http://2013.igem.org/wiki/index.php?title=Special:Contributions/Rahmilale&feed=atom&limit=50&target=Rahmilale&year=&month=2013.igem.org - User contributions [en]2024-03-29T14:25:42ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:NTNU-Trondheim/PressTeam:NTNU-Trondheim/Press2013-10-04T08:46:58Z<p>Rahmilale: </p>
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<p style="text-align:center; color:black; "> The project </p> </div><br />
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<p>Vesicles, fluorescence protein dimers.... </p><br />
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<div class="col4" style="background-color:#CBBAE9;><a href="https://2013.igem.org/Team:NTNU-Trondheim/Project#Vesicles"> <img src="https://static.igem.org/mediawiki/2013/b/b2/Marialab.jpg" div class="circular" width="303"><br />
<p style="text-align:center; color:black; "> Vesicle project </p> </div><br />
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<div class="col4"style="background-color:#CBBAE9;> <a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" div class="circular"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/2011/2/26/Subtitlepic3headerhover.jpg'" <br />
onmouseout="this.src='https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg'" width="230"> </a><br />
<p style="text-align:center; color:black;"> Fluorescence protein dimers </p></div><br />
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<div class="col4" style="background-color:#CBBAE9"><a href="https://2011.igem.org/Team:Berkeley/Project#StressPromoters"><img src="https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg" div class="circular"<br />
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<p style="text-align:center; color:black;"> PmXylS promoter </p> </div><br />
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<p style="text-align:center; color:black; "> About us</p> </div><br />
<p>The team from NTNU consists of five students. bla bla bla</p><br />
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{{Team:NTNU-Trondheim/Press/navigation}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/PressTeam:NTNU-Trondheim/Press2013-10-04T08:46:39Z<p>Rahmilale: </p>
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<p style="text-align:center; color:black; "> The project </p> </div><br />
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<p>Vesicles, fluorescence protein dimers.... </p><br />
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<div class="col4" style="background-color:#CBBAE9;><a href="https://2013.igem.org/Team:NTNU-Trondheim/Project#Vesicles"> <img src="https://static.igem.org/mediawiki/2013/b/b2/Marialab.jpg" div class="circular" width="303"><br />
<p style="text-align:center; color:black; "> Vesicle project </p> </div><br />
<br />
<div class="col4"style="background-color:#CBBAE9;> <a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" div class="circular"<br />
onmouseover="this.src='https://static.igem.org/mediawiki/2011/2/26/Subtitlepic3headerhover.jpg'" <br />
onmouseout="this.src='https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg'" width="230"> </a><br />
<p style="text-align:center; color:black;"> Fluorescence protein dimers </p></div><br />
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<div class="col4" style="background-color:#CBBAE9"><a href="https://2011.igem.org/Team:Berkeley/Project#StressPromoters"><img src="https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg" div class="circular"<br />
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onmouseout="this.src='https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg'" width="230"></a><br />
<p style="text-align:center; color:black;"> PmXylS promoter </p> </div><br />
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<p style="text-align:center; color:black; "> About us</p> </div><br />
<p>The team from NTNU consists of five students. bla bla bla</p><br />
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{{Team:NTNU-Trondheim/Press/navigation}}</div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-10-04T08:43:34Z<p>Rahmilale: </p>
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-10-04T08:42:21Z<p>Rahmilale: </p>
<hr />
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</html></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/ExamplePageTeam:NTNU-Trondheim/ExamplePage2013-10-04T08:41:38Z<p>Rahmilale: </p>
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<div>{{Team:NTNU-Trondheim/Press/header}}<br />
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</head><br />
<br />
<div id="top-section3"><center><img src="https://static.igem.org/mediawiki/2011/4/48/NavigationBar_Berkeley.jpg" alt="header" border="0" usemap="#igemmap"></center></div><br />
<map name="igemmap"><br />
<area shape="circle" coords="480,40,40" alt="Mercury" href="https://2011.igem.org" onfocus="this.blur()" /><br />
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<br />
<div class= "layout-1000" ><br />
<div class="row"><br />
<div ><br />
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<div id='cssmenu'><br />
<ul><br />
<li class='active'><a href='index.html'><span>Home</span></a></li><br />
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<li><a href='#'><span>BioBrick Parts</span></a></li><br />
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<li><a href='#'><span>Notebooks</span></a></li><br />
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<li><a href='#'><span>Protocols</span></a></li><br />
<li class='last'><a href='#'><span>Safety</span></a></li><br />
</ul><br />
</li><br />
<li class='has-sub'><a href='#'><span>Team</span></a><br />
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<div class="col12"><a name="ProjectTitle"></a> <img src="https://static.igem.org/mediawiki/2011/3/36/Projectdetectr.jpg" width="980"> </div><br />
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<a name="Project Motivation"></a> <img src="https://static.igem.org/mediawiki/2011/a/a2/ProjectsubHeader1.jpg" width="980"> </div><br />
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<br />
<div align="justify"><br />
<br />
The question of how cells detect specific molecules has always been important to biology, but is becoming an increasingly useful question to ask as we begin to engineer life. The applications of biosensing are widespread. There is a demand for biological diagnostic tools in medical or environmental applications, like detecting diseases or environmental contaminants. Sensing also has applications in biosecurity systems, when bioengineers try to develop strains that are stably dependent on the presence certain molecules. Finally, developing sensing systems for specific molecules is invaluable in metabolic engineering, because it allows for construction of metabolite-specific selection schemes. </p><br />
<center><br />
<p><img src="https://static.igem.org/mediawiki/2011/d/dc/Wiki_-_Biosensors4.png" width="500" align ="middle" /></p><br />
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<p><br />
Traditionally, most sensing systems in synthetic constructs have been borrowed from nature. However, very often researchers want to detect a ligand that does not have a receptor in the existing list of characterized sensors. Scanning natural systems for proteins that bind to a specific molecules is difficult and time-consuming, when it is not impossible. As synthetic biology realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.</p><p><br />
<center><br />
<img src="https://static.igem.org/mediawiki/2011/6/6b/Wiki_-_modular_biosensor_4.png" width="500" align ="middle" /></p><br />
</center><br />
<br />
<p>Here at Berkeley we were motivated to address this problem by constructing a biosensing mechanism that could be readily adapted to the ligand of choice. We wanted to build a modular biosensing device that could be modified by swapping binding domains. This approach would provide a standard method for engineering biosensors that could make use of both natural binding domains, optimized by evolution, as well as engineered domains that respond to molecules outside the range of natural receptors.</p><p><br />
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ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of ToxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association passes through the membrane, bringing the cytoplasmic domains of ToxR together to form an active homodimer. The active ToxR dimers drive transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.</p><p><br />
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ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.</p><br><br><br />
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<p> Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). ToxS detects changes in the environment, interacts with, and dimerizes ToxR's periplasmic domain. Dimerization of the periplasmic domain of ToxR results in dimerization of the cytoplasmic domain, which activates transcription of the ctx promoter. The ToxR domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by further engineering this dimerization-dependent transcriptional activation feature of the ToxR system to be ligand dependent. </p><p><br />
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We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the periplasmic domain of ToxR. The periplasmic domain will dimerize only in the presence of a particular ligand. The ligand dependent dimerization of the periplasmic domain of the ToxR fusion will cause the cytoplasmic domain of ToxR to dimerize, activating the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.</p><br><br />
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<p>The first step was to validate that a ToxR based two-hybrid system could be expressed in E. coli and that we could cause dimer-dependent transcriptional activation using a chimeric ToxR with periplasmic domains of our choosing. We started by truncating ToxR to eliminate the periplasmic domain because the dimerization state of ToxR's cytoplasmic domain controls transcription. Next, we built a translational fusion of the constitutively dimerizing leucine zipper protein IILK to the ToxR truncation. The constitutively dimerizing protein IILK portion of the translational fusion is located in the periplasm, while the ToxR cytoplasmic domain remains in the cytoplasm. The constitutively dimerizing IILK periplasmic domain should dimerize, causing the cytoplasmic domain of ToxR to dimerize, activating transcription from the ctx promoter.</p><br />
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The initial ToxR chimeras under the pBAD promoter were toxic to the cell, were difficult to express, and behaved unpredictably. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well. The graphs below show ToxR fused to Lambda-Rep, a different constitutive dimerizing protein (similar in behavior to IILK with ToxR fusions) that we used as the periplasmic domain in ToxR chimeras. The left graph demonstrates decreased growth and the right demonstrates low fluorescence with increasing arabinose. Both poor growth and low fluorescence are a result of toxicity from ToxR based chimeras. </p><br />
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<p>In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for stress-based regulatory elements. We wanted to find a promoter that would drive transcription while the cell was happy and health, but would shut down in response to cellular stress, behaving as a self-regulating expression system. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without toxicity to the cell. </p><br><br />
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<p> Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress-responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating its expression to produce the maximum amount of ToxR chimera without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the ToxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.</p><br />
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In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 potential stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP.</p><br><br><br />
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<p>The construct was transformed into E. coli and subjected to general stress conditions. We measured the fluorescence with a tecan plate reader and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a few did show a significant change in fluorescence, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.</p><br><br><br />
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<p>Since some of the stress promoters displayed the desired behavior, we proceeded to incorporate the stress promoter pool into a stress response system with ToxR. In our stress feedback system, expression of the ToxR chimera activates the ctx promoter (Pctx), driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating ToxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of ToxR chimera expression without toxicity.</p><br><br><br />
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In order to ensure nonlethal expression of the ToxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of ToxR-IILK. We transformed the plasmid into cells containing Pctx.RBS.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.</p><br><br><br />
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<p>From our combinatorial approach towards the construction of the stress-responsive feedback system, we were able to obtain big, healthy green colonies growing on our plates. The observation of big colonies indicated that our cells were healthy and not experiencing any apparent cellular stress. The high levels of GFP observed in the colonies indicated that cells were expressing enough ToxR chimera to strongly drive transcription of the ctx promoter (Pctx). Since we saw colonies that were big and green, we claim to have successfully resolved the toxicity of ToxR fusion proteins enabling strong transcriptional output while minimizing cellular stress.</p><br><br><br />
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We picked big green colonies and found that cultures showed improved fluorescence and OD with respect to pBAD promoters. In other experiments, we found that Lambda-Rep provided a better fluorescent signal so we replaced the IILK domain with Lambda-Rep in both the pBAD and Pstress, grew them in the same arabinose conditions and measured OD600 and RFU with a Tecan plate reader every five minutes. The as with IILK, ToxR-Lambda-Rep chimeras show improved fluorescence, reduced toxcity, and cells harboring this plasmid behave much more predictably.<br />
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<p>From our results thus far, we were able to:</p><br />
<b>-Characterize a library of stress promoters from the E. coli genome, providing data that they function as stress-responsive downregulators<br><br />
-Create an off-the-shelf negative feedback expression system using our stress promoters that enables the expression of toxic proteins to their highest non-toxic levels, solving our problem of ToxR chimera toxicity<br><br />
-Demonstrated our proof of principle biosensing system by creating constitutively dimerizing ToxR chimeras that drive transcription off of the ctx promoter (Pctx)</p></b><br />
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<p>Having achieved our goal of testing our proof of principle, we were ready to construct inducible homodimerizing systems.</p><br><br><br />
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Once we had successfully expressed a ToxR chimera that demonstrated constitutive transcription off Pctx we were ready to design a system that could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer, and after an extensive literature search, we decided to use the Estrogen Receptor. We chose the Estrogen receptor because it fulfilled the criteria of being a homodimer and dependant on Estradiol for dimerization. Furthermore, it has been previously used in a two-hybrid assay as a chimera attached to GAL4 parts to measure ß-galactosidase activity (Peters 1999). In addition to its structural and mechanical advantages it would also make a useful biosensor since estradiol poses numerous hazards to the environment. Therefore the ability to sense it cheaply and effectively would have great implications.</p><br />
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The Estrogen Receptor is made up of 6 domains: A, B, C, D, E, and F. Domains A and B serve as a transcriptional factor, domain C recognizes and binds to specific DNA sequences and is also involved in dimerization (Peters 1999). Domain E is the Ligand Binding Domain (LBD); when it binds to estrogen it homodimerizes to another Estrogen Receptor LBD. Domain D’s function is unknown. Domain F has been shown to inhibit dimerization and/or binding of estrogen and is thus undesirable for our project. In a wild type system these domains interact to bind Estrogen with Domain E, confer conformation so that C can bind a specific DNA sequence, dimerize with another estrogen receptor and act as a transcription factor to promote transcription downstream of its binding.</p><br><br><br />
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After studying the function of each of the domains as well as the findings of Peters 1999 paper we began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these estrogen receptor truncations were attached to the 3' end of ToxR and tested under various Estradiol levels. However, these truncations were non-responsive with and without estradiol. We hypothesized that different estrogen receptor truncations may be unable to express in the membrane, and the LBD by itself does not homodimerize as it didn't in the Peters' 1999 study. So, in order to mitigate both possibilities we made 8 more truncations that spanned the range between Domain A and Domain C.</p><br><br><br />
