http://2013.igem.org/wiki/index.php?title=Special:Contributions/Arneckelmann&feed=atom&limit=50&target=Arneckelmann&year=&month=2013.igem.org - User contributions [en]2024-03-28T22:07:55ZFrom 2013.igem.orgMediaWiki 1.16.5http://2013.igem.org/Team:UC_DavisTeam:UC Davis2013-12-05T22:43:08Z<p>Arneckelmann: </p>
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<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br /><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><img src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png" class="checkmark"></a><p>We also made the Anderson promoter family controllable through induction with RiboTALs.</p><br />
</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
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<!--<img src="https://static.igem.org/mediawiki/2013/f/fd/UCDavisMeettheteam.gif" width="200" height="300" align="left" class="genpic">--><br />
Welcome to the 2013 UC Davis iGEM Wiki!<br></br>We have created a novel class of transcription factors known as RiboTALs. We sought to address the constraints placed on circuit design by the limited number of well characterized promoters at our disposal, and their respective transcription factors. Our device is a hybrid system composed of two parts: Transcriptional Activator-Like (TAL) effectors which can be engineered to bind to and repress any sequence of interest and riboswitches that can respond to any inducer molecule due to their engineerable and modular aptamer binding domains. <br></br> We also designed and implemented a Biobrick characterization database called The Depot to promote sharing and openness in iGEM.</p><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_DavisTeam:UC Davis2013-10-29T03:55:45Z<p>Arneckelmann: </p>
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<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br /><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><img src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png" class="checkmark"></a><p>We also made the Anderson promoter family controllable through induction with RiboTALs.</p><br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
<br />
<br />
</tr><br />
</table><br />
<br />
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<!--<img src="https://static.igem.org/mediawiki/2013/f/fd/UCDavisMeettheteam.gif" width="200" height="300" align="left" class="genpic">--><br />
Welcome to the 2013 UC Davis iGEM Wiki!<br></br>We have a created a novel class of transcription factors known as RiboTALs. We sought to address the constraints placed on circuit design by the limited number of well characterized promoters at our disposal, and their respective transcription factors. Our device is a hybrid system composed of two parts: Transcriptional Activator-Like (TAL) effectors which can be engineered to bind to and repress any sequence of interest and riboswitches that can respond to any inducer molecule due to their engineerable and modular aptamer binding domains. <br></br> We also designed and implemented a Biobrick characterization database called The Depot to promote sharing and openness in iGEM.</p><br />
</div><br />
<br />
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</html></div>Arneckelmannhttp://2013.igem.org/File:UCD_iGEM_equation_pic_2013.pngFile:UCD iGEM equation pic 2013.png2013-10-29T03:54:05Z<p>Arneckelmann: </p>
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<div></div>Arneckelmannhttp://2013.igem.org/Team:UC_DavisTeam:UC Davis2013-10-29T03:47:07Z<p>Arneckelmann: </p>
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<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br /><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoter"><img src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png" class="checkmark"></a><p>We also made the Anderson promoter family controllable through induction with RiboTALs.</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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<h1>Welcome</h1><br />
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<img src="https://static.igem.org/mediawiki/2013/2/2a/IGEM_2013_Group_Photo.png" width="200" height="300" align="left" class="genpic"><br />
<!--<img src="https://static.igem.org/mediawiki/2013/f/fd/UCDavisMeettheteam.gif" width="200" height="300" align="left" class="genpic">--><br />
Welcome to the 2013 UC Davis iGEM Wiki!<br></br>We have a created a novel class of transcription factors known as RiboTALs. We sought to address the constraints placed on circuit design by the limited number of well characterized promoters at our disposal, and their respective transcription factors. Our device is a hybrid system composed of two parts: Transcriptional Activator-Like (TAL) effectors which can be engineered to bind to and repress any sequence of interest and riboswitches that can respond to any inducer molecule due to their engineerable and modular aptamer binding domains. <br></br> We also designed and implemented a Biobrick characterization database called The Depot to promote sharing and openness in iGEM.</p><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T03:46:19Z<p>Arneckelmann: </p>
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>See how we engineered the constitutive Anderson promoters into a family of inducible promoters with RiboTALs. <br /><br />
Also, see the secondary data page, <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">here</a>.<br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#graph2">Promoter Strength</a></li><br />
<li><a href="#graph3">RiboTAL Control</a></li><br />
<li><a href="#widget">KO3D</a></li><br />
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<h1 id="section1">Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
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<img src="https://static.igem.org/mediawiki/2013/f/fb/UCDavis_And_modelscheme.png" width= 620 height=195 class="centerimg" /><br />
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Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
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<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br><p><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
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<h1 id="graph2">Repression is modulated by theophylline and promoter strength<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br></br><p><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
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<h1 id="graph3">Control of RiboTALe transcript levels reliably alters system response<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. </p><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br></br><p><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
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<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
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<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
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<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
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<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
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<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
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<a id="ref"></a><h3>References</h3><br />
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<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
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</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/ModelingTeam:UC Davis/Modeling2013-10-29T03:28:08Z<p>Arneckelmann: </p>
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<h1>Equations</h1><br />
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The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
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<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#equations">Equations</a></li><br />
<li><a href="#parameters">Parameters</a></li><br />
<li><a href="#MATLABsim">MATLAB Simulation</a></li><br />
<li><a href="#anderson">Anderson Promoters</a></li><br />
</ul><br />
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<a id="parameters"></a><br />
<h1>Parameters <a href="#top" class="to_top">Return to Top</a></h1><br />
Included here are the parameters used in this model. Please refer to the <a href="#References">References</a> section of this page for the source of each parameter value. <br />
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<center><img src="https://static.igem.org/mediawiki/2013/5/5a/Ucdavivs_Parameter1.jpg" class="genpic"></center><br />
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<h1>MATLAB Simulation-- <i>See the Code:</i><a href="https://static.igem.org/mediawiki/2013/e/e7/Model2alt10_17.m">(1)</a> <i>and</i> <a href="https://static.igem.org/mediawiki/2013/a/a9/UCDavis_Callode.m">(2)</a><i>!</i><a href="#top" class="to_top">Return to Top</a></h1><br />
<br />
<h3>TALe Binding Site K<sub>D</sub> As a Source of Tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/0/08/UCDavis_Kdcomp.png" class="genpic" height=312 width=864></center><br />
<br>It should be noted that "less effective" does not mean that RiboTALe 1 is an inferior part, merely that it generates a distinct system response and displays kinematic behavior that may be specifically needed in a future circuit design. We have demonstrated that through their engineerable tunability RiboTALes are capable of achieving a broad range of system responses, a conclusion that is supported by this model. </br><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 8 with a K<sub>D</sub> of 1.3 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is, in fact, responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/e/ed/UCDavis_Theocomparison.png"class="genpic" height=312 width=864></center><br />
<br><br />
The expected effect of theophylline induction levels on the expression of the gene of interest can be calculated and plotted. The expression levels have been normalized to the expected expression of GFP under conditions of 1% arabinose and 10 mM theophylline for RiboTALe 8.</br><br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/63/UCDavis_Theoeffect.png" class="genpic" height=312 width=864></center> <br />
<br>Future work with this model may involve the simulation of RiboTALe activity under different non-theophylline riboswitches and an investigation of the orthogonality achievable when using multiple riboswitches in a system.</br><br />
