia cells with V. fisheri and use the plate reader to show that co-cultures will keep V. fisheri from emitting light at given ODs. </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins style="color: red; font-weight: bold; text-decoration: none;">4. '''Isolate and submit NucB''': Isolate NucB and standardize it to biobrick format.</ins></div></td></tr>
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</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=259272&oldid=prevJacksoag at 19:20, 3 October 20122012-10-03T19:20:54Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Aiia-eats-AHL.jpg]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:Aiia-eats-AHL.jpg]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>Inhibiting quorum sensing in bacteria has been shown to not only inhibit the transcription of pathogenesis factors, but also to keep biofilms from forming. In the history of iGEM teams have used secreted enzymes to digest biofilms that have already been made, but our use of quorum sensing inhibitors have gone a step further, and are being tested in their ability to keep bacteria from making biofilms in the first place.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>Inhibiting quorum sensing in bacteria has been shown to not only inhibit the transcription of pathogenesis factors, but also to keep biofilms from forming. In the history of iGEM<ins class="diffchange diffchange-inline">, </ins>teams have used secreted enzymes to digest biofilms that have already been made, but our use of quorum sensing inhibitors have gone a step further, and are being tested in their ability to keep bacteria from making biofilms in the first place.</div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div> </div></td></tr>
<tr><td colspan="2"> </td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div><ins class="diffchange diffchange-inline">We also identified and obtained a novel biofilm degradation enzyme, NucB. NucB is a deoxyribonuclease synthesized by <i>Bacillus subtilis</i>, a sporulating gram-positive bacteria. When <i>B. subtilis</i> goes through sporulation, it divides asymmetrically and generates two compartments of unequal size and different developmental fates. The smaller compartment eventually develops into a mature spore, while the larger compartment serves to protect and nurture the developing spore (Eichenberger et al., 2003). NucB is part of a large operon that responds to regulator molecules activated in the large compartment during sporulation. Its task is to degrade nucleic acids present in surrounding biofilm in order for the spore to escape and travel to a new location. DNA in biofilms is responsible for increased viscoelastic and adhesive properties. Since our goal is to prevent biofilm formation, we wanted to express NucB as an additional method of prevention. </ins></div></td></tr>
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</table>Jacksoaghttp://2012.igem.org/wiki/index.php?title=Team:CU-Boulder/Project&diff=259196&oldid=prevJacksoag at 19:07, 3 October 20122012-10-03T19:07:52Z<p></p>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:VF-characterization.jpg]]</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>[[File:VF-characterization.jpg]]</div></td></tr>
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<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The figure above shows the plate reader data characterizing <del class="diffchange diffchange-inline">Allivibrio </del>fisheri's luminosity at a specific OD. The luminosity starts to increase at OD .23, and then after the A. fisheri becomes stationary, the luminescence sharply decreases. This experiment was read on a plate reader every 20min with 5 trials. A. fisheri was grown up for 4 hours in a room temperature shaker. Then pelleted and washed three times with LB + NaCl. Diluted to an OD of .05 and placed in the plate reader.<br></div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The figure above shows the plate reader data characterizing <ins class="diffchange diffchange-inline"><i>Aliivibrio </ins>fisheri's<ins class="diffchange diffchange-inline"></i> </ins>luminosity at a specific OD. The luminosity starts to increase at OD .23, and then after the A. fisheri becomes stationary, the luminescence sharply decreases. This experiment was read on a plate reader every 20min with 5 trials. A. fisheri was grown up for 4 hours in a room temperature shaker. Then pelleted and washed three times with LB + NaCl. Diluted to an OD of .05 and placed in the plate reader.<br></div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Additionally, LuxC,D,E, and G were isolated from WH1 VF and standardized to biobrick format. LuxA and B were isolated from VF WH1 and contained internal EcoRI/PstI cut sites. We used site directed mutagenesis to modify these sites in order to obtain biobrick format, but had a difficult time getting the mutagenesis to work. </div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>Additionally, LuxC,D,E, and G were isolated from WH1 VF and standardized to biobrick format. LuxA and B were isolated from VF WH1 and contained internal EcoRI/PstI cut sites. We used site directed mutagenesis to modify these sites in order to obtain biobrick format, but had a difficult time getting the mutagenesis to work. </div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Quorum Sensing in LuxR containing Bacteria</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Quorum Sensing in LuxR containing Bacteria</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>This