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Preliminary data suggests that truncations 5, 6, and 8 all showed constitutive ToxR dimerization and resulting fluorescence. However, none seemed to be inducible in response to Estrogen. Nevertheless, we believe we are getting very close to successfully expressing an inducible chimera because by showing activity it proves the estrogen receptor is not broken. Peters 1999 showed that the estrogen receptor can show dimer inducibility as a chimera in the presence of estrogen, and we think that trying a few more strategies will allow our ToxR chimera system to be inducible. We are going to try several more truncations, as well as various linkers, and the co-expression of different estrogen receptor truncations as was shown to work in the Peters paper.</p><br><br><br />
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One issue that could be affecting the Estrogen Receptor is the physiology of the periplasm that causes its cysteines in the LBD to cross react. This could potentially throw off the structure and therefore the functionality of the ER. Ee sought to correct this problem by growing the cells in a reducing environment that would prevent such bonds from forming. Our preliminary results below show that Trunc 8 is greatly stabalized by the addition of beta-mercaptoethanol (BME), effectively creating a reducing environment sensor.</p><br><br><br />
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Since the above system, although clearly stabalized in the periplasm, was not estradiol inducible we decided to look deeper into its ability to sense reducing environments. The first experiment showed how it grew in a reducing agent, while our next experiment below showed how it would respond (after growth saturation) to introduction into a buffered and non-buffered reducing environment. Once again Trunc 8 showed impressive inducibility in a reducing environment but only if the solution was buffered. </p><br><br><br />
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We believe that the right combination of buffer, reducing media, and truncation size will allow us to create a functioning estrogen biosensor, but in the meantime we have a proof of principle for our biosensor platform in the form of a reducing environment sensor. </p><br><br><br />
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All of the composite parts submitted to the registry and experiments discussed on this wiki were created and performed by the 2011 iGEM team Berkeley undergraduates. Some of the basic parts (RBSes, etc.) were obtained through the Anderson lab, for which they have our thanks.</p><br />
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<div class="col4" style="background-color:#CBBAE9;><a href="https://2013.igem.org/Team:NTNU-Trondheim/Project#Vesicles"> <img src="https://static.igem.org/mediawiki/2013/b/b2/Marialab.jpg" div class="circular" width="303"><br />
<p style="text-align:center; color:black; "> Vesicle project </p> </div><br />
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<div class="col4"style="background-color:#CBBAE9;> <a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" div class="circular"<br />
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<p style="text-align:center; color:black;"> Fluorescence protein dimers </p></div><br />
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<div class="col4" style="background-color:#CBBAE9"><a href="https://2011.igem.org/Team:Berkeley/Project#StressPromoters"><img src="https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg" div class="circular"<br />
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<p style="text-align:center; color:black;"> PmXylS promoter </p> </div><br />
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<p>The team from NTNU consists of five students. bla bla bla</p><br />
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The question of how cells detect specific molecules has always been important to biology, but is becoming an increasingly useful question to ask as we begin to engineer life. The applications of biosensing are widespread. There is a demand for biological diagnostic tools in medical or environmental applications, like detecting diseases or environmental contaminants. Sensing also has applications in biosecurity systems, when bioengineers try to develop strains that are stably dependent on the presence certain molecules. Finally, developing sensing systems for specific molecules is invaluable in metabolic engineering, because it allows for construction of metabolite-specific selection schemes. </p><br />
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Traditionally, most sensing systems in synthetic constructs have been borrowed from nature. However, very often researchers want to detect a ligand that does not have a receptor in the existing list of characterized sensors. Scanning natural systems for proteins that bind to a specific molecules is difficult and time-consuming, when it is not impossible. As synthetic biology realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.</p><p><br />
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<p>Here at Berkeley we were motivated to address this problem by constructing a biosensing mechanism that could be readily adapted to the ligand of choice. We wanted to build a modular biosensing device that could be modified by swapping binding domains. This approach would provide a standard method for engineering biosensors that could make use of both natural binding domains, optimized by evolution, as well as engineered domains that respond to molecules outside the range of natural receptors.</p><p><br />
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ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of ToxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association passes through the membrane, bringing the cytoplasmic domains of ToxR together to form an active homodimer. The active ToxR dimers drive transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.</p><p><br />
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ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.</p><br><br><br />
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<p> Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). ToxS detects changes in the environment, interacts with, and dimerizes ToxR's periplasmic domain. Dimerization of the periplasmic domain of ToxR results in dimerization of the cytoplasmic domain, which activates transcription of the ctx promoter. The ToxR domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by further engineering this dimerization-dependent transcriptional activation feature of the ToxR system to be ligand dependent. </p><p><br />
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We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the periplasmic domain of ToxR. The periplasmic domain will dimerize only in the presence of a particular ligand. The ligand dependent dimerization of the periplasmic domain of the ToxR fusion will cause the cytoplasmic domain of ToxR to dimerize, activating the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.</p><br><br />
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<p>The first step was to validate that a ToxR based two-hybrid system could be expressed in E. coli and that we could cause dimer-dependent transcriptional activation using a chimeric ToxR with periplasmic domains of our choosing. We started by truncating ToxR to eliminate the periplasmic domain because the dimerization state of ToxR's cytoplasmic domain controls transcription. Next, we built a translational fusion of the constitutively dimerizing leucine zipper protein IILK to the ToxR truncation. The constitutively dimerizing protein IILK portion of the translational fusion is located in the periplasm, while the ToxR cytoplasmic domain remains in the cytoplasm. The constitutively dimerizing IILK periplasmic domain should dimerize, causing the cytoplasmic domain of ToxR to dimerize, activating transcription from the ctx promoter.</p><br />
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The initial ToxR chimeras under the pBAD promoter were toxic to the cell, were difficult to express, and behaved unpredictably. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well. The graphs below show ToxR fused to Lambda-Rep, a different constitutive dimerizing protein (similar in behavior to IILK with ToxR fusions) that we used as the periplasmic domain in ToxR chimeras. The left graph demonstrates decreased growth and the right demonstrates low fluorescence with increasing arabinose. Both poor growth and low fluorescence are a result of toxicity from ToxR based chimeras. </p><br />
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<p>In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for stress-based regulatory elements. We wanted to find a promoter that would drive transcription while the cell was happy and health, but would shut down in response to cellular stress, behaving as a self-regulating expression system. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without toxicity to the cell. </p><br><br />
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<p> Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress-responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating its expression to produce the maximum amount of ToxR chimera without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the ToxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.</p><br />
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In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 potential stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP.</p><br><br><br />
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<p>The construct was transformed into E. coli and subjected to general stress conditions. We measured the fluorescence with a tecan plate reader and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a few did show a significant change in fluorescence, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.</p><br><br><br />
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<p>Since some of the stress promoters displayed the desired behavior, we proceeded to incorporate the stress promoter pool into a stress response system with ToxR. In our stress feedback system, expression of the ToxR chimera activates the ctx promoter (Pctx), driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating ToxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of ToxR chimera expression without toxicity.</p><br><br><br />
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In order to ensure nonlethal expression of the ToxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of ToxR-IILK. We transformed the plasmid into cells containing Pctx.RBS.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.</p><br><br><br />
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<p>From our combinatorial approach towards the construction of the stress-responsive feedback system, we were able to obtain big, healthy green colonies growing on our plates. The observation of big colonies indicated that our cells were healthy and not experiencing any apparent cellular stress. The high levels of GFP observed in the colonies indicated that cells were expressing enough ToxR chimera to strongly drive transcription of the ctx promoter (Pctx). Since we saw colonies that were big and green, we claim to have successfully resolved the toxicity of ToxR fusion proteins enabling strong transcriptional output while minimizing cellular stress.</p><br><br><br />
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We picked big green colonies and found that cultures showed improved fluorescence and OD with respect to pBAD promoters. In other experiments, we found that Lambda-Rep provided a better fluorescent signal so we replaced the IILK domain with Lambda-Rep in both the pBAD and Pstress, grew them in the same arabinose conditions and measured OD600 and RFU with a Tecan plate reader every five minutes. The as with IILK, ToxR-Lambda-Rep chimeras show improved fluorescence, reduced toxcity, and cells harboring this plasmid behave much more predictably.<br />
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<p>From our results thus far, we were able to:</p><br />
<b>-Characterize a library of stress promoters from the E. coli genome, providing data that they function as stress-responsive downregulators<br><br />
-Create an off-the-shelf negative feedback expression system using our stress promoters that enables the expression of toxic proteins to their highest non-toxic levels, solving our problem of ToxR chimera toxicity<br><br />
-Demonstrated our proof of principle biosensing system by creating constitutively dimerizing ToxR chimeras that drive transcription off of the ctx promoter (Pctx)</p></b><br />
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<p>Having achieved our goal of testing our proof of principle, we were ready to construct inducible homodimerizing systems.</p><br><br><br />
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Once we had successfully expressed a ToxR chimera that demonstrated constitutive transcription off Pctx we were ready to design a system that could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer, and after an extensive literature search, we decided to use the Estrogen Receptor. We chose the Estrogen receptor because it fulfilled the criteria of being a homodimer and dependant on Estradiol for dimerization. Furthermore, it has been previously used in a two-hybrid assay as a chimera attached to GAL4 parts to measure ß-galactosidase activity (Peters 1999). In addition to its structural and mechanical advantages it would also make a useful biosensor since estradiol poses numerous hazards to the environment. Therefore the ability to sense it cheaply and effectively would have great implications.</p><br />
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The Estrogen Receptor is made up of 6 domains: A, B, C, D, E, and F. Domains A and B serve as a transcriptional factor, domain C recognizes and binds to specific DNA sequences and is also involved in dimerization (Peters 1999). Domain E is the Ligand Binding Domain (LBD); when it binds to estrogen it homodimerizes to another Estrogen Receptor LBD. Domain D’s function is unknown. Domain F has been shown to inhibit dimerization and/or binding of estrogen and is thus undesirable for our project. In a wild type system these domains interact to bind Estrogen with Domain E, confer conformation so that C can bind a specific DNA sequence, dimerize with another estrogen receptor and act as a transcription factor to promote transcription downstream of its binding.</p><br><br><br />
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After studying the function of each of the domains as well as the findings of Peters 1999 paper we began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these estrogen receptor truncations were attached to the 3' end of ToxR and tested under various Estradiol levels. However, these truncations were non-responsive with and without estradiol. We hypothesized that different estrogen receptor truncations may be unable to express in the membrane, and the LBD by itself does not homodimerize as it didn't in the Peters' 1999 study. So, in order to mitigate both possibilities we made 8 more truncations that spanned the range between Domain A and Domain C.</p><br><br><br />
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Preliminary data suggests that truncations 5, 6, and 8 all showed constitutive ToxR dimerization and resulting fluorescence. However, none seemed to be inducible in response to Estrogen. Nevertheless, we believe we are getting very close to successfully expressing an inducible chimera because by showing activity it proves the estrogen receptor is not broken. Peters 1999 showed that the estrogen receptor can show dimer inducibility as a chimera in the presence of estrogen, and we think that trying a few more strategies will allow our ToxR chimera system to be inducible. We are going to try several more truncations, as well as various linkers, and the co-expression of different estrogen receptor truncations as was shown to work in the Peters paper.</p><br><br><br />
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One issue that could be affecting the Estrogen Receptor is the physiology of the periplasm that causes its cysteines in the LBD to cross react. This could potentially throw off the structure and therefore the functionality of the ER. Ee sought to correct this problem by growing the cells in a reducing environment that would prevent such bonds from forming. Our preliminary results below show that Trunc 8 is greatly stabalized by the addition of beta-mercaptoethanol (BME), effectively creating a reducing environment sensor.</p><br><br><br />
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Since the above system, although clearly stabalized in the periplasm, was not estradiol inducible we decided to look deeper into its ability to sense reducing environments. The first experiment showed how it grew in a reducing agent, while our next experiment below showed how it would respond (after growth saturation) to introduction into a buffered and non-buffered reducing environment. Once again Trunc 8 showed impressive inducibility in a reducing environment but only if the solution was buffered. </p><br><br><br />
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<div class="row"><br />
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<br><br><p><br />
We believe that the right combination of buffer, reducing media, and truncation size will allow us to create a functioning estrogen biosensor, but in the meantime we have a proof of principle for our biosensor platform in the form of a reducing environment sensor. </p><br><br><br />
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<div class="col12-2"><br />
<a name="Attribs"></a> <img src="https://static.igem.org/mediawiki/2011/3/3e/AttributesSubHeader.jpg" width="980"> </div><br><br><br />