<br></br><br />
<h3>Amplifying System Response Through Transcript Induction</h3> <br />
<br>To investigate the effects of increasing GFP transcript while maintaining constant levels of arabinose and theophylline, the dynamic response of the system under RiboTALe 8 repression was simulated for aTc levels of 0, 25 ng/mL, and 100 ng/mL, where aTC is the inducer of the GFP transcript. The model results show the expected behavior: at higher concentrations of aTc GFP reaches greater peak concentration before repression by the RiboTALe becomes evident. Moreover, this event occurs later in the simulation under conditions of 100 ng/mL of aTc than it does for the other two simulated responses.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/c/c5/UCDavis_ATccomparison.png" height=312 width=864></center><br />
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This model can be further developed to take into account riboswitch leakiness and system stochasticity, and the parameters fine-tuned. It is, however, a useful model in that it provides a mathematical basis that supports the functionality of our RiboTALe devices and shows the wide variety of system responses achievable through the modulation of the engineerable and tunable elements of our construct. We tested combinations of two TAL repressors and two theophylline riboswitches. With this model we will be able to predict the response of a library of RiboTALes, composed a much greater variety of riboswitches and TAL repressors, and perhaps identify with which combination and under what induction conditions a desired system response may be achieved. <br />
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<h1>Anderson Promoter Model <a href="#top" class="to_top">Return to Top</a></h1><br />
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<center><img src="https://static.igem.org/mediawiki/2013/f/fb/UCDavis_And_modelscheme.png" width= 620 height=195></center><br />
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To determine whether our synthetic transcription factors would effectively repress the constitutive family of Anderson promoters, we placed the a TALe target sequence downstream of a number of Anderson promoters. The model presented above for the GFP testing construct holds for the repressible Anderson promoter constructs. However, instead of the pTET term we now deal with simply 'P', the relative promoter strength of the promoter in question. Solving the model at steady state, the following expression for GFP concentration is derived. </br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/7/7a/Andersonmod.png"></center><br />
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Given arabinose concentration data, theophylline concentration data, and corresponding GFP fluorescence levels, a nonlinear regression may be performed in order to estimate the parameters of the model. This model is intended to estimate relative levels of GFP fluorescence given the relative promoter strength and the induction levels of arabinose and theophylline. To this end, the derived expression may be simplified as follows.<br />
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<center><img src="https://static.igem.org/mediawiki/2013/8/8d/Andersonsimpl.png"></center><br />
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For simplicity, the activity of the pBAD promoter has been modeled as an activator-controlled promoter in the expression above. After performing a nonlinear regression using relative GFP values (fluorescence divided by maximum fluorescence under the case of no repression), the following parameter estimates for A, B, C, D, and E were generated.<br />
<center><table class="black"><br />
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<th>Parameter</th><br />
<th>Value</th><br />
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<td>A</td><br />
<td>1.0338</td><br />
</tr><br />
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<td>B</td><br />
<td>0.9669</td><br />
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<tr><th></th><br />
<td>C</td><br />
<td>0.0129</td><br />
</tr><br />
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<td>D</td><br />
<td>3.273*10<sup>3</sup></td><br />
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<td>E</td><br />
<td>5.8811</td><br />
</tr><br />
</table> </center><br />
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These parameter estimates were generated from data for the RiboTALe under control of Riboswitch 2 and expressing TALe 8. Plugging these parameter estimates back into the model, the following expected relative GFP fluorescence levels were calculated for five different relative promoter strengths across ranges of both theophylline and arabinose induction levels. <br />
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<center><img src ="https://static.igem.org/mediawiki/2013/2/25/UCDavis_Pamfit_theo.png" height = 312 width=864></center><br />
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<center><img src ="https://static.igem.org/mediawiki/2013/3/39/UCDavis_Pamfit_arab.png" height = 312 width=864></center><br />
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With the parameter estimates generated from a nonlinear regression, the model displays a decrease in GFP fluorescence as induction levels of arabinose and theophylline are increased. Furthermore, the effect of the relative promoter strength is accurately reflected in the graphs generated by the model. A small relative promoter strength results in a a lower baseline fluorescence under conditions of no repression, and lower fluorescence levels across all ranges of arabinose and theophylline. This model may be used to approximate the behavior of systems under control of Anderson promoters that have been engineered to be repressible by our synthetic transcription factors. <br />
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<div class="floatboxwide" id="References"><br />
<h1>References<a href="#top" class="to_top">Return to Top</a></h1><br />
<br>[1] <a href="http://bionumbers.hms.harvard.edu/KeyNumbers.aspx?redirect=false">"Key Numbers for Cell Biologists." Bionumbers: The Database of Useful Biological Numbers</a></h1></br><br />
<br>[2] <a href="http://openwetware.org/images/5/5b/NORgate%2BChemWires_SuppInfo_nature09565-s1.pdf">Tamsir et al. 'Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’: Supplementary Information'. Nature 469, 212–215 (13 January 2011)</a></hi></br><br />
<br>[3] <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a></br><br />
<br>[4] <a href="http://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1330.full">Wachsmuth M., Findeiss S., Weissheimer N., Stadler P., Morl M., "De novo design of a synthetic riboswitch that regulates transcription termination," Nucleic Acids Res, 2012</a></hi> </br><br />
<br>[5] <a href="http://parts.igem.org/Part:BBa_K750000">"Part:BBa_K750000" The Registry of Standard Biological Parts </a></hi></br> <br />
<br>[6] <a href="http://www.ncbi.nlm.nih.gov/pubmed/8707053">Cormack et al. 'FACS-optimized mutants of the green fluorescent protein (GFP).' Gene. 1996;173(1 Spec No):33-8.</a></hi></br><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/OutreachTeam:UC Davis/Outreach2013-10-29T03:22:45Z<p>Arneckelmann: </p>
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<h1>NorCal iGEM Meetup</h1><br />
<p>Over the summer, UC Davis organized an iGEM meetup with UCSF, UC Berkeley, and the Stanford-Brown teams. We started off the day giving a brief talk on each of our projects. After each talk, we had a chance to ask more questions about their project and offer possible constructive criticism. From this, we all received a different perspective on some of the tough problems we were trying to solve with our projects. After the meetup, we all had lunch and got to talk even more about synthetic biology among other things. To change things up, we also went bowling.</p><br />
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<h1>Other Efforts in Human Outreach</h1><br />
<ul><li>Provided DNA extractions for E.coli expressing RFP for the biotechnology class at Davis Senior High School.</li><br />
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<li>Worked with classmate Nicholas Armstrong to create a Synthetic Biology Club at UC Davis<br />
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<li>Grew to over 20 members after the first meeting.</li><br />
<li>We promoted the club on campus during the Activities Fair.</li><br />
<li>We began writing a lab proposal and a Club Finance Counsel grant for the club.</li><br />
<li>Team members Alex Neckelmann and Aura Ferreiro took the roles of Activities Chair and Secretary, respectively.</li><br />
<li>Check out the club's Facebook page for more information by searching "Synthetic Biology Club at UC Davis" or clicking <a href="https://www.facebook.com/groups/739965502695620/">here</a>.</li><br />
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</head></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T02:40:41Z<p>Arneckelmann: </p>
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
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<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br /><br />
Also, see the secondary data page, <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">here</a>.<br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#graph2">Promoter Strength</a></li><br />
<li><a href="#graph3">RiboTAL Control</a></li><br />
<li><a href="#widget">KO3D</a></li><br />
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<h1 id="section1">Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
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<div class="floatboxwide"><br />
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br><p><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
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<h1 id="graph2">Repression is modulated by theophylline and promoter strength<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br></br><p><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
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<h1 id="graph3">Control of RiboTALe transcript levels reliably alters system response<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. </p><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br></br><p><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
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<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
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<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