year the CU iGEM team has harnessed the quorum sensing factors endogenous in the Salmonella enterica serovar typhimurium LT2 strain to detect AHLs produced by other bacteria. Neither Salmonella nor E. coli have been shown to produce detectable levels of AHLs, so they were model organisms for the detection of other AHL producing bacteria such as <del class="diffchange diffchange-inline">Vibrio fisheri</del>, or pathogenic bacteria such as Yersinia pestis. AHLs are small molecules synthesized by most gram negative bacteria, and are able to freely diffuse throughout the membrane. When the concentration of AHL producing bacteria in a specific area increases, the total concentration of AHLs diffusing into the cytoplasm also increases. Once a threshold level of AHLs are in the cytosol, transcription factors like the <del class="diffchange diffchange-inline">Vibrio fisheri </del>LuxR or Salmonella enterica SdiA activate gene synthesis for quorum factors to be produced. In pathogenic bacteria, these signals have been shown to activate transcription of pathogenic factors.</div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>This year the CU iGEM team has harnessed the quorum sensing factors endogenous in the <ins class="diffchange diffchange-inline"><i></ins>Salmonella enterica serovar typhimurium<ins class="diffchange diffchange-inline"></i> </ins>LT2 strain to detect AHLs produced by other bacteria. Neither <ins class="diffchange diffchange-inline"><i></ins>Salmonella<ins class="diffchange diffchange-inline"></i> </ins>nor <ins class="diffchange diffchange-inline"><i></ins>E. coli<ins class="diffchange diffchange-inline"></i> </ins>have been shown to produce detectable levels of AHLs, so they were model organisms for the detection of other AHL producing bacteria such as <ins class="diffchange diffchange-inline">VF</ins>, or pathogenic bacteria such as <ins class="diffchange diffchange-inline"><i></ins>Yersinia pestis<ins class="diffchange diffchange-inline"></i></ins>. AHLs are small molecules synthesized by most gram negative bacteria, and are able to freely diffuse throughout the membrane. When the concentration of AHL producing bacteria in a specific area increases, the total concentration of AHLs diffusing into the cytoplasm also increases. Once a threshold level of AHLs are in the cytosol, transcription factors like the <ins class="diffchange diffchange-inline">VF </ins>LuxR or <ins class="diffchange diffchange-inline"><i></ins>Salmonella enterica<ins class="diffchange diffchange-inline"></i> </ins>SdiA activate gene synthesis for quorum factors to be produced. In pathogenic bacteria, these signals have been shown to activate transcription of pathogenic factors.</div></td></tr>
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<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Detection and silencing of AHL producing bacteria'''</div></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"><div>'''Detection and silencing of AHL producing bacteria'''</div></td></tr>
<tr><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td><td class='diff-marker'> </td><td style="background: #eee; color:black; font-size: smaller;"></td></tr>
<tr><td class='diff-marker'>-</td><td style="background: #ffa; color:black; font-size: smaller;"><div>The SdiA transcription factor has been shown to be more sensitive to lower concentrations and a greater diversity of AHLs than its LuxR homolog. We took advantage of this extra-sensitivity to create a detection system for AHL producing bacteria, while additionally attaching the detection system to the secretion of potent AHLases such as the Aiia enzyme, and a reporter protein RFP. Using this construct we tested whether we could disrupt the quorum sensing signal AHLs in order to keep them from reaching the threshold concentration to activate the less sensitive LuxR receptor. The <del class="diffchange diffchange-inline">V. fisheri </del>species and other pathogenic bacteria use the LuxR receptor, therefore inhibition of AHLs would keep <del class="diffchange diffchange-inline">V. fisheri </del>from producing light. Keeping the <del class="diffchange diffchange-inline">V. fisheri </del>from producing light is an analogous model to keeping pathogenic bacteria from being stimulated by AHLs to release their pathogenesis factors. </div></td><td class='diff-marker'>+</td><td style="background: #cfc; color:black; font-size: smaller;"><div>The SdiA transcription factor has been shown to be more sensitive to lower concentrations and a greater diversity of AHLs than its LuxR homolog. We took advantage of this extra-sensitivity to create a detection system for AHL producing bacteria, while additionally attaching the detection system to the secretion of potent AHLases such as the Aiia enzyme, and a reporter protein RFP. Using this construct we tested whether we could disrupt the quorum sensing signal AHLs in order to keep them from reaching the threshold concentration to activate the less sensitive LuxR receptor. The <ins class="diffchange diffchange-inline">VF </ins>species and other pathogenic bacteria use the LuxR receptor, therefore inhibition of AHLs would keep <ins class="diffchange diffchange-inline">VF </ins>from producing light. Keeping the <ins class="diffchange diffchange-inline">VF </ins>from producing light is an analogous model to keeping pathogenic bacteria from being stimulated by AHLs to release their pathogenesis factors. </div></td></tr>
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</table>Jacksoag