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<p><br />
All of the composite parts submitted to the registry and experiments discussed on this wiki were created and performed by the 2011 iGEM team Berkeley undergraduates. Some of the basic parts (RBSes, etc.) were obtained through the Anderson lab, for which they have our thanks.</p><br />
<div class="row-end"> </div><br />
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-10-04T08:32:11Z<p>Rahmilale: </p>
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-10-04T08:31:43Z<p>Rahmilale: </p>
<hr />
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-10-04T08:31:02Z<p>Rahmilale: </p>
<hr />
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-10-04T08:30:31Z<p>Rahmilale: </p>
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</html></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/ExamplePageTeam:NTNU-Trondheim/ExamplePage2013-10-04T08:29:21Z<p>Rahmilale: </p>
<hr />
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<div id="top-section3"><center><img src="https://static.igem.org/mediawiki/2011/4/48/NavigationBar_Berkeley.jpg" alt="header" border="0" usemap="#igemmap"></center></div><br />
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<br />
The question of how cells detect specific molecules has always been important to biology, but is becoming an increasingly useful question to ask as we begin to engineer life. The applications of biosensing are widespread. There is a demand for biological diagnostic tools in medical or environmental applications, like detecting diseases or environmental contaminants. Sensing also has applications in biosecurity systems, when bioengineers try to develop strains that are stably dependent on the presence certain molecules. Finally, developing sensing systems for specific molecules is invaluable in metabolic engineering, because it allows for construction of metabolite-specific selection schemes. </p><br />
<center><br />
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<p><br />
Traditionally, most sensing systems in synthetic constructs have been borrowed from nature. However, very often researchers want to detect a ligand that does not have a receptor in the existing list of characterized sensors. Scanning natural systems for proteins that bind to a specific molecules is difficult and time-consuming, when it is not impossible. As synthetic biology realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.</p><p><br />
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<br />
<p>Here at Berkeley we were motivated to address this problem by constructing a biosensing mechanism that could be readily adapted to the ligand of choice. We wanted to build a modular biosensing device that could be modified by swapping binding domains. This approach would provide a standard method for engineering biosensors that could make use of both natural binding domains, optimized by evolution, as well as engineered domains that respond to molecules outside the range of natural receptors.</p><p><br />
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ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of ToxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association passes through the membrane, bringing the cytoplasmic domains of ToxR together to form an active homodimer. The active ToxR dimers drive transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.</p><p><br />
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ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.</p><br><br><br />
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<p> Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). ToxS detects changes in the environment, interacts with, and dimerizes ToxR's periplasmic domain. Dimerization of the periplasmic domain of ToxR results in dimerization of the cytoplasmic domain, which activates transcription of the ctx promoter. The ToxR domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by further engineering this dimerization-dependent transcriptional activation feature of the ToxR system to be ligand dependent. </p><p><br />
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We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the periplasmic domain of ToxR. The periplasmic domain will dimerize only in the presence of a particular ligand. The ligand dependent dimerization of the periplasmic domain of the ToxR fusion will cause the cytoplasmic domain of ToxR to dimerize, activating the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.</p><br><br />
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<p>The first step was to validate that a ToxR based two-hybrid system could be expressed in E. coli and that we could cause dimer-dependent transcriptional activation using a chimeric ToxR with periplasmic domains of our choosing. We started by truncating ToxR to eliminate the periplasmic domain because the dimerization state of ToxR's cytoplasmic domain controls transcription. Next, we built a translational fusion of the constitutively dimerizing leucine zipper protein IILK to the ToxR truncation. The constitutively dimerizing protein IILK portion of the translational fusion is located in the periplasm, while the ToxR cytoplasmic domain remains in the cytoplasm. The constitutively dimerizing IILK periplasmic domain should dimerize, causing the cytoplasmic domain of ToxR to dimerize, activating transcription from the ctx promoter.</p><br />
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The initial ToxR chimeras under the pBAD promoter were toxic to the cell, were difficult to express, and behaved unpredictably. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well. The graphs below show ToxR fused to Lambda-Rep, a different constitutive dimerizing protein (similar in behavior to IILK with ToxR fusions) that we used as the periplasmic domain in ToxR chimeras. The left graph demonstrates decreased growth and the right demonstrates low fluorescence with increasing arabinose. Both poor growth and low fluorescence are a result of toxicity from ToxR based chimeras. </p><br />
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<p>In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for stress-based regulatory elements. We wanted to find a promoter that would drive transcription while the cell was happy and health, but would shut down in response to cellular stress, behaving as a self-regulating expression system. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without toxicity to the cell. </p><br><br />
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<p> Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress-responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating its expression to produce the maximum amount of ToxR chimera without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the ToxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.</p><br />
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In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 potential stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP.</p><br><br><br />
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<p>The construct was transformed into E. coli and subjected to general stress conditions. We measured the fluorescence with a tecan plate reader and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a few did show a significant change in fluorescence, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.</p><br><br><br />
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<p>Since some of the stress promoters displayed the desired behavior, we proceeded to incorporate the stress promoter pool into a stress response system with ToxR. In our stress feedback system, expression of the ToxR chimera activates the ctx promoter (Pctx), driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating ToxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of ToxR chimera expression without toxicity.</p><br><br><br />
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In order to ensure nonlethal expression of the ToxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of ToxR-IILK. We transformed the plasmid into cells containing Pctx.RBS.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.</p><br><br><br />
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<p>From our combinatorial approach towards the construction of the stress-responsive feedback system, we were able to obtain big, healthy green colonies growing on our plates. The observation of big colonies indicated that our cells were healthy and not experiencing any apparent cellular stress. The high levels of GFP observed in the colonies indicated that cells were expressing enough ToxR chimera to strongly drive transcription of the ctx promoter (Pctx). Since we saw colonies that were big and green, we claim to have successfully resolved the toxicity of ToxR fusion proteins enabling strong transcriptional output while minimizing cellular stress.</p><br><br><br />
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We picked big green colonies and found that cultures showed improved fluorescence and OD with respect to pBAD promoters. In other experiments, we found that Lambda-Rep provided a better fluorescent signal so we replaced the IILK domain with Lambda-Rep in both the pBAD and Pstress, grew them in the same arabinose conditions and measured OD600 and RFU with a Tecan plate reader every five minutes. The as with IILK, ToxR-Lambda-Rep chimeras show improved fluorescence, reduced toxcity, and cells harboring this plasmid behave much more predictably.<br />
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<p>From our results thus far, we were able to:</p><br />
<b>-Characterize a library of stress promoters from the E. coli genome, providing data that they function as stress-responsive downregulators<br><br />
-Create an off-the-shelf negative feedback expression system using our stress promoters that enables the expression of toxic proteins to their highest non-toxic levels, solving our problem of ToxR chimera toxicity<br><br />
-Demonstrated our proof of principle biosensing system by creating constitutively dimerizing ToxR chimeras that drive transcription off of the ctx promoter (Pctx)</p></b><br />
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<p>Having achieved our goal of testing our proof of principle, we were ready to construct inducible homodimerizing systems.</p><br><br><br />
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Once we had successfully expressed a ToxR chimera that demonstrated constitutive transcription off Pctx we were ready to design a system that could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer, and after an extensive literature search, we decided to use the Estrogen Receptor. We chose the Estrogen receptor because it fulfilled the criteria of being a homodimer and dependant on Estradiol for dimerization. Furthermore, it has been previously used in a two-hybrid assay as a chimera attached to GAL4 parts to measure ß-galactosidase activity (Peters 1999). In addition to its structural and mechanical advantages it would also make a useful biosensor since estradiol poses numerous hazards to the environment. Therefore the ability to sense it cheaply and effectively would have great implications.</p><br />
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The Estrogen Receptor is made up of 6 domains: A, B, C, D, E, and F. Domains A and B serve as a transcriptional factor, domain C recognizes and binds to specific DNA sequences and is also involved in dimerization (Peters 1999). Domain E is the Ligand Binding Domain (LBD); when it binds to estrogen it homodimerizes to another Estrogen Receptor LBD. Domain D’s function is unknown. Domain F has been shown to inhibit dimerization and/or binding of estrogen and is thus undesirable for our project. In a wild type system these domains interact to bind Estrogen with Domain E, confer conformation so that C can bind a specific DNA sequence, dimerize with another estrogen receptor and act as a transcription factor to promote transcription downstream of its binding.</p><br><br><br />
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After studying the function of each of the domains as well as the findings of Peters 1999 paper we began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these estrogen receptor truncations were attached to the 3' end of ToxR and tested under various Estradiol levels. However, these truncations were non-responsive with and without estradiol. We hypothesized that different estrogen receptor truncations may be unable to express in the membrane, and the LBD by itself does not homodimerize as it didn't in the Peters' 1999 study. So, in order to mitigate both possibilities we made 8 more truncations that spanned the range between Domain A and Domain C.</p><br><br><br />
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Preliminary data suggests that truncations 5, 6, and 8 all showed constitutive ToxR dimerization and resulting fluorescence. However, none seemed to be inducible in response to Estrogen. Nevertheless, we believe we are getting very close to successfully expressing an inducible chimera because by showing activity it proves the estrogen receptor is not broken. Peters 1999 showed that the estrogen receptor can show dimer inducibility as a chimera in the presence of estrogen, and we think that trying a few more strategies will allow our ToxR chimera system to be inducible. We are going to try several more truncations, as well as various linkers, and the co-expression of different estrogen receptor truncations as was shown to work in the Peters paper.</p><br><br><br />
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One issue that could be affecting the Estrogen Receptor is the physiology of the periplasm that causes its cysteines in the LBD to cross react. This could potentially throw off the structure and therefore the functionality of the ER. Ee sought to correct this problem by growing the cells in a reducing environment that would prevent such bonds from forming. Our preliminary results below show that Trunc 8 is greatly stabalized by the addition of beta-mercaptoethanol (BME), effectively creating a reducing environment sensor.</p><br><br><br />
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Since the above system, although clearly stabalized in the periplasm, was not estradiol inducible we decided to look deeper into its ability to sense reducing environments. The first experiment showed how it grew in a reducing agent, while our next experiment below showed how it would respond (after growth saturation) to introduction into a buffered and non-buffered reducing environment. Once again Trunc 8 showed impressive inducibility in a reducing environment but only if the solution was buffered. </p><br><br><br />
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We believe that the right combination of buffer, reducing media, and truncation size will allow us to create a functioning estrogen biosensor, but in the meantime we have a proof of principle for our biosensor platform in the form of a reducing environment sensor. </p><br><br><br />
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All of the composite parts submitted to the registry and experiments discussed on this wiki were created and performed by the 2011 iGEM team Berkeley undergraduates. Some of the basic parts (RBSes, etc.) were obtained through the Anderson lab, for which they have our thanks.</p><br />
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The question of how cells detect specific molecules has always been important to biology, but is becoming an increasingly useful question to ask as we begin to engineer life. The applications of biosensing are widespread. There is a demand for biological diagnostic tools in medical or environmental applications, like detecting diseases or environmental contaminants. Sensing also has applications in biosecurity systems, when bioengineers try to develop strains that are stably dependent on the presence certain molecules. Finally, developing sensing systems for specific molecules is invaluable in metabolic engineering, because it allows for construction of metabolite-specific selection schemes. </p><br />
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Traditionally, most sensing systems in synthetic constructs have been borrowed from nature. However, very often researchers want to detect a ligand that does not have a receptor in the existing list of characterized sensors. Scanning natural systems for proteins that bind to a specific molecules is difficult and time-consuming, when it is not impossible. As synthetic biology realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.</p><p><br />
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<p>Here at Berkeley we were motivated to address this problem by constructing a biosensing mechanism that could be readily adapted to the ligand of choice. We wanted to build a modular biosensing device that could be modified by swapping binding domains. This approach would provide a standard method for engineering biosensors that could make use of both natural binding domains, optimized by evolution, as well as engineered domains that respond to molecules outside the range of natural receptors.</p><p><br />
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ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of ToxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association passes through the membrane, bringing the cytoplasmic domains of ToxR together to form an active homodimer. The active ToxR dimers drive transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.</p><p><br />
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ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.</p><br><br><br />