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<div class="playme"><br />
<p id="play">Play With Me</p><br />
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<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
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<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T02:37:48Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
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<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br /><br />
Also, see the secondary data page, <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">here</a>.<br />
</p><br />
</td> <br />
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</table><br />
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<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#graph2">Promoter Strength</a></li><br />
<li><a href="#graph3">RiboTAL Control</a></li><br />
<li><a href="#widget">KO3D</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1 id="section1">Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
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</center><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br><p><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
</p><br />
</div><br />
<div class="floatboxwide"><br />
<h1 id="graph2">Repression is modulated by theophylline and promoter strength<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br><p><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
</p><br />
</div><br />
<div class="floatboxwide"><br />
<h1 id="graph3">Control of RiboTALe transcript levels reliably alters system response<a href="#top" class="to_top">Return to Top</a></h1><br />
<br><p>The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. </p><br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br><p><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">3</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
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<div class="playme"><br />
<p id="play">Play With Me</p><br />
</div><br />
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<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
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<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T02:21:43Z<p>Arneckelmann: </p>
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<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
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<div class="floatbox"><br />
<table class = "showbox"><br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br /><br />
Also, see the secondary data page, <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">here</a>.<br />
</p><br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#graph2">Promoter Strength</a></li><br />
<li><a href="#graph3">RiboTAL Control</a></li><br />
<li><a href="#widget">KO3D</a></li><br />
</ul><br />
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<div class="floatbox"><br />
<h1 id="section1">Targeting the Anderson Promoters</h1><br><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
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<div class="floatboxwide"><br />
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br><p><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
</p><br />
</div><br />
<div class="floatboxwide"><br />
<h1 id="graph2">Repression is modulated by theophylline and promoter strength<a href="#top" class="to_top">Return to Top</a></h1><br><br />
<p>After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br><p><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
</p><br />
</div><br />
<div class="floatboxwide"><br />
<h1 id="graph3">Control of RiboTALe transcript levels reliably alters system response<a href="#top" class="to_top">Return to Top</a></h1><br />
<br><p>The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. </p><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br><p><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
</p><br />
</div><br />
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<br />
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<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">3</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
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</div><br />
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<div class="playme"><br />
<p id="play">Play With Me</p><br />
</div><br />
</div><br />
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<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
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</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T01:31:35Z<p>Arneckelmann: </p>
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<div>{{Team:UC_Davis/Head}}<br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
</p><br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#mutantwidget">KO3D</a></li><br />
</ul><br />
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<h1 id="section1">Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
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<div class="floatboxwide"><br />
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br></br><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
<br></br><br />
<h1>Repression is modulated by theophylline</h1><br />
After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. <br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br></br><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
<h1>Control of RiboTALe transcript levels reliably alters system response</h1><br />
The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. <br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br></br><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
<br />
<h1 id="graph1">TAL Binding Site alters Anderson Promoter Activity<a href="#top" class="to_top">Return to Top</a></h1><br />
<center><br />
<table class="showbox1"><br />
<tr><br />
<td> Promoter </td><br />
<td> Relative Strength </td><br />
<td> Fluorescence/OD </td><br />
<td> Expected Fluorescence/OD </td><br />
<td><br />
</tr><br />
<tr><br />
<td> J23100 </td><br />
<td> 1</td><br />
<td> 76048.39 </td><br />
<td> 76048.39</td><br />
</tr><br />
<tr><br />
<td> J23101 </td><br />
<td> 0.7 </td><br />
<td>65469.43</td><br />
<td>53233.88</td><br />
</tr><br />
<tr> <br />
<td>J23105 </td><br />
<td> 0.24</td><br />
<td>8812.306</td><br />
<td>18251.61</td><br />
</tr><br />
<tr><br />
<td>J23106</td><br />
<td>0.47 </td><br />
<td>1341.74 </td><br />
<td>35742.74</td><br />
</tr><br />
<tr><br />
<td>J23109</td><br />
<td>0.04 </td><br />
<td>925.0912</td><br />
<td>3041.936</td><br />
</tr><br />
<br />
</table><br />
<br />
<p>With consideration of data in the Registry, it appears that the TAL Binding Site has some sort of effect on the relative strengths of these promoters. Supposedly, the </p><br />
</div><br />
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<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
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<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
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/* Hiding default wiki displays: Credits to TU-Delft and Groningen for the code! */<br />
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.blur:hover<br />
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<br />
<br />
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<br />
/*End Blur CSS */<br />
<br />
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}<br />
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{ border-radius:4px;<br />
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margin-bottom:10px;<br />
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}<br />
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{ border-radius:4px;<br />
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float:right;<br />
margin-bottom:10px;<br />
}<br />
.clear {clear:both;}<br />
<br />
<br />
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.partstable<br />
{width:700px;<br />
}<br />
<br />
/*end parts table css*/<br />
<br />
/*back to top links*/<br />
a.to_top {float:right;<br />
font-size:12pt;}<br />
<br />
<br />
</style><br />
<br />
<br />
<br />
<br />
</head><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T00:41:15Z<p>Arneckelmann: </p>
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<table class = "showbox"><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#mutantwidget">KO3D</a></li><br />
</ul><br />
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<h1 id="section1">Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
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Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
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<div class="floatboxwide"><br />
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br></br><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
<br></br><br />
<h1>Repression is modulated by theophylline</h1><br />
After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. <br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br></br><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
<h1>Control of RiboTALe transcript levels reliably alters system response</h1><br />
The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. <br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br></br><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
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<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
</p><br />
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<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
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<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
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</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-29T00:37:17Z<p>Arneckelmann: </p>
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<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#section1">Anderson Promoters</a></li><br />
<li><a href="#graph1">Inducible Repression</a></li><br />
<li><a href="#mutantwidget">KO3D</a></li><br />
</ul><br />
</div><br />
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<div class="floatbox"><br />
<h1 id="section1">Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
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<div class="floatboxwide"><br />
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">^back to top</a></h1><br />
<p>We subjected our constructs to a no induction condition with no arabinose or theophylline added, which would result in maximal GFP expression for each promoter. We also subjected our constructs to the induction condition of 1% arabinose and 10mM Theophylline, which would result in maximal production RiboTALe transcript and TAL repressor proteins. Thus, difference in fluorescence between no induction and induction conditions would be due to the RiboTALe repression activity. We measured the fluorescence of our constructs in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the Protocols page for details on our culture preparation and Tecan testing parameters. In the graphs shown here, error bars indicate the range over which the average fluorescence values were calculated.<br />
</p><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center><br />
<br></br><br />
The data show clear repression of the reporter under conditions of maximal induction of the RiboTALe transcription factor, indicating that gene expression is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are now repressor-controlled by the RiboTALe device. The data also show that the promoters maintain, qualitatively, their relative strengths. The value listed under each promoter is it's relative promoter strength as listed in the Registry of Standard Biological Parts. The fold-decrease in fluorescence achieved by maximum induction of the RiboTALe was 56.8, 21.7, 3.99, and 1.38 for J23100, J23101, J23105, and J23109, respectively. <br />
<br></br><br />
<h1>Repression is modulated by theophylline</h1><br />
After ascertaining that the new promoter constructs were in fact repressible with addition of the RiboTALe inducers, the sensitivity to different levels of theophylline induction was investigated for each promoter construct. The promoters were subjected to theophylline induction levels ranging from 0 mM to 10 mM. Below are the results of these experiments, measured at the RiboTALe transcript induction level of 0.1% arabinose. <br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center><br />
<br></br><br />
These data show greater repression at greater levels of theophylline induction, for all promoter constructs. The data indicate that the repression activity of the RiboTALe device is sensitive to the level of theophylline added. A RiboTALe transcription factor may be used to reliably modulate the expression of genes of interest by means of the riboswitch ligand. <br />
<h1>Control of RiboTALe transcript levels reliably alters system response</h1><br />
The sensitivity of the system to levels of RiboTALe transcript was investigated by subjecting the testing constructs to arabinose induction levels ranging from 0.01% to 0.50%, where arabinose is the inducer for the RiboTALe transcript. The constructs were also subjected to the previous range of theophylline induction levels to ascertain continued repression activity. The image below presents the results of this experiment for the J23101 promoter construct. <br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/20/UCDavis_andArabdep_n.png" height = 525 width=800></center><br />
<br></br><br />
The data show a faster system response and greater repression at larger arabinose levels. Furthermore, theophylline sensitivity is maintained across different levels of RiboTALe transcript. The amount of RiboTALe transcript available is a controllable element of the repression system that affects gene expression in a consistent and predictable manner. The relative promoter strengths of the original constitutive promoter continues to be apparent, though each of the promoters has been shown to be repressor-controlled by the RiboTALe transcription factors. <br />
</div><br />
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<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view. The maximum Y value of the 2D plot can also be changed with the buttons in the upper left corner of the 2D plot.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
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<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
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<div class="playme"><br />
<p id="play">Play With Me</p><br />
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<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
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<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/ModelingTeam:UC Davis/Modeling2013-10-29T00:35:29Z<p>Arneckelmann: </p>
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<div>{{Team:UC_Davis/Head}}<br />
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<a id="equations"> </a><br />
<h1>Equations</h1><br />
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<center><img src="https://static.igem.org/mediawiki/2013/3/35/UCDavis_Testing_modelscheme_n.png" width= 620 height=195></center><br />
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The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
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<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
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<img src="https://static.igem.org/mediawiki/2013/4/42/UCDavis_Probofactivecomplex.png" width=1019 height=330><br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#equations">Equations</a></li><br />
<li><a href="#parameters">Parameters</a></li><br />
<li><a href="#MATLABsim">MATLAB Simulation</a></li><br />
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<a id="parameters"></a><br />
<h1>Parameters <a href="#top" class="to_top">^back to top</a></h1><br />
Included here are the parameters used in this model. Please refer to the <a href="#References">References</a> section of this page for the source of each parameter value. <br />
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<center><img src="https://static.igem.org/mediawiki/2013/4/4e/Ucdavisparameters1.jpg" class="genpic"></center><br />
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<a id="MATLABsim"></a><br />
<h1>MATLAB Simulation<a href="https://static.igem.org/mediawiki/2013/8/8b/UCD2013RiboTALe.m"><i>See the Code!</i></a><a href="#top" class="to_top">^back to top</a></h1><br />
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<h3>TALe Binding Site K<sub>D</sub> As a Source of Tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/0/0a/Ucdavismodel2.png" class="genpic"></center><br />
<br>It should be noted that "less effective" does not mean that RiboTALe 1 is an inferior part, merely that it generates a distinct system response and displays kinematic behavior that may be specifically needed in a future circuit design. We have demonstrated that through their engineerable tunability RiboTALes are capable of achieving a broad range of system responses, a conclusion that is supported by this model. </br><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 1 with a K<sub>D</sub> of 240 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is, in fact, responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/2/2c/Ucdavismodel3.png"class="genpic"></center><br />
<br>Future work with this model may involve the simulation of RiboTALe activity under different non-theophylline riboswitches and an investigation of the orthogonality achievable when using multiple riboswitches in a system.</br><br />
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<h3>Amplifying System Response Through Transcript Induction</h3> <br />
<br>To investigate the effects of increasing GFP transcript while maintaining constant levels of arabinose and theophylline, the dynamic response of the system under RiboTALe 1 repression was simulated for aTc levels of 0, 25 ng/mL, and 100 ng/mL, where aTC is the inducer of the GFP transcript. The model results show the expected behavior: at higher concentrations of aTc GFP reaches greater peak concentration before repression by the RiboTALe becomes evident. Moreover, this event occurs later in the simulation under conditions of 100 ng/mL of aTc than it does for the other two simulated responses.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/c/c9/Ucdavismodel4.png"></center><br />
<br></br><br />
This model can be further developed to take into account riboswitch leakiness and system stochasticity, and the parameters fine-tuned. It is, however, a useful model in that it provides a mathematical basis that supports the functionality of our RiboTALe devices and shows the wide variety of system responses achievable through the modulation of the engineerable and tunable elements of our construct. We tested combinations of two TAL repressors and two theophylline riboswitches. With this model we will be able to predict the response of a library of RiboTALes, composed a much greater variety of riboswitches and TAL repressors, and perhaps identify with which combination and under what induction conditions a desired system response may be achieved. <br />
</div><br />
<br />
<br />
<div class="floatboxwide" id="References"><br />
<h1>References<a href="#top" class="to_top">^back to top</a></h1><br />
<br>[1] <a href="http://bionumbers.hms.harvard.edu/KeyNumbers.aspx?redirect=false">"Key Numbers for Cell Biologists." Bionumbers: The Database of Useful Biological Numbers</a></h1></br><br />
<br>[2] <a href="http://openwetware.org/images/5/5b/NORgate%2BChemWires_SuppInfo_nature09565-s1.pdf">Tamsir et al. 'Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’: Supplementary Information'. Nature 469, 212–215 (13 January 2011)</a></hi></br><br />
<br>[3] <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a></br><br />
<br>[4] Adjusted from <a href="http://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1330.full">literature</a></hi> to fit our data.</br><br />
<br>[5] <a href="http://parts.igem.org/Part:BBa_K750000">"Part:BBa_K750000" The Registry of Standard Biological Parts </a></hi></br> <br />
<br>[6] <a href="http://www.ncbi.nlm.nih.gov/pubmed/8707053">Cormack et al. 'FACS-optimized mutants of the green fluorescent protein (GFP).' Gene. 1996;173(1 Spec No):33-8.</a></hi></br><br />
</div> <br />
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</html></div>Arneckelmannhttp://2013.igem.org/Template:Team:UC_Davis/KO3DRPUTemplate:Team:UC Davis/KO3DRPU2013-10-28T23:50:10Z<p>Arneckelmann: Created page with "<html> <script src="http://code.jquery.com/jquery.min.js"></script> <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.10.2/jquery.min.js"></script> <!--script src='http:..."</p>
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// RGB <-> HSL conversions based on equations from wikipedia.<br />