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<p> Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). ToxS detects changes in the environment, interacts with, and dimerizes ToxR's periplasmic domain. Dimerization of the periplasmic domain of ToxR results in dimerization of the cytoplasmic domain, which activates transcription of the ctx promoter. The ToxR domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by further engineering this dimerization-dependent transcriptional activation feature of the ToxR system to be ligand dependent. </p><p><br />
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We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the periplasmic domain of ToxR. The periplasmic domain will dimerize only in the presence of a particular ligand. The ligand dependent dimerization of the periplasmic domain of the ToxR fusion will cause the cytoplasmic domain of ToxR to dimerize, activating the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.</p><br><br />
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<p>The first step was to validate that a ToxR based two-hybrid system could be expressed in E. coli and that we could cause dimer-dependent transcriptional activation using a chimeric ToxR with periplasmic domains of our choosing. We started by truncating ToxR to eliminate the periplasmic domain because the dimerization state of ToxR's cytoplasmic domain controls transcription. Next, we built a translational fusion of the constitutively dimerizing leucine zipper protein IILK to the ToxR truncation. The constitutively dimerizing protein IILK portion of the translational fusion is located in the periplasm, while the ToxR cytoplasmic domain remains in the cytoplasm. The constitutively dimerizing IILK periplasmic domain should dimerize, causing the cytoplasmic domain of ToxR to dimerize, activating transcription from the ctx promoter.</p><br />
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The initial ToxR chimeras under the pBAD promoter were toxic to the cell, were difficult to express, and behaved unpredictably. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well. The graphs below show ToxR fused to Lambda-Rep, a different constitutive dimerizing protein (similar in behavior to IILK with ToxR fusions) that we used as the periplasmic domain in ToxR chimeras. The left graph demonstrates decreased growth and the right demonstrates low fluorescence with increasing arabinose. Both poor growth and low fluorescence are a result of toxicity from ToxR based chimeras. </p><br />
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<p>In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for stress-based regulatory elements. We wanted to find a promoter that would drive transcription while the cell was happy and health, but would shut down in response to cellular stress, behaving as a self-regulating expression system. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without toxicity to the cell. </p><br><br />
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<p> Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress-responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating its expression to produce the maximum amount of ToxR chimera without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the ToxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.</p><br />
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In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 potential stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP.</p><br><br><br />
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<p>The construct was transformed into E. coli and subjected to general stress conditions. We measured the fluorescence with a tecan plate reader and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a few did show a significant change in fluorescence, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.</p><br><br><br />
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<p>Since some of the stress promoters displayed the desired behavior, we proceeded to incorporate the stress promoter pool into a stress response system with ToxR. In our stress feedback system, expression of the ToxR chimera activates the ctx promoter (Pctx), driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating ToxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of ToxR chimera expression without toxicity.</p><br><br><br />
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In order to ensure nonlethal expression of the ToxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of ToxR-IILK. We transformed the plasmid into cells containing Pctx.RBS.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.</p><br><br><br />
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<p>From our combinatorial approach towards the construction of the stress-responsive feedback system, we were able to obtain big, healthy green colonies growing on our plates. The observation of big colonies indicated that our cells were healthy and not experiencing any apparent cellular stress. The high levels of GFP observed in the colonies indicated that cells were expressing enough ToxR chimera to strongly drive transcription of the ctx promoter (Pctx). Since we saw colonies that were big and green, we claim to have successfully resolved the toxicity of ToxR fusion proteins enabling strong transcriptional output while minimizing cellular stress.</p><br><br><br />
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We picked big green colonies and found that cultures showed improved fluorescence and OD with respect to pBAD promoters. In other experiments, we found that Lambda-Rep provided a better fluorescent signal so we replaced the IILK domain with Lambda-Rep in both the pBAD and Pstress, grew them in the same arabinose conditions and measured OD600 and RFU with a Tecan plate reader every five minutes. The as with IILK, ToxR-Lambda-Rep chimeras show improved fluorescence, reduced toxcity, and cells harboring this plasmid behave much more predictably.<br />
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<p>From our results thus far, we were able to:</p><br />
<b>-Characterize a library of stress promoters from the E. coli genome, providing data that they function as stress-responsive downregulators<br><br />
-Create an off-the-shelf negative feedback expression system using our stress promoters that enables the expression of toxic proteins to their highest non-toxic levels, solving our problem of ToxR chimera toxicity<br><br />
-Demonstrated our proof of principle biosensing system by creating constitutively dimerizing ToxR chimeras that drive transcription off of the ctx promoter (Pctx)</p></b><br />
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<p>Having achieved our goal of testing our proof of principle, we were ready to construct inducible homodimerizing systems.</p><br><br><br />
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Once we had successfully expressed a ToxR chimera that demonstrated constitutive transcription off Pctx we were ready to design a system that could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer, and after an extensive literature search, we decided to use the Estrogen Receptor. We chose the Estrogen receptor because it fulfilled the criteria of being a homodimer and dependant on Estradiol for dimerization. Furthermore, it has been previously used in a two-hybrid assay as a chimera attached to GAL4 parts to measure ß-galactosidase activity (Peters 1999). In addition to its structural and mechanical advantages it would also make a useful biosensor since estradiol poses numerous hazards to the environment. Therefore the ability to sense it cheaply and effectively would have great implications.</p><br />
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The Estrogen Receptor is made up of 6 domains: A, B, C, D, E, and F. Domains A and B serve as a transcriptional factor, domain C recognizes and binds to specific DNA sequences and is also involved in dimerization (Peters 1999). Domain E is the Ligand Binding Domain (LBD); when it binds to estrogen it homodimerizes to another Estrogen Receptor LBD. Domain D’s function is unknown. Domain F has been shown to inhibit dimerization and/or binding of estrogen and is thus undesirable for our project. In a wild type system these domains interact to bind Estrogen with Domain E, confer conformation so that C can bind a specific DNA sequence, dimerize with another estrogen receptor and act as a transcription factor to promote transcription downstream of its binding.</p><br><br><br />
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After studying the function of each of the domains as well as the findings of Peters 1999 paper we began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these estrogen receptor truncations were attached to the 3' end of ToxR and tested under various Estradiol levels. However, these truncations were non-responsive with and without estradiol. We hypothesized that different estrogen receptor truncations may be unable to express in the membrane, and the LBD by itself does not homodimerize as it didn't in the Peters' 1999 study. So, in order to mitigate both possibilities we made 8 more truncations that spanned the range between Domain A and Domain C.</p><br><br><br />
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Preliminary data suggests that truncations 5, 6, and 8 all showed constitutive ToxR dimerization and resulting fluorescence. However, none seemed to be inducible in response to Estrogen. Nevertheless, we believe we are getting very close to successfully expressing an inducible chimera because by showing activity it proves the estrogen receptor is not broken. Peters 1999 showed that the estrogen receptor can show dimer inducibility as a chimera in the presence of estrogen, and we think that trying a few more strategies will allow our ToxR chimera system to be inducible. We are going to try several more truncations, as well as various linkers, and the co-expression of different estrogen receptor truncations as was shown to work in the Peters paper.</p><br><br><br />
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One issue that could be affecting the Estrogen Receptor is the physiology of the periplasm that causes its cysteines in the LBD to cross react. This could potentially throw off the structure and therefore the functionality of the ER. Ee sought to correct this problem by growing the cells in a reducing environment that would prevent such bonds from forming. Our preliminary results below show that Trunc 8 is greatly stabalized by the addition of beta-mercaptoethanol (BME), effectively creating a reducing environment sensor.</p><br><br><br />
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Since the above system, although clearly stabalized in the periplasm, was not estradiol inducible we decided to look deeper into its ability to sense reducing environments. The first experiment showed how it grew in a reducing agent, while our next experiment below showed how it would respond (after growth saturation) to introduction into a buffered and non-buffered reducing environment. Once again Trunc 8 showed impressive inducibility in a reducing environment but only if the solution was buffered. </p><br><br><br />
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We believe that the right combination of buffer, reducing media, and truncation size will allow us to create a functioning estrogen biosensor, but in the meantime we have a proof of principle for our biosensor platform in the form of a reducing environment sensor. </p><br><br><br />
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All of the composite parts submitted to the registry and experiments discussed on this wiki were created and performed by the 2011 iGEM team Berkeley undergraduates. Some of the basic parts (RBSes, etc.) were obtained through the Anderson lab, for which they have our thanks.</p><br />
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The question of how cells detect specific molecules has always been important to biology, but is becoming an increasingly useful question to ask as we begin to engineer life. The applications of biosensing are widespread. There is a demand for biological diagnostic tools in medical or environmental applications, like detecting diseases or environmental contaminants. Sensing also has applications in biosecurity systems, when bioengineers try to develop strains that are stably dependent on the presence certain molecules. Finally, developing sensing systems for specific molecules is invaluable in metabolic engineering, because it allows for construction of metabolite-specific selection schemes. </p><br />
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Traditionally, most sensing systems in synthetic constructs have been borrowed from nature. However, very often researchers want to detect a ligand that does not have a receptor in the existing list of characterized sensors. Scanning natural systems for proteins that bind to a specific molecules is difficult and time-consuming, when it is not impossible. As synthetic biology realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.</p><p><br />
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<p>Here at Berkeley we were motivated to address this problem by constructing a biosensing mechanism that could be readily adapted to the ligand of choice. We wanted to build a modular biosensing device that could be modified by swapping binding domains. This approach would provide a standard method for engineering biosensors that could make use of both natural binding domains, optimized by evolution, as well as engineered domains that respond to molecules outside the range of natural receptors.</p><p><br />
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ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of ToxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association passes through the membrane, bringing the cytoplasmic domains of ToxR together to form an active homodimer. The active ToxR dimers drive transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.</p><p><br />
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ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.</p><br><br><br />
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<p> Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). ToxS detects changes in the environment, interacts with, and dimerizes ToxR's periplasmic domain. Dimerization of the periplasmic domain of ToxR results in dimerization of the cytoplasmic domain, which activates transcription of the ctx promoter. The ToxR domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by further engineering this dimerization-dependent transcriptional activation feature of the ToxR system to be ligand dependent. </p><p><br />
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We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the periplasmic domain of ToxR. The periplasmic domain will dimerize only in the presence of a particular ligand. The ligand dependent dimerization of the periplasmic domain of the ToxR fusion will cause the cytoplasmic domain of ToxR to dimerize, activating the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.</p><br><br />
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<p>The first step was to validate that a ToxR based two-hybrid system could be expressed in E. coli and that we could cause dimer-dependent transcriptional activation using a chimeric ToxR with periplasmic domains of our choosing. We started by truncating ToxR to eliminate the periplasmic domain because the dimerization state of ToxR's cytoplasmic domain controls transcription. Next, we built a translational fusion of the constitutively dimerizing leucine zipper protein IILK to the ToxR truncation. The constitutively dimerizing protein IILK portion of the translational fusion is located in the periplasm, while the ToxR cytoplasmic domain remains in the cytoplasm. The constitutively dimerizing IILK periplasmic domain should dimerize, causing the cytoplasmic domain of ToxR to dimerize, activating transcription from the ctx promoter.</p><br />
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The initial ToxR chimeras under the pBAD promoter were toxic to the cell, were difficult to express, and behaved unpredictably. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well. The graphs below show ToxR fused to Lambda-Rep, a different constitutive dimerizing protein (similar in behavior to IILK with ToxR fusions) that we used as the periplasmic domain in ToxR chimeras. The left graph demonstrates decreased growth and the right demonstrates low fluorescence with increasing arabinose. Both poor growth and low fluorescence are a result of toxicity from ToxR based chimeras. </p><br />
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<p>In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for stress-based regulatory elements. We wanted to find a promoter that would drive transcription while the cell was happy and health, but would shut down in response to cellular stress, behaving as a self-regulating expression system. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without toxicity to the cell. </p><br><br />
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<p> Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress-responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating its expression to produce the maximum amount of ToxR chimera without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the ToxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.</p><br />
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In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 potential stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP.</p><br><br><br />
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<p>The construct was transformed into E. coli and subjected to general stress conditions. We measured the fluorescence with a tecan plate reader and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a few did show a significant change in fluorescence, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.</p><br><br><br />