// If they're wrong, don't blame me!<br />
function RGB2HSL(r,g,b)<br />
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var h = Math.atan2(beta, alpha);<br />
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function HSL2RGB(h,s,l)<br />
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var C = (1-Math.abs(2*l-1))*s;<br />
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var X = C*(1 - Math.abs(Hprime%2-1));<br />
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var r = X, g = C, b = 0;<br />
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var r = 0, g = X, b = C;<br />
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var r = X, g = 0, b = C;<br />
} else if(h < 6) {<br />
var r = C, g = 0, b = X;<br />
}<br />
m = l - C/2;<br />
r = r + m;<br />
g = g + m;<br />
b = b + m;<br />
return {r:r, g:g, b:b}<br />
}<br />
<br />
//Bilinear interpolation<br />
// y1-*z0 - *z1<br />
// fr1<br />
// +x,y<br />
// fr2<br />
// y2-*z2 - *z3<br />
// | |<br />
// x1 x2<br />
function interp2(x1, y1, x2, y2, z0, z1, z2, z3, x, y)<br />
{<br />
fr1 = (x-x1)/(x2-x1)*z1 + (x2-x)/(x2-x1)*z0;<br />
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{<br />
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}<br />
return ((y-y1)/(y2-y1)*fr2 + (y2-y)/(y2-y1)*fr1);<br />
}<br />
<br />
function colorRamp(value, colormap)<br />
{<br />
if(value > 1)<br />
{<br />
value = 1;<br />
}<br />
if(value < 0)<br />
{<br />
value = 0;<br />
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var maxval = colormap.length-1;<br />
var newval = value*maxval;<br />
var highval = Math.ceil(newval);<br />
var lowval = highval - 1;<br />
if(highval == 0)<br />
{<br />
lowval = 0;<br />
highval = 1;<br />
}<br />
<br />
lowcolor = RGB2HSL(colormap[lowval][0], colormap[lowval][1], colormap[lowval][2]);<br />
highcolor = RGB2HSL(colormap[highval][0], colormap[highval][1], colormap[highval][2]);<br />
var h = lowcolor.h*(highval - newval) + highcolor.h*(newval - lowval);<br />
var s = lowcolor.s*(highval - newval) + highcolor.s*(newval - lowval);<br />
var v = lowcolor.l*(highval - newval) + highcolor.l*(newval - lowval);<br />
//s = lowcolor.s;<br />
//v = lowcolor.l;<br />
<br />
newcolor = HSL2RGB(h,s,v);<br />
<br />
newcolor.r = colormap[lowval][0]*(highval - newval) + colormap[highval][0]*(newval - lowval);<br />
newcolor.g = colormap[lowval][1]*(highval - newval) + colormap[highval][1]*(newval - lowval);<br />
newcolor.b = colormap[lowval][2]*(highval - newval) + colormap[highval][2]*(newval - lowval);<br />
<br />
return [newcolor.r, newcolor.g, newcolor.b];<br />
}<br />
<br />
cool_color_ramp = [[0.00000, 0.00000, 0.50000],<br />
[0.00000, 0.00000, 0.56349],<br />
[0.00000, 0.00000, 0.62698],<br />
[0.00000, 0.00000, 0.69048],<br />
[0.00000, 0.00000, 0.75397],<br />
[0.00000, 0.00000, 0.81746],<br />
[0.00000, 0.00000, 0.88095],<br />
[0.00000, 0.00000, 0.94444],<br />
[0.00000, 0.00794, 1.00000],<br />
[0.00000, 0.07143, 1.00000],<br />
[0.00000, 0.13492, 1.00000],<br />
[0.00000, 0.19841, 1.00000],<br />
[0.00000, 0.26190, 1.00000],<br />
[0.00000, 0.32540, 1.00000],<br />
[0.00000, 0.38889, 1.00000],<br />
[0.00000, 0.45238, 1.00000],<br />
[0.00000, 0.51587, 1.00000],<br />
[0.00000, 0.57937, 1.00000],<br />
[0.00000, 0.64286, 1.00000],<br />
[0.00000, 0.70635, 1.00000],<br />
[0.00000, 0.76984, 1.00000],<br />
[0.00000, 0.83333, 1.00000],<br />
[0.00000, 0.89683, 1.00000],<br />
[0.00000, 0.96032, 1.00000],<br />
[0.02381, 1.00000, 0.97619],<br />
[0.08730, 1.00000, 0.91270],<br />
[0.15079, 1.00000, 0.84921],<br />
[0.21429, 1.00000, 0.78571],<br />
[0.27778, 1.00000, 0.72222],<br />
[0.34127, 1.00000, 0.65873],<br />
[0.40476, 1.00000, 0.59524],<br />
[0.46825, 1.00000, 0.53175],<br />
[0.53175, 1.00000, 0.46825],<br />
[0.59524, 1.00000, 0.40476],<br />
[0.65873, 1.00000, 0.34127],<br />
[0.72222, 1.00000, 0.27778],<br />
[0.78571, 1.00000, 0.21429],<br />
[0.84921, 1.00000, 0.15079],<br />
[0.91270, 1.00000, 0.08730],<br />
[0.97619, 1.00000, 0.02381],<br />
[1.00000, 0.96032, 0.00000],<br />
[1.00000, 0.89683, 0.00000],<br />
[1.00000, 0.83333, 0.00000],<br />
[1.00000, 0.76984, 0.00000],<br />
[1.00000, 0.70635, 0.00000],<br />
[1.00000, 0.64286, 0.00000],<br />
[1.00000, 0.57937, 0.00000],<br />
[1.00000, 0.51587, 0.00000],<br />
[1.00000, 0.45238, 0.00000],<br />
[1.00000, 0.38889, 0.00000],<br />
[1.00000, 0.32540, 0.00000],<br />
[1.00000, 0.26190, 0.00000],<br />
[1.00000, 0.19841, 0.00000],<br />
[1.00000, 0.13492, 0.00000],<br />
[1.00000, 0.07143, 0.00000],<br />
[1.00000, 0.00794, 0.00000],<br />
[0.94444, 0.00000, 0.00000],<br />
[0.88095, 0.00000, 0.00000],<br />
[0.81746, 0.00000, 0.00000],<br />
[0.75397, 0.00000, 0.00000],<br />
[0.69048, 0.00000, 0.00000],<br />
[0.62698, 0.00000, 0.00000],<br />
[0.56349, 0.00000, 0.00000],<br />
[0.50000, 0.00000, 0.00000]];<br />
<br />
//cool_color_ramp = [[0,0,0], [0,0,1], [1,1,1]];<br />
<br />
var WIDTH = 650;<br />
var HEIGHT = 350;<br />
var VIEW_ANGLE = 45,<br />
ASPECT = WIDTH/HEIGHT,<br />
NEAR = 0.1,<br />
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mutantcolors = ["white", "#ff6666", "yellow", "#66ff66", "#66ffff", "#ddaaff", "pink"];<br />
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$('#mw_3dplot #navbuttons3d td').eq(1).mousedown(function(){theta += Math.PI/8;rerender();})<br />
$('#mw_3dplot').toggle();<br />
$('#mutantwidget').parent().append('<div id="mw_sequencearea"/>');<br />
$('#mutantwidget span').hide();<br />
$('#mutantwidget .sequence').appendTo($('#mw_sequencearea'));<br />
$('#mutantwidget').append("<div class='yaxis'>RPU</div>");<br />
$('#mutantwidget').append("<div class='xaxis'>Theophylline (mM)</div>");<br />
<br />
$('#mw_selectmenu td').mouseover(function()<br />
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$('#mw_zoommenu td').mouseover(function()<br />
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if(!$(this).hasClass("selected"))<br />
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<br />
});<br />
$('#mw_selectmenu td').mouseout(function()<br />
{<br />
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});<br />
<br />
$('#mw_zoommenu td').mouseout(function()<br />
{<br />
if(!$(this).hasClass("selected"))<br />
{<br />
$(this).dequeue().animate({"opacity":.3, color:"rgb(225, 225, 225)"}, 500);<br />
}<br />
});<br />
<br />
$('#mw_zoommenu td').click(<br />
function() {<br />
if(!$(this).hasClass("selected"))<br />
{ $('#mw_zoommenu td').removeClass("selected");<br />
$(this).addClass("selected");<br />
$('#mw_zoommenu td').mouseout();<br />
dataMax = $(this).text();<br />
}<br />
$('#mw_optionsmenu td').removeClass("selected");<br />
if (widget_mode == "2D")<br />
{$('#mw_optionsmenu td').eq(0).mouseover().click();}<br />
else<br />
{}<br />
});<br />
<br />
$('#mw_optionsmenu td').click(<br />
function() {<br />
menuitem = $(this).parent().index();<br />
if(!$(this).hasClass("selected"))<br />
{<br />
$('#mw_optionsmenu td').removeClass("selected");<br />
$(this).addClass("selected");<br />
$('#mw_optionsmenu td').mouseout();<br />
if($(this).text() == "2D Plot")<br />
{<br />
$('#mw_3dplot').hide();<br />
$('#mw_plot').show();<br />
$('.yaxis').show();<br />
$('.xaxis').show();<br />
$('#mw_zoommenu').show();<br />
widget_mode = "2D";<br />
dataseries = [];<br />
$('#mw_selectmenu td').each(<br />
function(index)<br />
{<br />
if($(this).hasClass("selected"))<br />
{<br />
if(data[index])<br />
{ <br />
dataseries.push(data[index]);<br />
dataseries.push(errbars_2d[index]);<br />
}<br />
}<br />
}<br />
);<br />
if(!dataseries[0])<br />
{<br />
dataseries.push([[0,0]]);<br />
}<br />
$.plot($("#mw_plot"), dataseries, <br />
{ <br />
xaxis: {max:10, min:0},<br />
yaxis: {max: dataMax, min:0},<br />
grid: { },<br />
points: {show: true}, <br />
lines: {show: true}<br />
});<br />
}<br />
if($(this).text() == "3D Plot")<br />
{<br />
$('#mw_plot').hide();<br />
$('#mw_3dplot').show();<br />
$('.yaxis').hide();<br />
$('.xaxis').hide();<br />
$('#mw_zoommenu').hide();<br />
widget_mode = "3D";<br />
update3DPlotMeshes();<br />
}<br />
}<br />
<br />
}<br />
);<br />
$('#mw_optionsmenu td').eq(0).mouseover().click();<br />
$('#mw_selectmenu td').click(<br />
function() {<br />
if($(this).hasClass("selected"))<br />
{<br />
$(this).removeClass("selected");<br />
$(this).mouseout();<br />
$('#mw_sequencearea').children().eq($(this).parent().index()).hide();<br />
} else<br />
{<br />
$(this).dequeue().css({opacity:1});<br />
$(this).addClass("selected");<br />
$('#mw_sequencearea').children().hide();<br />
// $('#mw_sequencearea').children().eq($(this).parent().index()).show();<br />
}<br />
if(widget_mode == "2D")<br />
{<br />
dataseries = [];<br />
$('#mw_selectmenu td').each(<br />
function(index)<br />
{<br />
if($(this).hasClass("selected"))<br />
{<br />
if(data[index])<br />
{ <br />
dataseries.push(data[index]);<br />
dataseries.push(errbars_2d[index]);<br />
}<br />
}<br />
}<br />
);<br />
if(!dataseries[0])<br />
{<br />
dataseries.push([[0,0]]);<br />
}<br />
$.plot($("#mw_plot"), dataseries, <br />
{ <br />
xaxis: {max:10, min:0},<br />
yaxis: {max:dataMax, min:0},<br />
grid: { },<br />
points: {show: true}, <br />
lines: {show: true}<br />
});<br />
}else if(widget_mode == "3D")<br />
{<br />
update3DPlotMeshes();<br />
}<br />
} <br />
);<br />
//Split sequences<br />
$('#mutantwidget .sequence').each(<br />
function(index) {<br />
var curloc = 0;<br />
var curseq = $(this).text();<br />
$(this).html('');<br />
while(curloc < curseq.length)<br />
{<br />
oldloc = curloc;<br />
$(this).append(curseq.substr(oldloc, 15)).append(" ");<br />
curloc += 10;<br />
}<br />
}<br />
);<br />
<br />
//Extract plotting data<br />
dataMax = $('#mutantwidget .dataMax').text();<br />
if (dataMax.length == 0)<br />
{ dataMax = 90000; }<br />
axisdimensions = [0, 1, 0, 10, 0, dataMax];<br />
data = [];<br />
errbars_2d = [];<br />
$('#mutantwidget .xdata').each(<br />
function(index)<br />
{<br />
data.push({color:mutantcolors[index], data:[]});<br />
errbars_2d.push({color:"#ff0000", data:[], points:{show:false}});<br />
var thisdatax = $(this).text().split(',');<br />
var thisdatay = $('#mutantwidget .ydata').eq(index).text().split(',');<br />
var thisdataerr = $('#mutantwidget .stdevs').eq(index).text().split(',');<br />
var i;<br />
for(i=0; i < thisdatay.length; ++i)<br />
{<br />
thisdatay[i] = parseFloat(thisdatay[i]);<br />
thisdataerr[i] = parseFloat(thisdataerr[i]);<br />
}<br />
for(i=0; i < thisdatax.length; ++i)<br />
{<br />
thisdatax[i] = parseFloat(thisdatax[i]);<br />