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<p>Since some of the stress promoters displayed the desired behavior, we proceeded to incorporate the stress promoter pool into a stress response system with ToxR. In our stress feedback system, expression of the ToxR chimera activates the ctx promoter (Pctx), driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating ToxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of ToxR chimera expression without toxicity.</p><br><br><br />
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In order to ensure nonlethal expression of the ToxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of ToxR-IILK. We transformed the plasmid into cells containing Pctx.RBS.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.</p><br><br><br />
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<p>From our combinatorial approach towards the construction of the stress-responsive feedback system, we were able to obtain big, healthy green colonies growing on our plates. The observation of big colonies indicated that our cells were healthy and not experiencing any apparent cellular stress. The high levels of GFP observed in the colonies indicated that cells were expressing enough ToxR chimera to strongly drive transcription of the ctx promoter (Pctx). Since we saw colonies that were big and green, we claim to have successfully resolved the toxicity of ToxR fusion proteins enabling strong transcriptional output while minimizing cellular stress.</p><br><br><br />
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We picked big green colonies and found that cultures showed improved fluorescence and OD with respect to pBAD promoters. In other experiments, we found that Lambda-Rep provided a better fluorescent signal so we replaced the IILK domain with Lambda-Rep in both the pBAD and Pstress, grew them in the same arabinose conditions and measured OD600 and RFU with a Tecan plate reader every five minutes. The as with IILK, ToxR-Lambda-Rep chimeras show improved fluorescence, reduced toxcity, and cells harboring this plasmid behave much more predictably.<br />
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<p>From our results thus far, we were able to:</p><br />
<b>-Characterize a library of stress promoters from the E. coli genome, providing data that they function as stress-responsive downregulators<br><br />
-Create an off-the-shelf negative feedback expression system using our stress promoters that enables the expression of toxic proteins to their highest non-toxic levels, solving our problem of ToxR chimera toxicity<br><br />
-Demonstrated our proof of principle biosensing system by creating constitutively dimerizing ToxR chimeras that drive transcription off of the ctx promoter (Pctx)</p></b><br />
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<p>Having achieved our goal of testing our proof of principle, we were ready to construct inducible homodimerizing systems.</p><br><br><br />
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Once we had successfully expressed a ToxR chimera that demonstrated constitutive transcription off Pctx we were ready to design a system that could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer, and after an extensive literature search, we decided to use the Estrogen Receptor. We chose the Estrogen receptor because it fulfilled the criteria of being a homodimer and dependant on Estradiol for dimerization. Furthermore, it has been previously used in a two-hybrid assay as a chimera attached to GAL4 parts to measure ß-galactosidase activity (Peters 1999). In addition to its structural and mechanical advantages it would also make a useful biosensor since estradiol poses numerous hazards to the environment. Therefore the ability to sense it cheaply and effectively would have great implications.</p><br />
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The Estrogen Receptor is made up of 6 domains: A, B, C, D, E, and F. Domains A and B serve as a transcriptional factor, domain C recognizes and binds to specific DNA sequences and is also involved in dimerization (Peters 1999). Domain E is the Ligand Binding Domain (LBD); when it binds to estrogen it homodimerizes to another Estrogen Receptor LBD. Domain D’s function is unknown. Domain F has been shown to inhibit dimerization and/or binding of estrogen and is thus undesirable for our project. In a wild type system these domains interact to bind Estrogen with Domain E, confer conformation so that C can bind a specific DNA sequence, dimerize with another estrogen receptor and act as a transcription factor to promote transcription downstream of its binding.</p><br><br><br />
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After studying the function of each of the domains as well as the findings of Peters 1999 paper we began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these estrogen receptor truncations were attached to the 3' end of ToxR and tested under various Estradiol levels. However, these truncations were non-responsive with and without estradiol. We hypothesized that different estrogen receptor truncations may be unable to express in the membrane, and the LBD by itself does not homodimerize as it didn't in the Peters' 1999 study. So, in order to mitigate both possibilities we made 8 more truncations that spanned the range between Domain A and Domain C.</p><br><br><br />
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Preliminary data suggests that truncations 5, 6, and 8 all showed constitutive ToxR dimerization and resulting fluorescence. However, none seemed to be inducible in response to Estrogen. Nevertheless, we believe we are getting very close to successfully expressing an inducible chimera because by showing activity it proves the estrogen receptor is not broken. Peters 1999 showed that the estrogen receptor can show dimer inducibility as a chimera in the presence of estrogen, and we think that trying a few more strategies will allow our ToxR chimera system to be inducible. We are going to try several more truncations, as well as various linkers, and the co-expression of different estrogen receptor truncations as was shown to work in the Peters paper.</p><br><br><br />
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One issue that could be affecting the Estrogen Receptor is the physiology of the periplasm that causes its cysteines in the LBD to cross react. This could potentially throw off the structure and therefore the functionality of the ER. Ee sought to correct this problem by growing the cells in a reducing environment that would prevent such bonds from forming. Our preliminary results below show that Trunc 8 is greatly stabalized by the addition of beta-mercaptoethanol (BME), effectively creating a reducing environment sensor.</p><br><br><br />
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Since the above system, although clearly stabalized in the periplasm, was not estradiol inducible we decided to look deeper into its ability to sense reducing environments. The first experiment showed how it grew in a reducing agent, while our next experiment below showed how it would respond (after growth saturation) to introduction into a buffered and non-buffered reducing environment. Once again Trunc 8 showed impressive inducibility in a reducing environment but only if the solution was buffered. </p><br><br><br />
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We believe that the right combination of buffer, reducing media, and truncation size will allow us to create a functioning estrogen biosensor, but in the meantime we have a proof of principle for our biosensor platform in the form of a reducing environment sensor. </p><br><br><br />
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All of the composite parts submitted to the registry and experiments discussed on this wiki were created and performed by the 2011 iGEM team Berkeley undergraduates. Some of the basic parts (RBSes, etc.) were obtained through the Anderson lab, for which they have our thanks.</p><br />
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<p>Biosensors have widespread applications ranging from diagnostics to environmental monitoring. Vibrio cholerae's ToxR system can be used as a component in biological sensing devices. ToxS causes ToxR homodimerization, activating transcription of the ctx promoter. By replacing the periplasmic domain of ToxR with existing or engineered ligand-dependent homodimers, we hope to link ToxR dimerization (and gene expression) to the presence of specific ligands. Initially, ToxR constructs proved toxic to E. coli. We built a stress-regulated transcription system that drives relatively high expression of toxic proteins. This allowed us to further engineer ToxR chimeras. We fused an estrogen-dependent dimer with ToxR hoping to create an estrogen biosensor. We observed a range of constitutive phenotypes and plan more experiments to engineer a dose-dependent transcriptional response to estrogen. By fusing existing or engineered ligand dependent homodimers to ToxR, this modular system can be used to build new biosensors. </p><br />
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<div class="col3-2"style="background-color:#101a4d;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxR"> <img src="https://static.igem.org/mediawiki/igem.org/1/10/Subtitlepic1.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> A protein with great potential as a general biosensor system.</p> </div><br />
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<div class="col3-2"style="background-color:#185f73;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Chimeric proteins that drive translation off of the Pctx promoter.</p></div><br />
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<div class="col3-2" style="background-color:#303285"><a href="https://2011.igem.org/Team:Berkeley/Project#StressPromoters"><img src="https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Our method for expressing interesting (but toxic) proteins.</p> </div><br />
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<p style="text-align:center; color:#CECECE;"> Bacteria engineered to detect the presence of estrogen. <br></p></div><br />
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<p>We are Team Berkeley, a cohesive unit of 7 undergraduates and 3 advisers. Earlier this year we planned a complex and risky project, given the short amount of time iGEM made available. We quickly learned each others strengths and weaknesses and developed standard systems of organizational management in order to synchronize our efforts for the many parallel tasks at hand. We created protocols, shared them with one another, and worked together on troubleshooting. Using google docs to keep up with the status of cloning projects, the results of assays, material logistics, or the final steps required to complete a project ensured that through the months of hard work, we fine-tuned our ability to work together. As a team, we have learned firsthand how the synthetic biology community relies on the goal-oriented cooperation of skilled individuals from very different backgrounds and with very different skill sets. Some of us have strong engineering backgrounds while others of us have strong biology backgrounds, but we all share a desire to build synthetic biological systems that solve human problems. We are proud of the project that we have created which we will present at the Jamboree together in October.</p><br />
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The question of how cells detect specific molecules has always been important to biology, but is becoming an increasingly useful question to ask as we begin to engineer life. The applications of biosensing are widespread. There is a demand for biological diagnostic tools in medical or environmental applications, like detecting diseases or environmental contaminants. Sensing also has applications in biosecurity systems, when bioengineers try to develop strains that are stably dependent on the presence certain molecules. Finally, developing sensing systems for specific molecules is invaluable in metabolic engineering, because it allows for construction of metabolite-specific selection schemes. </p><br />
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Traditionally, most sensing systems in synthetic constructs have been borrowed from nature. However, very often researchers want to detect a ligand that does not have a receptor in the existing list of characterized sensors. Scanning natural systems for proteins that bind to a specific molecules is difficult and time-consuming, when it is not impossible. As synthetic biology realizes more and more unnatural functions, we will want to be able to design cells that can detect molecules that life has never before responded to. This is something we cannot achieve using natural receptors.</p><p><br />
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<p>Here at Berkeley we were motivated to address this problem by constructing a biosensing mechanism that could be readily adapted to the ligand of choice. We wanted to build a modular biosensing device that could be modified by swapping binding domains. This approach would provide a standard method for engineering biosensors that could make use of both natural binding domains, optimized by evolution, as well as engineered domains that respond to molecules outside the range of natural receptors.</p><p><br />
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ToxR is a characterized transmembrane transcription factor from Vibrio cholerae with active domains in both the periplasm and cytoplasm. Natively, it is involved in the transcription of a number of virulence factors, including the two subunits of the cholera toxin ctxAB and the TCP pilus. It is activated in trans by ToxS, a membrane-anchored periplasmic protein coded by the gene directly downstream of ToxR. Active ToxS in the periplasm stabilizes the dimerization of the periplasmic domain of ToxR. The association passes through the membrane, bringing the cytoplasmic domains of ToxR together to form an active homodimer. The active ToxR dimers drive transcription from the ctx promoter by binding to TTTGAT repeats and recruiting transcription machinery.</p><p><br />
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ToxR elegantly allows Vibrio cholerae rapid sensing and the activation of pathogenic functions. Virulence factors such as those controlled by this system are often clustered in the genome within pathogenicity islands, remnants from a past horizontal gene transfer. The simplicity and orthogonality of the systems contained in these small virulence cassettes make them an ideal source for modular biosynthetic tools. Indeed, one of the reasons ToxR is so interesting is that it single handedly achieves the task of the standard two-component signal transduction pathway: it is activated in the periplasm and directly promotes transcription in the cytoplasm. The simplicity of this regulation system and the scope of this single protein make it an ideal starting point for synthetic design.</p><br><br><br />
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<p> Dimerization of ToxR has been found to activate the ctx promoter in Vibrio cholerae by binding directly to the DNA. DiRita et. al. (1991). ToxS detects changes in the environment, interacts with, and dimerizes ToxR's periplasmic domain. Dimerization of the periplasmic domain of ToxR results in dimerization of the cytoplasmic domain, which activates transcription of the ctx promoter. The ToxR domains are all on one peptide, so signals can be quickly relayed from the periplasm to the cytoplasm. This unique aspect of ToxR makes it a good part for building a biosensor. We aim to make a biosensor by further engineering this dimerization-dependent transcriptional activation feature of the ToxR system to be ligand dependent. </p><p><br />
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We hypothesized that we could create a biosensor if we attach ligand dependent dimerizing proteins to the periplasmic domain of ToxR. The periplasmic domain will dimerize only in the presence of a particular ligand. The ligand dependent dimerization of the periplasmic domain of the ToxR fusion will cause the cytoplasmic domain of ToxR to dimerize, activating the ctx promoter. By putting GFP after the ctx promoter, we can then detect if the ctx promoter is activated.</p><br><br />
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<p>The first step was to validate that a ToxR based two-hybrid system could be expressed in E. coli and that we could cause dimer-dependent transcriptional activation using a chimeric ToxR with periplasmic domains of our choosing. We started by truncating ToxR to eliminate the periplasmic domain because the dimerization state of ToxR's cytoplasmic domain controls transcription. Next, we built a translational fusion of the constitutively dimerizing leucine zipper protein IILK to the ToxR truncation. The constitutively dimerizing protein IILK portion of the translational fusion is located in the periplasm, while the ToxR cytoplasmic domain remains in the cytoplasm. The constitutively dimerizing IILK periplasmic domain should dimerize, causing the cytoplasmic domain of ToxR to dimerize, activating transcription from the ctx promoter.</p><br />
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The initial ToxR chimeras under the pBAD promoter were toxic to the cell, were difficult to express, and behaved unpredictably. Even at low concentrations of arabinose, there was high toxicity and cells did not grow well. The graphs below show ToxR fused to Lambda-Rep, a different constitutive dimerizing protein (similar in behavior to IILK with ToxR fusions) that we used as the periplasmic domain in ToxR chimeras. The left graph demonstrates decreased growth and the right demonstrates low fluorescence with increasing arabinose. Both poor growth and low fluorescence are a result of toxicity from ToxR based chimeras. </p><br />
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<p>In order to resolve the toxicity of ToxR, we constructed a new design principle. Stress is a natural regulatory cue in bacteria that dictates changes in behavior or expression levels. We decided to look into the E. coli genome for stress-based regulatory elements. We wanted to find a promoter that would drive transcription while the cell was happy and health, but would shut down in response to cellular stress, behaving as a self-regulating expression system. Toxic over expression of ToxR would be down regulated with this negative feedback system. With this method, we are able to express ToxR chimeras at optimal levels without toxicity to the cell. </p><br><br />
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<p> Stress is a common regulatory cue that allows organisms to adjust to variable conditions in the environment. As a result, stress-responsive regulatory elements are prevalent motifs in nature. Due to the toxicity caused by the toxR chimeras in E. coli, a method of regulating its expression to produce the maximum amount of ToxR chimera without killing the cell was needed. We hypothesized that the E. coli genome likely contains such stress responsive regulatory systems from which regulatory feedback systems can be constructed. Expression of the ToxR chimera to toxic levels by stress promoters can be downregulated by stress in a feedback loop. This maintains a high but nonlethal level of expression of the toxic gene.</p><br />
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In order to construct a feedback system, we analyzed microarray data of the E. coli genome for regulatory elements downregulated by stress. Moen, et. al. (2009) subjected E. coli to generalized stresses and measured the global mRNA levels of all genome elements for stress response. From their data, we identified 35 potential stress promoters that were downregulated under all stress conditions tested for further characterization. We isolated the region of the genome upstream of the ORF through PCR and placed the resulting pool of 35 stress promoters in front of GFP.</p><br><br><br />
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<p>The construct was transformed into E. coli and subjected to general stress conditions. We measured the fluorescence with a tecan plate reader and looked for decreased fluorescence. While not all promoters responded by downregulating GFP expression, a few did show a significant change in fluorescence, with the cold condition yielding the best data. The fluorescence measurements from the Tecan plate reader were confirmed by flow cytometry.</p><br><br><br />
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<p>Since some of the stress promoters displayed the desired behavior, we proceeded to incorporate the stress promoter pool into a stress response system with ToxR. In our stress feedback system, expression of the ToxR chimera activates the ctx promoter (Pctx), driving GFP production, and creates cellular stress. The stress promoter responds to stress by downregulating ToxR-IILK expression to a nonlethal level, allowing the cell to maintain a high level of ToxR chimera expression without toxicity.</p><br><br><br />
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In order to ensure nonlethal expression of the ToxR chimera, we created an rbs library for each of the 35 stress promoters to construct a promoter-rbs library in front of ToxR-IILK. We transformed the plasmid into cells containing Pctx.RBS.GFP and grew them up to assay the efficacy of the stress responsive feedback system. In our growth assay, we looked for large, healthy colonies as well as strong fluorescence, indicators of robust growth and desired expression levels.</p><br><br><br />
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<p>From our combinatorial approach towards the construction of the stress-responsive feedback system, we were able to obtain big, healthy green colonies growing on our plates. The observation of big colonies indicated that our cells were healthy and not experiencing any apparent cellular stress. The high levels of GFP observed in the colonies indicated that cells were expressing enough ToxR chimera to strongly drive transcription of the ctx promoter (Pctx). Since we saw colonies that were big and green, we claim to have successfully resolved the toxicity of ToxR fusion proteins enabling strong transcriptional output while minimizing cellular stress.</p><br><br><br />
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We picked big green colonies and found that cultures showed improved fluorescence and OD with respect to pBAD promoters. In other experiments, we found that Lambda-Rep provided a better fluorescent signal so we replaced the IILK domain with Lambda-Rep in both the pBAD and Pstress, grew them in the same arabinose conditions and measured OD600 and RFU with a Tecan plate reader every five minutes. The as with IILK, ToxR-Lambda-Rep chimeras show improved fluorescence, reduced toxcity, and cells harboring this plasmid behave much more predictably.<br />
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<p>From our results thus far, we were able to:</p><br />
<b>-Characterize a library of stress promoters from the E. coli genome, providing data that they function as stress-responsive downregulators<br><br />
-Create an off-the-shelf negative feedback expression system using our stress promoters that enables the expression of toxic proteins to their highest non-toxic levels, solving our problem of ToxR chimera toxicity<br><br />
-Demonstrated our proof of principle biosensing system by creating constitutively dimerizing ToxR chimeras that drive transcription off of the ctx promoter (Pctx)</p></b><br />
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<p>Having achieved our goal of testing our proof of principle, we were ready to construct inducible homodimerizing systems.</p><br><br><br />
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Once we had successfully expressed a ToxR chimera that demonstrated constitutive transcription off Pctx we were ready to design a system that could be inducible, and thus a more useful biosensor. We set out to find a ligand dependent homodimer, and after an extensive literature search, we decided to use the Estrogen Receptor. We chose the Estrogen receptor because it fulfilled the criteria of being a homodimer and dependant on Estradiol for dimerization. Furthermore, it has been previously used in a two-hybrid assay as a chimera attached to GAL4 parts to measure ß-galactosidase activity (Peters 1999). In addition to its structural and mechanical advantages it would also make a useful biosensor since estradiol poses numerous hazards to the environment. Therefore the ability to sense it cheaply and effectively would have great implications.</p><br />
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The Estrogen Receptor is made up of 6 domains: A, B, C, D, E, and F. Domains A and B serve as a transcriptional factor, domain C recognizes and binds to specific DNA sequences and is also involved in dimerization (Peters 1999). Domain E is the Ligand Binding Domain (LBD); when it binds to estrogen it homodimerizes to another Estrogen Receptor LBD. Domain D’s function is unknown. Domain F has been shown to inhibit dimerization and/or binding of estrogen and is thus undesirable for our project. In a wild type system these domains interact to bind Estrogen with Domain E, confer conformation so that C can bind a specific DNA sequence, dimerize with another estrogen receptor and act as a transcription factor to promote transcription downstream of its binding.</p><br><br><br />
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After studying the function of each of the domains as well as the findings of Peters 1999 paper we began by making two different ToxR chimeras with two Estrogen Receptor truncations: ER∆F (no F domain) and the ER-LBD alone. Both of these estrogen receptor truncations were attached to the 3' end of ToxR and tested under various Estradiol levels. However, these truncations were non-responsive with and without estradiol. We hypothesized that different estrogen receptor truncations may be unable to express in the membrane, and the LBD by itself does not homodimerize as it didn't in the Peters' 1999 study. So, in order to mitigate both possibilities we made 8 more truncations that spanned the range between Domain A and Domain C.</p><br><br><br />
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Preliminary data suggests that truncations 5, 6, and 8 all showed constitutive ToxR dimerization and resulting fluorescence. However, none seemed to be inducible in response to Estrogen. Nevertheless, we believe we are getting very close to successfully expressing an inducible chimera because by showing activity it proves the estrogen receptor is not broken. Peters 1999 showed that the estrogen receptor can show dimer inducibility as a chimera in the presence of estrogen, and we think that trying a few more strategies will allow our ToxR chimera system to be inducible. We are going to try several more truncations, as well as various linkers, and the co-expression of different estrogen receptor truncations as was shown to work in the Peters paper.</p><br><br><br />
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One issue that could be affecting the Estrogen Receptor is the physiology of the periplasm that causes its cysteines in the LBD to cross react. This could potentially throw off the structure and therefore the functionality of the ER. Ee sought to correct this problem by growing the cells in a reducing environment that would prevent such bonds from forming. Our preliminary results below show that Trunc 8 is greatly stabalized by the addition of beta-mercaptoethanol (BME), effectively creating a reducing environment sensor.</p><br><br><br />
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Since the above system, although clearly stabalized in the periplasm, was not estradiol inducible we decided to look deeper into its ability to sense reducing environments. The first experiment showed how it grew in a reducing agent, while our next experiment below showed how it would respond (after growth saturation) to introduction into a buffered and non-buffered reducing environment. Once again Trunc 8 showed impressive inducibility in a reducing environment but only if the solution was buffered. </p><br><br><br />
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We believe that the right combination of buffer, reducing media, and truncation size will allow us to create a functioning estrogen biosensor, but in the meantime we have a proof of principle for our biosensor platform in the form of a reducing environment sensor. </p><br><br><br />
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All of the composite parts submitted to the registry and experiments discussed on this wiki were created and performed by the 2011 iGEM team Berkeley undergraduates. Some of the basic parts (RBSes, etc.) were obtained through the Anderson lab, for which they have our thanks.</p><br />
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</html></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/PressTeam:NTNU-Trondheim/Press2013-09-30T21:24:10Z<p>Rahmilale: </p>
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<p>Biosensors have widespread applications ranging from diagnostics to environmental monitoring. Vibrio cholerae's ToxR system can be used as a component in biological sensing devices. ToxS causes ToxR homodimerization, activating transcription of the ctx promoter. By replacing the periplasmic domain of ToxR with existing or engineered ligand-dependent homodimers, we hope to link ToxR dimerization (and gene expression) to the presence of specific ligands. Initially, ToxR constructs proved toxic to E. coli. We built a stress-regulated transcription system that drives relatively high expression of toxic proteins. This allowed us to further engineer ToxR chimeras. We fused an estrogen-dependent dimer with ToxR hoping to create an estrogen biosensor. We observed a range of constitutive phenotypes and plan more experiments to engineer a dose-dependent transcriptional response to estrogen. By fusing existing or engineered ligand dependent homodimers to ToxR, this modular system can be used to build new biosensors. </p><br />
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<div class="col3-2"style="background-color:#101a4d;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxR"> <img src="https://static.igem.org/mediawiki/igem.org/1/10/Subtitlepic1.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> A protein with great potential as a general biosensor system.</p> </div><br />
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<div class="col3-2"style="background-color:#185f73;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Chimeric proteins that drive translation off of the Pctx promoter.</p></div><br />
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<div class="col3-2" style="background-color:#303285"><a href="https://2011.igem.org/Team:Berkeley/Project#StressPromoters"><img src="https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg" <br />
onmouseover="this.src='https://static.igem.org/mediawiki/2011/3/32/Subtitlepic2headerhover.jpg'" <br />
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<p style="text-align:center; color:#CECECE;"> Our method for expressing interesting (but toxic) proteins.</p> </div><br />
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<div class="col3-2"style="background-color:#499394"><a href="https://2011.igem.org/Team:Berkeley/Project#EstrogenBiosensor"><img src="https://static.igem.org/mediawiki/2011/c/c8/Subtitlepic4.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Bacteria engineered to detect the presence of estrogen. <br></p></div><br />
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<p>We are Team Berkeley, a cohesive unit of 7 undergraduates and 3 advisers. Earlier this year we planned a complex and risky project, given the short amount of time iGEM made available. We quickly learned each others strengths and weaknesses and developed standard systems of organizational management in order to synchronize our efforts for the many parallel tasks at hand. We created protocols, shared them with one another, and worked together on troubleshooting. Using google docs to keep up with the status of cloning projects, the results of assays, material logistics, or the final steps required to complete a project ensured that through the months of hard work, we fine-tuned our ability to work together. As a team, we have learned firsthand how the synthetic biology community relies on the goal-oriented cooperation of skilled individuals from very different backgrounds and with very different skill sets. Some of us have strong engineering backgrounds while others of us have strong biology backgrounds, but we all share a desire to build synthetic biological systems that solve human problems. We are proud of the project that we have created which we will present at the Jamboree together in October.</p><br />
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<b># of parts submitted:</b><br />
<ul><br />
<li>35 Regulatory promoters</li><br />
<li>2 Ribosome Binding Sites</li><br />
<li>2 Composite expression modules</li><br />
<li>2 Coding proteins</li><br />
</ul><br />
<b>Total:</b> 42 Parts <br> <br><br />
<b>Assembly Method:</b> BglBricks <br><br><br />
<b>Accomplishments:</b> <br><br />
<ul><br />
<li>All 42 parts have been submitted to the registry and can be found <a href="http://partsregistry.org/cgi/partsdb/pgroup.cgi?pgroup=iGEM2011&group=Berkeley">HERE</a></li><br />
<li>We submitted two new well-characterized BioBrick Parts in vector pSB1AC3 according to RFC10</li><br />
<li>Compiled a set of 35 stress promoters that future iGEMers can utilize as regulatory elements</li><br />
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<h3>Function</h3><br />
Stress promoters respond differently to various types of stress that is placed on the cell. We wanted a promoter that would be downregulated in the presence of membrane stress caused by the overexpression of ToxR fusion proteins. The design principle was to express ToxR fusion proteins under this promoter such that the stress it caused would downregulate its production.<br />
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<h3>Assembly</h3><br />
The 34 stress promoters, identified from microarray data (Moen, 2009), were PCRed from E. coli MG1655 Genomic DNA. Basic parts were made in plasmids with pUC origins. A constitutive promoter (Pcon) was also cloned into a basic part as a control for characterization.<br />
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<h3>Characterization</h3><br />
<p>The pool of 35 promoters was assembled in front of a rbs.sfGFP reporter gene, and the construct was transformed into E. coli MC1061. The transformed cells were subjected to generalized stresses (heat, cold, acid, base, salt) overnight, and their resulting fluorescence was measured.</p> <br />
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<h3>Data</h3><br />
<p>Downregulation in response to stress, resulting in decreased fluorescence, was desired. Tecan plate measurements showed initial downregulation in some of the stress promoeters, with the cold condition yielding the best results. Further flow cytometry experiments confirmed the stress-responsive downregulation shown by the Tecan. In the figures below, we show fluorescence relative to that of the constitutive promoter in both conditions. Here we show our cold condition (25 degrees C) vs our control condition (37 degrees C). We find that that these chosen 4 promoters have downregulated activity from the environmental stress we put on the cells.</p><br />
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<h3>Function</h3><br />
The expression plasmid consists of a stress promoter regulating the production of ToxR. This construct allows us to express enough ToxR to allow for the functionality we desire and at the same time have minimal affect on bacterial growth rate.<br />