data[index].data.push([thisdatax[i], thisdatay[i]]);<br />
}<br />
var errbarwidth = Math.max.apply(null,thisdatax)/150;<br />
for(i=0; i < thisdatax.length; ++i)<br />
{<br />
errbars_2d[index].data.push([thisdatax[i], thisdatay[i]+thisdataerr[i]]);<br />
errbars_2d[index].data.push([thisdatax[i], thisdatay[i]-thisdataerr[i]]);<br />
errbars_2d[index].data.push([null, null]);<br />
errbars_2d[index].data.push([thisdatax[i]-errbarwidth, thisdatay[i]+thisdataerr[i]]);<br />
errbars_2d[index].data.push([thisdatax[i]+errbarwidth, thisdatay[i]+thisdataerr[i]]);<br />
errbars_2d[index].data.push([null, null]);<br />
errbars_2d[index].data.push([thisdatax[i]-errbarwidth, thisdatay[i]-thisdataerr[i]]);<br />
errbars_2d[index].data.push([thisdatax[i]+errbarwidth, thisdatay[i]-thisdataerr[i]]);<br />
errbars_2d[index].data.push([null, null]);<br />
}<br />
<br />
}<br />
);<br />
$.plot($("#mw_plot"), [[0,0]], <br />
{ <br />
xaxis: {max:10, min:0},<br />
yaxis: {max:dataMax, min:0},<br />
grid: { },<br />
});<br />
<br />
});<br />
<br />
sphereMaterial = new THREE.MeshBasicMaterial(<br />
{<br />
color:0xCC0000<br />
}<br />
);<br />
plotMaterial = new THREE.MeshFaceMaterial(<br />
// {<br />
// color:0xCC0000,<br />
// opacity:.4,<br />
// vertexColors:true<br />
// }<br />
);<br />
pointMaterial = new THREE.ParticleCanvasMaterial( { color: 0xffffff, program: function(context)<br />
{<br />
context.beginPath();<br />
context.arc( 0, 0, .5, 0, Math.PI * 2, true );<br />
context.fill();<br />
} } );<br />
<br />
axisMat = new THREE.LineBasicMaterial(<br />
{<br />
color:0xffffff,<br />
opacity:1,<br />
linewidth:1<br />
}<br />
);<br />
axisdimensions = [0, 1, 0, 10, 0, dataMax];<br />
var numxpoints = 30;<br />
var numypoints = 30;<br />
<br />
var lastMouseX, lastMouseY;<br />
<br />
var data_3d;<br />
isdragging = false;<br />
<br />
<br />
$(document).ready(function() {<br />
data_3d = [];<br />
errorbars_3d = [];<br />
particles = [];<br />
meshchildren = [];<br />
//Parse datas!<br />
$('.zdata_3d').each(<br />
function(index)<br />
{<br />
particles.push(new THREE.Geometry());<br />
thisdataz = $(this).text().split(',');<br />
thisdatax = $('.xdata_3d').eq(index).text().split(',');<br />
thisdatay = $('.ydata_3d').eq(index).text().split(',');<br />
thisdataerr = $('.stdevs_3d').eq(index).text().split(',');<br />
<br />
<br />
//numxpoints = thisdatax.length;<br />
//numypoints = thisdatay.length;<br />
meshchildren.push(new THREE.Object3D());<br />
errorbars_3d.push(new THREE.Object3D());<br />
data_3d.push(new THREE.PlaneGeometry(100,100,numxpoints-1, numypoints-1));<br />
var i;<br />
for(i=0; i < thisdatax.length; ++i)<br />
{<br />
thisdatax[i] = parseFloat(thisdatax[i]);<br />
}<br />
for(i=0; i < thisdatay.length; ++i)<br />
{<br />
thisdatay[i] = parseFloat(thisdatay[i]);<br />
}<br />
for(i=0; i < thisdataz.length; ++i)<br />
{<br />
thisdataz[i] = parseFloat(thisdataz[i]);<br />
<br />
/*<br />
var particle = new THREE.Vertex(<br />
new THREE.Vector3(thisdatax[i%thisdatax.length]/axisdimensions[1]*100, <br />
thisdatay[Math.floor(i/thisdatax.length)]/axisdimensions[3]*100, <br />
thisdataz[i]/axisdimensions[5]*100)<br />
);<br />
particles[index].vertices.push(particle);<br />
var newparticle = new THREE.Particle(pointMaterial)<br />
newparticle.position.x = particle.position.x;<br />
newparticle.position.y = particle.position.y;<br />
newparticle.position.z = particle.position.z;<br />
meshchildren[index].addChild(newparticle);<br />
*/<br />
<br />
<br />
}<br />
var lineMat = new THREE.LineBasicMaterial(<br />
{<br />
color:mutantcolors_hex[index],<br />
opacity:1,<br />
linewidth:2<br />
}<br />
);<br />
for(i=0; i < thisdataerr.length; i++)<br />
{<br />
thisdataerr[i] = parseFloat(thisdataerr[i]);<br />
var errbargeom = new THREE.Geometry();<br />
errbargeom.vertices.push(new THREE.Vertex(new THREE.Vector3(<br />
thisdatax[i%thisdatax.length]/axisdimensions[1]*100,<br />
thisdatay[Math.floor(i/thisdatax.length)]/axisdimensions[3]*100,<br />
thisdataz[i]/axisdimensions[5]*100-thisdataerr[i]/axisdimensions[5]*100<br />
)));<br />
errbargeom.vertices.push(new THREE.Vertex(new THREE.Vector3(<br />
thisdatax[i%thisdatax.length]/axisdimensions[1]*100,<br />
thisdatay[Math.floor(i/thisdatax.length)]/axisdimensions[3]*100,<br />
thisdataz[i]/axisdimensions[5]*100+thisdataerr[i]/axisdimensions[5]*100<br />
)));<br />
var errbar = new THREE.Line(errbargeom, lineMat);<br />
errorbars_3d[index].addChild(errbar);<br />
}<br />
//Particles look like ass.<br />
//Use lines instead.<br />
<br />
for(i=0; i < thisdatax.length*(thisdatay.length-1); i++)<br />
{<br />
<br />
var linegeom = new THREE.Geometry();<br />
linegeom.vertices.push(new THREE.Vertex(new THREE.Vector3(<br />
thisdatax[i%thisdatax.length]/axisdimensions[1]*100,<br />
thisdatay[Math.floor(i/thisdatax.length)]/axisdimensions[3]*100,<br />
thisdataz[i]/axisdimensions[5]*100<br />
)));<br />
linegeom.vertices.push(new THREE.Vertex(new THREE.Vector3(<br />
thisdatax[(i+thisdatax.length)%thisdatax.length]/axisdimensions[1]*100,<br />
thisdatay[Math.floor((i+thisdatax.length)/thisdatax.length)]/axisdimensions[3]*100,<br />
thisdataz[i+thisdatax.length]/axisdimensions[5]*100<br />
)));<br />
meshchildren[index].addChild(new THREE.Line(linegeom, lineMat));<br />
}<br />
for(i=0; i < (thisdatax.length-1)*(thisdatay.length); i++)<br />
{<br />
//Particles look like ass.<br />
//Use lines instead.<br />
var linegeom = new THREE.Geometry();<br />
linegeom.vertices.push(new THREE.Vertex(new THREE.Vector3(<br />
thisdatax[i%(thisdatax.length-1)]/axisdimensions[1]*100,<br />
thisdatay[Math.floor(i/(thisdatax.length-1))]/axisdimensions[3]*100,<br />
thisdataz[Math.floor(i/(thisdatax.length-1))+i]/axisdimensions[5]*100<br />
)));<br />
linegeom.vertices.push(new THREE.Vertex(new THREE.Vector3(<br />
thisdatax[i%(thisdatax.length-1)+1]/axisdimensions[1]*100,<br />
thisdatay[Math.floor(i/(thisdatax.length-1))]/axisdimensions[3]*100,<br />
thisdataz[Math.floor(i/(thisdatax.length-1))+i+1]/axisdimensions[5]*100<br />
)));<br />
meshchildren[index].addChild(new THREE.Line(linegeom, lineMat));<br />
}<br />
<br />
for(i=0; i < numxpoints; ++i)<br />
{<br />
for(var j=0; j < numypoints; ++j)<br />
{<br />
var thisx = thisdatax[thisdatax.length-1]*i/(numxpoints-1);<br />
var thisy = thisdatay[thisdatay.length-1]*j/(numypoints-1);<br />
data_3d[index].vertices[j*numxpoints+i].position.x = thisx/axisdimensions[1]*100;<br />
data_3d[index].vertices[j*numxpoints+i].position.y = thisy/axisdimensions[3]*100;<br />
//Find flanking x,y values<br />
var kx = ky = 0;<br />
while(thisx >= thisdatax[kx]) { <br />
kx=kx+1; <br />
}<br />
while(thisy >= thisdatay[ky]) { <br />
ky=ky+1; <br />
}<br />
if(kx > thisdatax.length-1)<br />
{<br />
kx = thisdatax.length-1;<br />
}<br />
if(ky > thisdatay.length-1)<br />
{<br />
ky = thisdatay.length-1;<br />
}<br />
kx--;<br />
ky--;<br />
<br />
kx = parseInt(kx);<br />
ky = parseInt(ky);<br />
var tli = kx+ky*thisdatax.length;<br />
var tri = kx+ky*thisdatax.length+1;<br />
var bli = kx+(ky+1)*thisdatax.length;<br />
var bri = kx+1+(ky+1)*thisdatax.length;<br />
if(!thisdataz[bli] || !thisdataz[tli] || !thisdataz[tri] || !thisdataz[bri])<br />
{<br />
alert("Porblem!\nkx:" + kx + "\nky:" + ky + "\nkx is a "+typeof(kx) + "\nky is a" +typeof(ky) + "\nset:" + index);<br />
}<br />
thisz = interp2(thisdatax[kx], <br />
thisdatay[ky], <br />
thisdatax[kx+1], <br />
thisdatay[ky+1], <br />
thisdataz[tli],<br />
thisdataz[tri],<br />
thisdataz[bli],<br />
thisdataz[bri], thisx, thisy)<br />
data_3d[index].vertices[j*numxpoints+i].position.z = thisz/axisdimensions[5]*100;<br />
}<br />
}<br />
data_3d[index].computeCentroids();<br />
//vertex colors<br />
/*<br />
var colors = [];<br />
for(i=0; i < thisdataz.length; ++i)<br />
{<br />
var color = new THREE.Color(0x550000);<br />
// color.setHSV(.125, 1, 0);<br />
colors.push(color);<br />
}<br />
*/<br />
materials = [];<br />
for(i=0; i < data_3d[index].faces.length; ++i)<br />
{<br />
thisfacecolor = (<br />
data_3d[index].vertices[data_3d[index].faces[i].a].position.z+<br />
data_3d[index].vertices[data_3d[index].faces[i].b].position.z+<br />
data_3d[index].vertices[data_3d[index].faces[i].c].position.z+<br />
data_3d[index].vertices[data_3d[index].faces[i].d].position.z)/4/100;<br />
materials.push([new THREE.MeshBasicMaterial(<br />
{<br />
color:thisfacecolor*0xffffff,<br />
opacity:.6<br />
}<br />
)]);<br />
if(!thisfacecolor)<br />
{<br />
alert("Something is terribly wrong!\na ("+data_3d[index].faces[i].a+"):"+data_3d[index].vertices[data_3d[index].faces[i].a].position.z+"\nb("+data_3d[index].faces[i].b+"):"+data_3d[index].vertices[data_3d[index].faces[i].b].position.z+"\nc("+data_3d[index].faces[i].c+"):"+data_3d[index].vertices[data_3d[index].faces[i].c].position.z+"\nd("+data_3d[index].faces[i].d+"):"+data_3d[index].vertices[data_3d[index].faces[i].a].position.z);<br />
}<br />
var newcolor = colorRamp(thisfacecolor, cool_color_ramp);<br />
materials[i][0].color.setRGB(newcolor[0], newcolor[1], newcolor[2]);<br />
data_3d[index].faces[i].materials = materials[i];<br />
}<br />
<br />
}<br />
);<br />
<br />
var origin = new THREE.Vertex(new THREE.Vector3(0,0,0));<br />
<br />
theta = -Math.PI/4;<br />
phi = Math.PI/8;<br />
<br />
//Axis labels<br />
var x = document.createElement("canvas");<br />
var xc = x.getContext("2d");<br />
x.width = 400;<br />
x.height = 40;<br />
xc.fillStyle = "#ffffff";<br />
xc.font = "20pt arial";<br />
xc.textBaseline = "top";<br />
xc.fillText("Arabinose (% w/v)", 10, 0);<br />
<br />
var xm = new THREE.MeshBasicMaterial({<br />
map: new THREE.Texture(x)<br />
});<br />
xm.map.needsUpdate = true;<br />
<br />
xaxislabel = new THREE.Mesh(new THREE.PlaneGeometry(400, 40, 2, 2), xm);<br />
xaxislabel.position.x = -50;<br />
xaxislabel.position.y = 0;<br />
xaxislabel.position.z = -48;<br />
xaxislabel.scale.x = 0.2;<br />
xaxislabel.scale.y = -0.2;<br />
xaxislabel.rotation.x = -Math.PI/2;<br />
xaxislabel.rotation.y = -Math.PI/2;<br />
xaxislabel.doubleSided = true;<br />
<br />
var x = document.createElement("canvas");<br />
var xc = x.getContext("2d");<br />
x.width = 400;<br />
x.height = 40;<br />
xc.fillStyle = "#ffffff";<br />
xc.font = "20pt arial";<br />
xc.textBaseline = "top";<br />
xc.fillText("Theophylline (mM)", 10, 0);<br />
<br />
var xm = new THREE.MeshBasicMaterial({<br />
map: new THREE.Texture(x)<br />
});<br />
xm.map.needsUpdate = true;<br />
<br />
yaxislabel = new THREE.Mesh(new THREE.PlaneGeometry(400, 40, 2, 2), xm);<br />
yaxislabel.position.x = 0;<br />
yaxislabel.position.y = -50;<br />
yaxislabel.position.z = -48;<br />
yaxislabel.scale.x = 0.2;<br />
yaxislabel.scale.y = -0.2;<br />
yaxislabel.rotation.x = -Math.PI/2;<br />
yaxislabel.doubleSided = true;<br />
<br />
var x = document.createElement("canvas");<br />
var xc = x.getContext("2d");<br />
x.width = 400;<br />
x.height = 40;<br />
xc.fillStyle = "#ffffff";<br />
xc.font = "20pt arial";<br />
xc.textBaseline = "top";<br />
xc.fillText("RPU", 10, 0);<br />
<br />
var xm = new THREE.MeshBasicMaterial({<br />
map: new THREE.Texture(x)<br />
});<br />
xm.map.needsUpdate = true;<br />
<br />
zaxislabel = new THREE.Mesh(new THREE.PlaneGeometry(400, 40, 2, 2), xm);<br />
zaxislabel.position.x = -50;<br />
zaxislabel.position.y = -52;<br />
zaxislabel.position.z = 0;<br />
zaxislabel.rotation.x = -Math.PI/2;<br />
zaxislabel.rotation.y = -Math.PI/2;<br />
zaxislabel.rotation.z = -Math.PI/2;<br />
zaxislabel.scale.x = 0.2;<br />
zaxislabel.scale.y = -0.2;<br />
<br />
zaxislabel.doubleSided = true;<br />
zaxislabel.updateMatrix();<br />
<br />
<br />
var $container = $('#mw_3dplot');<br />
$('body').mouseup(function(){<br />
isdragging = false;<br />
$container.unbind('mousemove');<br />
});<br />
$container.mousedown(function(event){<br />
isdragging = true;<br />
lastMouseX = event.pageX;<br />
lastMouseY = event.pageY;<br />
$container.mousemove(function(event)<br />
{<br />
theta += (lastMouseX - event.pageX)/70<br />
phi -= (lastMouseY - event.pageY)/70;<br />
lastMouseX = event.pageX;<br />
lastMouseY = event.pageY;<br />
rerender();<br />
});<br />
});<br />
renderer = new THREE.CanvasRenderer();<br />
//renderer.autoClear = false;<br />
camera = new THREE.OrthoCamera( -75, 75, 75, -75, - 2000, 1000 );<br />
<br />
scene = new THREE.Scene();<br />
camera.position.x = 250;<br />
camera.position.y = -150;<br />
camera.position.z = 75;<br />
camera.up.y = 0;<br />
camera.up.z = 1;<br />
// camera.rotation.y = -Math.PI/6;<br />
camera.position.x = 250*Math.cos(theta);<br />
camera.position.y = 250*Math.sin(theta);<br />
renderer.setSize(WIDTH, HEIGHT);<br />
$container.append(renderer.domElement);<br />
var radius = 50, segments = 16, rings = 16;<br />
<br />
sphere = new THREE.Mesh(<br />
new THREE.SphereGeometry(radius,<br />
segments,<br />
rings),<br />
sphereMaterial<br />
);<br />
sphere.position.x = 50;<br />
sphere.position.y = 50;<br />
sphere.position.z = 50;<br />
<br />
// add it to the scene<br />
plot_mesh = [];<br />
plot_points = [];<br />
for(var i = 0; i < data_3d.length; ++i)<br />
{<br />
plot_mesh.push(new THREE.Mesh(data_3d[i], plotMaterial));<br />
plot_mesh[i].doubleSided = true;<br />
<br />
plot_points.push(new THREE.ParticleSystem(<br />
particles[i],<br />
pointMaterial));<br />
}<br />
<br />
plot_obj = new THREE.Object3D();<br />
plot_obj.rotation.z = -Math.PI/2;<br />
plot_obj.scale.x = -1;<br />
//plot_mesh.addChild(sphere);<br />
<br />
<br />
axisgeom = new THREE.Geometry();<br />
axisgeom.vertices.push(origin);<br />
axisgeom.vertices.push(new THREE.Vertex(new THREE.Vector3(100,0,0)));<br />
axisgeom.vertices.push(new THREE.Vertex(new THREE.Vector3(98, 1, 0)));<br />
axisgeom.vertices.push(new THREE.Vertex(new THREE.Vector3(98, -1, 0)));<br />
axisgeom.vertices.push(new THREE.Vertex(new THREE.Vector3(100, 0, 0)));<br />
<br />
xaxis = new THREE.Line(<br />
axisgeom,<br />
axisMat<br />
);<br />
<br />
yaxis = new THREE.Line(<br />
axisgeom,<br />
axisMat<br />
);<br />
yaxis.rotation.z = 90*Math.PI/180;<br />
<br />
zaxis = new THREE.Line(<br />
axisgeom,<br />
axisMat<br />
);<br />
scene.addChild(xaxislabel);<br />
scene.addChild(yaxislabel);<br />
scene.addChild(zaxislabel);<br />
<br />
zaxis.rotation.y = -90*Math.PI/180;<br />
<br />
plot_obj.position.x = -50;<br />
plot_obj.position.z = -50;<br />
plot_obj.position.y = -50;<br />
<br />
zaxis.position.x = -50;<br />
zaxis.position.z = -50;<br />
zaxis.position.y = -50;<br />
<br />
xaxis.position.x = -50;<br />
xaxis.position.z = -50;<br />
xaxis.position.y = -50;<br />
<br />
yaxis.position.x = -50;<br />
yaxis.position.z = -50;<br />
yaxis.position.y = -50;<br />
<br />
//plot_obj.addChild(particleSystem);<br />
<br />
<br />
scene.addChild(xaxis);<br />
scene.addChild(yaxis);<br />
scene.addChild(zaxis);<br />
scene.addChild(plot_obj);<br />
// origin_object.addChild(sphere);<br />
<br />
<br />
//Grid<br />
var gridlinegeom = new THREE.Geometry();<br />
gridlinegeom.vertices.push(new THREE.Vertex(new THREE.Vector3(0,0,0)));<br />
gridlinegeom.vertices.push(new THREE.Vertex(new THREE.Vector3(100,0,0)));<br />
<br />
for(var i=1; i <= 5; i++)<br />
{<br />
var gridline = new THREE.Line(gridlinegeom, axisMat);<br />
gridline.position.y = i/5*100-50;<br />
gridline.position.x = -50;<br />
gridline.position.z = -50;<br />
scene.addChild(gridline);<br />
<br />
<br />
<br />
var gridline = new THREE.Line(gridlinegeom, axisMat);<br />
gridline.position.x = i/5*100-50;<br />
gridline.position.y = -50;<br />
gridline.position.z = -50;<br />
gridline.rotation.z = Math.PI/2;<br />
scene.addChild(gridline);<br />
}<br />
yticklabels = [];<br />
xticklabels = [];<br />
for(var i=1; i <= 5; i++)<br />
{<br />
var x = document.createElement("canvas");<br />
var xc = x.getContext("2d");<br />
x.width = 40;<br />
x.height = 40;<br />
xc.fillStyle = "#ffffff";<br />
xc.font = "12pt arial";<br />
xc.textBaseline = "top";<br />
xc.fillText(i/5*axisdimensions[1], 10, 0);<br />
<br />
var xm = new THREE.MeshBasicMaterial({<br />
map: new THREE.Texture(x)<br />
});<br />
xm.map.needsUpdate = true;<br />
var xtick = new THREE.Mesh(new THREE.PlaneGeometry(13/ASPECT, 13, 2, 2), xm);<br />
xtick.position.y = i/5*100-50;<br />
xtick.position.x = -50;<br />
xtick.position.z = -58;<br />
xtick.rotation.x = Math.PI/2;<br />
xtick.doubleSided = true;<br />
xtick.updateMatrix();<br />
scene.addChild(xtick);<br />
<br />
var x = document.createElement("canvas");<br />
var xc = x.getContext("2d");<br />
x.width = 80;<br />
x.height = 40;<br />
xc.fillStyle = "#ffffff";<br />
xc.font = "12pt arial";<br />
xc.textBaseline = "top";<br />
xc.fillText((i/5*axisdimensions[3]).toFixed(4), 10, 0);<br />
<br />
var xm = new THREE.MeshBasicMaterial({<br />
map: new THREE.Texture(x)<br />
});<br />
xm.map.needsUpdate = true;<br />
var ytick = new THREE.Mesh(new THREE.PlaneGeometry(26/ASPECT, 13, 2, 2), xm);<br />
ytick.position.x = i/5*100-50;<br />
ytick.position.y = -50;<br />
ytick.position.z = -58;<br />
ytick.rotation.x = Math.PI/2;<br />
ytick.doubleSided = true;<br />
ytick.updateMatrix();<br />
scene.addChild(ytick);<br />
<br />
xticklabels.push(xtick);<br />
yticklabels.push(ytick);<br />
}<br />
<br />
var particle = new THREE.Particle(pointMaterial);<br />
<br />
<br />
renderer.render(scene, camera);<br />
<br />
<br />
$('#mw_selectmenu td').eq(0).mouseover().click();<br />
$('#mw_selectmenu td').eq(1).mouseover().click();<br />
$('#mw_selectmenu td').eq(3).mouseover().click();<br />
$('#mw_optionstable td').eq(1).mouseover().click();<br />
$('#mw_zoomoptionstable td').eq(0).mouseover().click();<br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/ModelingTeam:UC Davis/Modeling2013-10-28T23:48:57Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3DRPU}}<br />
<html><br />
<head><br />
</head><br />
<body><br />
<br />
<div><br />
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</div><br />
<br />
<div class="floatbox"><br />
<a id="equations"> </a><br />
<h1>Equations</h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/3/35/UCDavis_Testing_modelscheme_n.png" width= 620 height=195></center><br />
<br></br><br />
<br><br />
The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/4/42/UCDavis_Probofactivecomplex.png" width=1019 height=330><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/c/cc/UCDavis_Testing_odes.png" width=938 height=520><br />
<br></br><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#equations">Equations</a></li><br />
<li><a href="#parameters">Parameters</a></li><br />
<li><a href="#MATLABsim">MATLAB Simulation</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="parameters"></a><br />
<h1>Parameters <a href="#top" class="to_top">^back to top</a></h1><br />
Included here are the parameters used in this model. Please refer to the <a href="#References">References</a> section of this page for the source of each parameter value. <br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4e/Ucdavisparameters1.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/d/de/Ucdavisparameters2.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/a/aa/Ucdavisparameters3.jpg" class="genpic"></center><br />
<br></br><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<a id="MATLABsim"></a><br />
<h1>MATLAB Simulation<a href="https://static.igem.org/mediawiki/2013/8/8b/UCD2013RiboTALe.m"><i>See the Code!</i></a><a href="#top" class="to_top">^back to top</a></h1><br />
<br />
<h3>TALe Binding Site K<sub>D</sub> As a Source of Tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0a/Ucdavismodel2.png" class="genpic"></center><br />
<br>It should be noted that "less effective" does not mean that RiboTALe 1 is an inferior part, merely that it generates a distinct system response and displays kinematic behavior that may be specifically needed in a future circuit design. We have demonstrated that through their engineerable tunability RiboTALes are capable of achieving a broad range of system responses, a conclusion that is supported by this model. </br><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 1 with a K<sub>D</sub> of 240 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is, in fact, responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2c/Ucdavismodel3.png"class="genpic"></center><br />
<br>Future work with this model may involve the simulation of RiboTALe activity under different non-theophylline riboswitches and an investigation of the orthogonality achievable when using multiple riboswitches in a system.</br><br />
<br></br><br />
<h3>Amplifying System Response Through Transcript Induction</h3> <br />
<br>To investigate the effects of increasing GFP transcript while maintaining constant levels of arabinose and theophylline, the dynamic response of the system under RiboTALe 1 repression was simulated for aTc levels of 0, 25 ng/mL, and 100 ng/mL, where aTC is the inducer of the GFP transcript. The model results show the expected behavior: at higher concentrations of aTc GFP reaches greater peak concentration before repression by the RiboTALe becomes evident. Moreover, this event occurs later in the simulation under conditions of 100 ng/mL of aTc than it does for the other two simulated responses.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/c/c9/Ucdavismodel4.png"></center><br />