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<h3>Assembly</h3><br />
The ToxR was PCR amplified out of V. cholerae genomic DNA, digested, and ligated in front of the leucine zipper IILK. A library of potential stress promoters and an RBS library were put in front of the ToxR fusion protein and the library was screened for healthy, transcriptionally active clones. We found a combination of the rffGH stress promoter and an rbs variant gave us the functionality we desired. This construct was then used as our template for our other ToxR fusion proteins. Note: for our ToxR chimeras, we switched from an IILK constitutive dimerizing periplasmic domain to Lambda-Rep and MukF constitutive dimerizing periplasmic domains after we identified the stress promoter because they provided higher levels fluorescence in our experiments. For this reason, we collected data using ToxR fusions of Lambda-Rep and MukF.<br />
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<br><br><center><img src="https://static.igem.org/mediawiki/2011/1/14/ToxRLamRep.png" height="120" align="center"></center><br />
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<h3>Characterization</h3><br />
For characterization, we made fusion proteins of ToxR with Lamda Repressor and MukF proteins. Both of these proteins dimerize in wild-type and we expect them to help ToxR dimerize as well. We transformed ToxR-LambdaRep driven by Pstress into our reporter strain (see below for more details). In parallel, we also transformed our original constructs of ToxR-lambdaRep driven by Pbad into our reporter strains. Each construct was grown in 100 mg/ml, 50 mg/ml, 25 mg/ml, 12 mg/ml, 6 mg/ml 3 mg/ml, 1.5 mg/ml, and 0 mg/ml arabinose. For our control, a known nontoxic plasmid was transformed into the cells that should not activate our reporter strain. <br />
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<h3>Data</h3><br />
As shown in the graph of Pbad-LambdaRep and Pbad-MukF, cells did not grow up well in the varying levels of arabinose. The relative fluorescence graphs also supported the conclusion that our constructs were toxic to the cells.<br />
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<br><br/><br />
Our solution to the high toxicity of our ToxR chimeras was to have a promoter that would down regulate transcription when the cell was under stress. As shown in the Pstress graphs of LambdaRep and MukF, cells grew to a much higher OD and fluorescence especially in Pstress-LambdaRep compared to the control. All levels of arabinose showed similar results.<br />
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<img src="https://static.igem.org/mediawiki/2011/2/26/Pstress_mukF.png" height="350"> <br><br />
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<h3>Function</h3><br />
The reporter construct give a fluorescent output that correlates to the amount of transcriptional activity ToxR promotes on Pctx (it's wild type promoter). Since transcriptional activity by ToxR dimers depends on the strength of dimerization, the level of fluorescence can be correlated to how tightly the ToxR dimerizes.<br />
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<h3>Assembly</h3><br />
ToxR’s native target, the ctx promoter (Pctx), was PCRed out of Vibrio cholerae genomic DNA and made into a basic part. The promoter was then placed upstream of an rbs.sfGFP part using 2AB assembly.<br />
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<br><br><center><img src="https://static.igem.org/mediawiki/2011/2/27/PctxGFP.png" height="100" align="center"></center><br />
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<h3>Characterization</h3><br />
By cotransforming this reporter plasmid and the expression plasmid (see above) we were able to measure the strength of ctx promoter (Pctx) induction by ToxR.<br />
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<h3>Data</h3><br />
See Expression Plasmid Data above.<br />
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<h3>Function</h3><br />
The human estrogen receptor is known to dimerize around estradiol. By fusing the human estrogen receptor to ToxR, we can create an estradiol biosensor and potentially an estradiol-inducible system in E. coli.<br />
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<h3>Assembly</h3><br />
The estrogen receptor was refactored to optimally express in E. coli and assembled using designed oligos and PCA (polymerase cycling assembly). The final protein was made into a basic part. Various sub-sections of the estrogen receptor were PCR amplified as basic parts in order to create truncations that might display different responses to estradiol (see figure). The estrogen receptor was fused to ToxR’s C-terminus with Gly-Ser linkers and thus exposed to the periplasm when expressed in the cell.<br />
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<br><br> <center><img src="https://static.igem.org/mediawiki/2011/2/29/EstrogenToxR.png" height="100"></center><br />
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<h3>Characterization</h3><br />
In order to characterize the system, the different truncations of estrogen receptor fused to ToxR were tested with a plate reader. In our control condition, no estrogen was added. For the experimental condition, 1uM of estradiol was added to the solution.<br />
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<center><img src="https://static.igem.org/mediawiki/2011/f/ff/EstrogenFigureFinal.png" height="370"></center><br />
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<h3>Data</h3><br />
Below are the results of the plate reader data for our control and experimental conditions described above. We find that there is no truncation of estrogen receptor that gives an estradiol-inducible system; however, we are optimistic about trying other truncations and ToxR fusions that might lead us to an estrogen biosensor.<br />
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<br><br> <center><img src="https://static.igem.org/mediawiki/2011/9/97/Estrogendata.png" width="800"></center><br />
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/cssTemplate:Team:NTNU-Trondheim/Press/css2013-09-30T21:18:13Z<p>Rahmilale: Created page with "<html> <style type="text/css"> /* grid layout with 4 columns */ div.layout-1000{ width: 1000px; margin: 10px auto;} div.row { height: 1%; } div.row-end { clear: both; font: 1px..."</p>
<hr />
<div><html><br />
<style type="text/css"><br />
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div.layout-1000{ width: 1000px; margin: 10px auto;}<br />
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</html></div>Rahmilalehttp://2013.igem.org/Template:Team:NTNU-Trondheim/Press/headerTemplate:Team:NTNU-Trondheim/Press/header2013-09-30T21:12:55Z<p>Rahmilale: Created page with "<html lang="en"> <style = "text/css" media="screen"> ul.topnav { list-style: none; padding: 0px 0px; margin: 0px 0px 0px -22px; width:1000px; float: left; } ul.topna..."</p>
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/*<br />
* jQuery Nivo Slider v2.6<br />
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effect: 'fade'<br />
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</html></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/Press/navigationTeam:NTNU-Trondheim/Press/navigation2013-09-30T21:08:13Z<p>Rahmilale: Created page with "<html> <script type="text/javascript"> /* * Slides, A Slideshow Plugin for jQuery * Intructions: http://slidesjs.com * By: Nathan Searles, http://nathansearles.com * Version: ..."</p>
<hr />
<div><html><br />
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<br />
/*<br />
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</html></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/Press/headerTeam:NTNU-Trondheim/Press/header2013-09-30T21:07:39Z<p>Rahmilale: Created page with "<html lang="en"> <head> <meta property="og:title" content="Berkeley iGEM 2013"/> <meta property="og:type" content="university"/> <meta property="og:description" con..."</p>
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<p>Biosensors have widespread applications ranging from diagnostics to environmental monitoring. Vibrio cholerae's ToxR system can be used as a component in biological sensing devices. ToxS causes ToxR homodimerization, activating transcription of the ctx promoter. By replacing the periplasmic domain of ToxR with existing or engineered ligand-dependent homodimers, we hope to link ToxR dimerization (and gene expression) to the presence of specific ligands. Initially, ToxR constructs proved toxic to E. coli. We built a stress-regulated transcription system that drives relatively high expression of toxic proteins. This allowed us to further engineer ToxR chimeras. We fused an estrogen-dependent dimer with ToxR hoping to create an estrogen biosensor. We observed a range of constitutive phenotypes and plan more experiments to engineer a dose-dependent transcriptional response to estrogen. By fusing existing or engineered ligand dependent homodimers to ToxR, this modular system can be used to build new biosensors. </p><br />
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<div class="col3-2"style="background-color:#101a4d;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxR"> <img src="https://static.igem.org/mediawiki/igem.org/1/10/Subtitlepic1.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> A protein with great potential as a general biosensor system.</p> </div><br />
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<div class="col3-2"style="background-color:#185f73;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Chimeric proteins that drive translation off of the Pctx promoter.</p></div><br />
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<p style="text-align:center; color:#CECECE;"> Our method for expressing interesting (but toxic) proteins.</p> </div><br />
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<p style="text-align:center; color:#CECECE;"> Bacteria engineered to detect the presence of estrogen. <br></p></div><br />
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<p>We are Team Berkeley, a cohesive unit of 7 undergraduates and 3 advisers. Earlier this year we planned a complex and risky project, given the short amount of time iGEM made available. We quickly learned each others strengths and weaknesses and developed standard systems of organizational management in order to synchronize our efforts for the many parallel tasks at hand. We created protocols, shared them with one another, and worked together on troubleshooting. Using google docs to keep up with the status of cloning projects, the results of assays, material logistics, or the final steps required to complete a project ensured that through the months of hard work, we fine-tuned our ability to work together. As a team, we have learned firsthand how the synthetic biology community relies on the goal-oriented cooperation of skilled individuals from very different backgrounds and with very different skill sets. Some of us have strong engineering backgrounds while others of us have strong biology backgrounds, but we all share a desire to build synthetic biological systems that solve human problems. We are proud of the project that we have created which we will present at the Jamboree together in October.</p><br />
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<p style="text-align:left; color:#CECECE; font-size:13px; font-weight:bold; padding:3px"> The UC Berkeley iGEM team would like to thank Autodesk and Agilent for their financial support and Synberc, for their administrative support. </p><br />
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{{Team:NTNU-Trondheim/Press/navigation}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/Press/cssTeam:NTNU-Trondheim/Press/css2013-09-30T21:02:10Z<p>Rahmilale: Created page with "<html> <style type="text/css"> /* grid layout with 4 columns */ div.layout-1000{ width: 1000px; margin: 10px auto;} div.row { height: 1%; } div.row-end { clear: both; font: 1px..."</p>
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</html></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/PressTeam:NTNU-Trondheim/Press2013-09-30T21:01:01Z<p>Rahmilale: </p>
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<p>Biosensors have widespread applications ranging from diagnostics to environmental monitoring. Vibrio cholerae's ToxR system can be used as a component in biological sensing devices. ToxS causes ToxR homodimerization, activating transcription of the ctx promoter. By replacing the periplasmic domain of ToxR with existing or engineered ligand-dependent homodimers, we hope to link ToxR dimerization (and gene expression) to the presence of specific ligands. Initially, ToxR constructs proved toxic to E. coli. We built a stress-regulated transcription system that drives relatively high expression of toxic proteins. This allowed us to further engineer ToxR chimeras. We fused an estrogen-dependent dimer with ToxR hoping to create an estrogen biosensor. We observed a range of constitutive phenotypes and plan more experiments to engineer a dose-dependent transcriptional response to estrogen. By fusing existing or engineered ligand dependent homodimers to ToxR, this modular system can be used to build new biosensors. </p><br />
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<div class="col3-2"style="background-color:#101a4d;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxR"> <img src="https://static.igem.org/mediawiki/igem.org/1/10/Subtitlepic1.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> A protein with great potential as a general biosensor system.</p> </div><br />
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<div class="col3-2"style="background-color:#185f73;"><a href="https://2011.igem.org/Team:Berkeley/Project#ToxRChimera"><img src="https://static.igem.org/mediawiki/2011/b/bb/Subtitlepic3header.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Chimeric proteins that drive translation off of the Pctx promoter.</p></div><br />
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<div class="col3-2" style="background-color:#303285"><a href="https://2011.igem.org/Team:Berkeley/Project#StressPromoters"><img src="https://static.igem.org/mediawiki/2011/2/2b/Subtitlepic2header.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Our method for expressing interesting (but toxic) proteins.</p> </div><br />
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<div class="col3-2"style="background-color:#499394"><a href="https://2011.igem.org/Team:Berkeley/Project#EstrogenBiosensor"><img src="https://static.igem.org/mediawiki/2011/c/c8/Subtitlepic4.jpg" <br />
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<p style="text-align:center; color:#CECECE;"> Bacteria engineered to detect the presence of estrogen. <br></p></div><br />
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<p>We are Team Berkeley, a cohesive unit of 7 undergraduates and 3 advisers. Earlier this year we planned a complex and risky project, given the short amount of time iGEM made available. We quickly learned each others strengths and weaknesses and developed standard systems of organizational management in order to synchronize our efforts for the many parallel tasks at hand. We created protocols, shared them with one another, and worked together on troubleshooting. Using google docs to keep up with the status of cloning projects, the results of assays, material logistics, or the final steps required to complete a project ensured that through the months of hard work, we fine-tuned our ability to work together. As a team, we have learned firsthand how the synthetic biology community relies on the goal-oriented cooperation of skilled individuals from very different backgrounds and with very different skill sets. Some of us have strong engineering backgrounds while others of us have strong biology backgrounds, but we all share a desire to build synthetic biological systems that solve human problems. We are proud of the project that we have created which we will present at the Jamboree together in October.</p><br />
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<p style="text-align:left; color:#CECECE; font-size:13px; font-weight:bold; padding:3px"> The UC Berkeley iGEM team would like to thank Autodesk and Agilent for their financial support and Synberc, for their administrative support. </p><br />
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{{Team:Berkeley/navigation}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/Templates/SponsorsTeam:NTNU-Trondheim/Templates/Sponsors2013-06-27T12:50:08Z<p>Rahmilale: </p>
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<p>Documentation of the testing of the components in our genetic circuit.</p><br />
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<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
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<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
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<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
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<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-27T12:49:02Z<p>Rahmilale: </p>
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<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>Documentation of the testing of the components in our genetic circuit.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8c/Ntnu_rhit_collaboration" alt=""><br />
</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We have collaborated with the Rose-Hulman Institute of Technology iGEM team.</p><br />
</div><br />
</div><br />
</div><br />
<a class="left carousel-control" href="#myCarousel" data-slide="prev">&lsaquo;</a><br />
<a class="right carousel-control" href="#myCarousel" data-slide="next">&rsaquo;</a><br />
</div><br />
<div class="row-fluid"><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><img alt="" src="https://static.igem.org/mediawiki/2012/b/b2/Project_illustration_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><img alt="" src="https://static.igem.org/mediawiki/2012/6/62/Achievements_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
</div><br />
</ul> <br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Experiments_results_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Collaboration_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach"><img alt="" src="https://static.igem.org/mediawiki/2012/7/70/Outreach_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Press"><img alt="" src="https://static.igem.org/mediawiki/2012/2/28/Press_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/ProtocolsTeam:NTNU-Trondheim/Protocols2013-06-20T12:50:01Z<p>Rahmilale: </p>
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== Protocols ==<br />
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== more protocols ==<br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/ProtocolsTeam:NTNU-Trondheim/Protocols2013-06-20T12:49:30Z<p>Rahmilale: </p>
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== Protocols ==<br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/ProtocolsTeam:NTNU-Trondheim/Protocols2013-06-20T12:49:19Z<p>Rahmilale: Created page with "{{:Team:NTNU-Trondheim/Templates/Header}} {{#calendar: year=2013 | title=Team:NTNU-Trondheim }} <html> </html> <div id="Default"> == Protocols == </div> {{:Team:NTNU..."</p>
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== Protocols ==<br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:58:40Z<p>Rahmilale: /* Notebook */</p>
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table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
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== Notebook ==<br />
{{:Team:NTNU-Trondheim/20_June_2013}}<br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/20_June_2013Team:NTNU-Trondheim/20 June 20132013-06-20T10:57:54Z<p>Rahmilale: Created page with "Today is June 20th"</p>
<hr />
<div>Today is June 20th</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:57:34Z<p>Rahmilale: </p>
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<div>{{:Team:NTNU-Trondheim/Templates/Header}}<br />
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table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
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== Notebook ==<br />
<br />
Here comes the notebook<br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:56:26Z<p>Rahmilale: </p>
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table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
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font-weight: bold;<br />
font-size: 8pt;<br />
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table.month a { background:none; padding:0 }<br />
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== Notebook ==<br />
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Here comes the notebook<br />
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</div><br />
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{{#calendar: year=2013 | title=Team:NTNU-Trondheim }}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:56:02Z<p>Rahmilale: </p>
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table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
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}<br />
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== Notebook ==<br />
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Here comes the notebook<br />
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</div></div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:55:44Z<p>Rahmilale: </p>
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table.calendar { margin: 100px; padding: 10px; }<br />
table.calendar td { margin: 100px; padding: 2px; vertical-align: top; }<br />
table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #eee;<br />
}<br />
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table.month a { background:none; padding:0 }<br />
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== Notebook ==<br />
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<br />
</div></div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-20T10:52:49Z<p>Rahmilale: </p>
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<br />
<!--<br />
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<div class="item active"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><br />
<img src="https://static.igem.org/mediawiki/2012/1/1d/Project_illustration_carousel_870x400_5.png" alt=""><br />
</a><br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Click to read more about our project, To be announced!</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><br />
<img src="https://static.igem.org/mediawiki/2012/a/ae/Achievements_870x400.png" alt=""><br />
</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/Experiments_results_870x400" alt=""><br />
</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>Documentation of the testing of the components in our genetic circuit.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8c/Ntnu_rhit_collaboration" alt=""><br />
</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We have collaborated with the Rose-Hulman Institute of Technology iGEM team.</p><br />
</div><br />
</div><br />
</div><br />
<a class="left carousel-control" href="#myCarousel" data-slide="prev">&lsaquo;</a><br />
<a class="right carousel-control" href="#myCarousel" data-slide="next">&rsaquo;</a><br />
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<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><img alt="" src="https://static.igem.org/mediawiki/2012/b/b2/Project_illustration_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><img alt="" src="https://static.igem.org/mediawiki/2012/6/62/Achievements_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
</div><br />
</ul> <br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Experiments_results_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Collaboration_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach"><img alt="" src="https://static.igem.org/mediawiki/2012/7/70/Outreach_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Press"><img alt="" src="https://static.igem.org/mediawiki/2012/2/28/Press_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-20T10:52:38Z<p>Rahmilale: </p>
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<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
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<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
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<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
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--/></div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-20T10:52:19Z<p>Rahmilale: </p>
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<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
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</li><br />
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</li><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
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<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-20T10:52:07Z<p>Rahmilale: </p>
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<p>Documentation of the testing of the components in our genetic circuit.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We have collaborated with the Rose-Hulman Institute of Technology iGEM team.</p><br />
</div><br />
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<a class="left carousel-control" href="#myCarousel" data-slide="prev">&lsaquo;</a><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
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<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Experiments_results_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
</div><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
</div><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Press"><img alt="" src="https://static.igem.org/mediawiki/2012/2/28/Press_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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--/></div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-20T10:51:40Z<p>Rahmilale: </p>
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<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><br />
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</a><br />
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<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>Documentation of the testing of the components in our genetic circuit.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
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<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We have collaborated with the Rose-Hulman Institute of Technology iGEM team.</p><br />
</div><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
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<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><img alt="" src="https://static.igem.org/mediawiki/2012/2/2d/Collaboration_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
</div><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach"><img alt="" src="https://static.igem.org/mediawiki/2012/7/70/Outreach_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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<p style="text-align: center;">Countdown to the <a href="https://2012.igem.org/Regions/Europe/Jamboree" target="_blank">European jamboree</a>:<br />
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{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-TrondheimTeam:NTNU-Trondheim2013-06-20T10:51:08Z<p>Rahmilale: </p>
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<div>{{:Team:NTNU-Trondheim/Templates/Header}}<br />
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</a><br />
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<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Click to read more about our project, To be announced!</p><br />
</div><br />
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<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements"><br />
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</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Project"><br />
<img src="https://static.igem.org/mediawiki/2012/d/d0/Experiments_results_870x400" alt=""><br />
</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>Documentation of the testing of the components in our genetic circuit.</p><br />
</div><br />
</div><br />
<div class="item"><br />
<a href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration"><br />
<img src="https://static.igem.org/mediawiki/2012/8/8c/Ntnu_rhit_collaboration" alt=""><br />
</a><br />
<div class="carousel-caption"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We have collaborated with the Rose-Hulman Institute of Technology iGEM team.</p><br />
</div><br />
</div><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-info-sign"></i> Our project</h4><br />
<p>Read the description of our project, Bacterial Anti-Cancer Kamikaze.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Project">Read more &raquo;</a></p><br />
</div><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-trophy"></i> Achievements</h4><br />
<p>See how we have fulfilled the judging criteria.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Achievements">Read more &raquo;</a></p><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-beaker"></i> Experiments and results</h4><br />
<p>See how we tested our components and what results we got.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Experiments_and_Results">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-globe"></i> Collaboration</h4><br />
<p>We collaborated with the RHIT iGEM team. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Collaboration">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
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<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Model"><img alt="" src="https://static.igem.org/mediawiki/2012/d/d0/Model_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-bar-chart"></i> Modelling</h4><br />
<p>We created a stochastic model of our genetic circuit.</p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Model">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker"><img alt="" src="https://static.igem.org/mediawiki/2012/c/c7/Matchmaker_thumb.png"></a><br />
</li><br />
<div class="span3"><br />
<h4><i class="icon-heart"></i> Matchmaker</h4><br />
<p>We created a tool to make it easy for all iGEM teams to find other teams to collaborate with. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Matchmaker">Read more &raquo;</a></p><br />
</div><br />
</ul><br />
<ul class="thumbnails"><br />
<li class="span3"><br />
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</li><br />
<div class="span3"><br />
<h4><i class="icon-eye-open"></i> Human practices</h4><br />
<p>We brought synthetic biology to high schools students at NTNU's Researchers' Night. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Outreach">Read more &raquo;</a></p><br />
</div><br />
<li class="span3"><br />
<a class="thumbnail" href="https://2012.igem.org/Team:NTNU_Trondheim/Press"><img alt="" src="https://static.igem.org/mediawiki/2012/2/28/Press_thumb.png"></a><br />
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<h4><i class="icon-camera"></i> Press coverage</h4><br />
<p>We have received attention in the media. </p><br />
<p><a class="btn" href="https://2012.igem.org/Team:NTNU_Trondheim/Press">Read more &raquo;</a></p><br />
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<p style="text-align: center;">Countdown to the <a href="https://2012.igem.org/Regions/Europe/Jamboree" target="_blank">European jamboree</a>:<br />
<div id="jamboreeCountdown"></div><br /><br /><br/><br />
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<a class="twitter-timeline" href="https://twitter.com/iGEM_NTNU" data-widget-id="346356078235246592">Tweets fra @iGEM_NTNU</a><br />
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<div class="fb-like-box" data-href="https://www.facebook.com/ntnuigem" data-width="292" data-show-faces="false" data-stream="false" data-show-border="false" data-header="false"></div><br />
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{{:Team:NTNU-Trondheim/Templates/Sponsors}}<html></div></div></html><br />
{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:50:16Z<p>Rahmilale: </p>
<hr />
<div>{{:Team:NTNU-Trondheim/Templates/Header}}<br />
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<html><br />
<br />
<style media="screen" type="text/css"><br />
<br />
table.calendar { margin: 100px; padding: 10px; }<br />
table.calendar td { margin: 100px; padding: 2px; vertical-align: top; }<br />
table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #eee;<br />
}<br />
<br />
table.month a { background:none; padding:0 }<br />
.day-active { color:#0000cc }<br />
.day-empty { color:#cc0000 }<br />
<br />
</style><br />
</html><br />
{{#calendar: year=2013 | title=Team:NTNU-Trondheim }}<br />
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{{:Team:NTNU-Trondheim/Templates/Sponsors}}<br />
{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:49:48Z<p>Rahmilale: </p>
<hr />
<div>{{:Team:NTNU-Trondheim/Templates/Header}}<br />
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<html><br />
<br />
<style media="screen" type="text/css"><br />
<br />
table.calendar { margin: 100; padding: 10px; }<br />
table.calendar td { margin: 100; padding: 2px; vertical-align: top; }<br />
table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #eee;<br />
}<br />
<br />
table.month a { background:none; padding:0 }<br />
.day-active { color:#0000cc }<br />
.day-empty { color:#cc0000 }<br />
<br />
</style><br />
</html><br />
{{#calendar: year=2013 | title=Team:NTNU-Trondheim }}<br />
<br />
<br />
{{:Team:NTNU-Trondheim/Templates/Sponsors}}<br />
{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:49:28Z<p>Rahmilale: </p>
<hr />
<div>{{:Team:NTNU-Trondheim/Templates/Header}}<br />
<br />
<html><br />
<br />
<style media="screen" type="text/css"><br />
<br />
table.calendar { margin: 0; padding: 10px; }<br />
table.calendar td { margin: 0; padding: 2px; vertical-align: top; }<br />
table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #eee;<br />
}<br />
<br />
table.month a { background:none; padding:0 }<br />
.day-active { color:#0000cc }<br />
.day-empty { color:#cc0000 }<br />
<br />
</style><br />
</html><br />
{{#calendar: year=2013 | title=Team:NTNU-Trondheim }}<br />
<br />
<br />
{{:Team:NTNU-Trondheim/Templates/Sponsors}}<br />
{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilalehttp://2013.igem.org/Team:NTNU-Trondheim/NotebookTeam:NTNU-Trondheim/Notebook2013-06-20T10:49:16Z<p>Rahmilale: </p>
<hr />
<div>{{:Team:NTNU-Trondheim/Templates/Header}}<br />
<br />
<html><br />
<br />
<style media="screen" type="text/css"><br />
<br />
table.calendar { margin: 0; padding: 10px; }<br />
table.calendar td { margin: 0; padding: 2px; vertical-align: top; }<br />
table.month .heading td { padding:2px; background-color:#d4d4d4; color:#aaa; text-align:center; font-size:120%; font-weight:bold; }<br />
table.month .dow td { color:#aaa; text-align:center; font-size:110%; }<br />
table.month td.today { background-color:#ddd; }<br />
table.month td {<br />
border: none;<br />
margin: 0;<br />
padding: 1pt 1.5pt;<br />
font-weight: bold;<br />
font-size: 8pt;<br />
text-align: right;<br />
background-color: #eee;<br />
}<br />
<br />
table.month a { background:none; padding:0 }<br />
.day-active { color:#0000cc }<br />
.day-empty { color:#cc0000 }<br />
<br />
</style><br />
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{{#calendar: year=2013 | title=Team:NTNU-Trondheim }}<br />
<br />
</html><br />
{{:Team:NTNU-Trondheim/Templates/Sponsors}}<br />
{{:Team:NTNU-Trondheim/Templates/Footer}}</div>Rahmilale