<br></br><br />
This model can be further developed to take into account riboswitch leakiness and system stochasticity, and the parameters fine-tuned. It is, however, a useful model in that it provides a mathematical basis that supports the functionality of our RiboTALe devices and shows the wide variety of system responses achievable through the modulation of the engineerable and tunable elements of our construct. We tested combinations of two TAL repressors and two theophylline riboswitches. With this model we will be able to predict the response of a library of RiboTALes, composed a much greater variety of riboswitches and TAL repressors, and perhaps identify with which combination and under what induction conditions a desired system response may be achieved. <br />
</div><br />
<br />
<div class="floatboxwide" id="References"><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">30</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1.16158623970914, 21.6609858203536,, 10.7238390098074, 0.0296612965982997</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1.16158623970914,<br />
1.38324321672162,<br />
1.56787760577646,<br />
3.44344634823164,<br />
28.1550374713881,<br />
1.12263448435245,<br />
1.23446651043791,<br />
1.37349621495727,<br />
2.91488533171903,<br />
21.6609858203536,<br />
0.681669891981254,<br />
1.2548618440377,<br />
1.55449707407215,<br />
4.34727847535614,<br />
19.9321910930205,<br />
0.838126720409999,<br />
0.7232594704811,<br />
1.67616077719149,<br />
6.75843601081123,<br />
21.5221383017144,<br />
0.761406719776734,<br />
0.955255402108518,<br />
0.700049421274729,<br />
10.7238390098074,<br />
0.485471902706462,<br />
0.0196404111837095,<br />
0.0296612965982997,<br />
0.0313199181732694,<br />
0.108325822385663,<br />
0.442737316405862,<br />
<br />
<br />
<!--106247.4518,104406.975,107697.0566,87605.53496,113026.3159,116238.2595,<br />
97390.65405,102272.0717,105314.5467,36495.57538,102836.5068,117957.4598,<br />
25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
1311.329197,4760.869684,27024.29053,1093.892416,4443.585781,38823.82162,<br />
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</span><br />
<span class='stdevs_3d'><br />
</span><br />
<br />
<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
<!--2369.383411,2422.018312,2387.445279,2327.26362,2303.398056,2254.056792,<br />
2345.828233,2307.888693,2292.350333,2258.230481,2233.40043,2104.700912,<br />
2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
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</span><br />
<span class='stdevs_3d'><br />
</span><br />
</div><br />
</div><br />
<br />
<div class="floatboxwide" id="References"><br />
<h1>References<a href="#top" class="to_top">^back to top</a></h1><br />
<br>[1] <a href="http://bionumbers.hms.harvard.edu/KeyNumbers.aspx?redirect=false">"Key Numbers for Cell Biologists." Bionumbers: The Database of Useful Biological Numbers</a></h1></br><br />
<br>[2] <a href="http://openwetware.org/images/5/5b/NORgate%2BChemWires_SuppInfo_nature09565-s1.pdf">Tamsir et al. 'Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’: Supplementary Information'. Nature 469, 212–215 (13 January 2011)</a></hi></br><br />
<br>[3] <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a></br><br />
<br>[4] Adjusted from <a href="http://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1330.full">literature</a></hi> to fit our data.</br><br />
<br>[5] <a href="http://parts.igem.org/Part:BBa_K750000">"Part:BBa_K750000" The Registry of Standard Biological Parts </a></hi></br> <br />
<br>[6] <a href="http://www.ncbi.nlm.nih.gov/pubmed/8707053">Cormack et al. 'FACS-optimized mutants of the green fluorescent protein (GFP).' Gene. 1996;173(1 Spec No):33-8.</a></hi></br><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/ModelingTeam:UC Davis/Modeling2013-10-28T23:46:14Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/c/cf/Modelingbanner_UCDavis.jpg" class="banner" width="967" height="226"><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/92/UCD_2013_Modeling_Bannerv2.PNG" class="banner" width="967" height="226"--><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="equations"> </a><br />
<h1>Equations</h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/3/35/UCDavis_Testing_modelscheme_n.png" width= 620 height=195></center><br />
<br></br><br />
<br><br />
The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/4/42/UCDavis_Probofactivecomplex.png" width=1019 height=330><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/c/cc/UCDavis_Testing_odes.png" width=938 height=520><br />
<br></br><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#equations">Equations</a></li><br />
<li><a href="#parameters">Parameters</a></li><br />
<li><a href="#MATLABsim">MATLAB Simulation</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="parameters"></a><br />
<h1>Parameters <a href="#top" class="to_top">^back to top</a></h1><br />
Included here are the parameters used in this model. Please refer to the <a href="#References">References</a> section of this page for the source of each parameter value. <br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4e/Ucdavisparameters1.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/d/de/Ucdavisparameters2.jpg" class="genpic"></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/a/aa/Ucdavisparameters3.jpg" class="genpic"></center><br />
<br></br><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<a id="MATLABsim"></a><br />
<h1>MATLAB Simulation<a href="https://static.igem.org/mediawiki/2013/8/8b/UCD2013RiboTALe.m"><i>See the Code!</i></a><a href="#top" class="to_top">^back to top</a></h1><br />
<br />
<h3>TALe Binding Site K<sub>D</sub> As a Source of Tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0a/Ucdavismodel2.png" class="genpic"></center><br />
<br>It should be noted that "less effective" does not mean that RiboTALe 1 is an inferior part, merely that it generates a distinct system response and displays kinematic behavior that may be specifically needed in a future circuit design. We have demonstrated that through their engineerable tunability RiboTALes are capable of achieving a broad range of system responses, a conclusion that is supported by this model. </br><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 1 with a K<sub>D</sub> of 240 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is, in fact, responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2c/Ucdavismodel3.png"class="genpic"></center><br />
<br>Future work with this model may involve the simulation of RiboTALe activity under different non-theophylline riboswitches and an investigation of the orthogonality achievable when using multiple riboswitches in a system.</br><br />
<br></br><br />
<h3>Amplifying System Response Through Transcript Induction</h3> <br />
<br>To investigate the effects of increasing GFP transcript while maintaining constant levels of arabinose and theophylline, the dynamic response of the system under RiboTALe 1 repression was simulated for aTc levels of 0, 25 ng/mL, and 100 ng/mL, where aTC is the inducer of the GFP transcript. The model results show the expected behavior: at higher concentrations of aTc GFP reaches greater peak concentration before repression by the RiboTALe becomes evident. Moreover, this event occurs later in the simulation under conditions of 100 ng/mL of aTc than it does for the other two simulated responses.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/c/c9/Ucdavismodel4.png"></center><br />
<br></br><br />
This model can be further developed to take into account riboswitch leakiness and system stochasticity, and the parameters fine-tuned. It is, however, a useful model in that it provides a mathematical basis that supports the functionality of our RiboTALe devices and shows the wide variety of system responses achievable through the modulation of the engineerable and tunable elements of our construct. We tested combinations of two TAL repressors and two theophylline riboswitches. With this model we will be able to predict the response of a library of RiboTALes, composed a much greater variety of riboswitches and TAL repressors, and perhaps identify with which combination and under what induction conditions a desired system response may be achieved. <br />
</div><br />
<br />
<div class="floatboxwide" id="References"><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">30</span><br />
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<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1.16158623970914, 21.6609858203536,, 10.7238390098074, 0.0296612965982997</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
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1.16158623970914,<br />
1.38324321672162,<br />
1.56787760577646,<br />
3.44344634823164,<br />
28.1550374713881,<br />
1.12263448435245,<br />
1.23446651043791,<br />
1.37349621495727,<br />
2.91488533171903,<br />
21.6609858203536,<br />
0.681669891981254,<br />
1.2548618440377,<br />
1.55449707407215,<br />
4.34727847535614,<br />
19.9321910930205,<br />
0.838126720409999,<br />
0.7232594704811,<br />
1.67616077719149,<br />
6.75843601081123,<br />
21.5221383017144,<br />
0.761406719776734,<br />
0.955255402108518,<br />
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10.7238390098074,<br />
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0.0296612965982997,<br />
0.0313199181732694,<br />
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0.442737316405862,<br />
<br />
<br />
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25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
1311.329197,4760.869684,27024.29053,1093.892416,4443.585781,38823.82162,<br />
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<br />
<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
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1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
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2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
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</span><br />
<span class='stdevs_3d'><br />
</span><br />
</div><br />
</div><br />
<br />
<div class="floatboxwide" id="References"><br />
<h1>References<a href="#top" class="to_top">^back to top</a></h1><br />
<br>[1] <a href="http://bionumbers.hms.harvard.edu/KeyNumbers.aspx?redirect=false">"Key Numbers for Cell Biologists." Bionumbers: The Database of Useful Biological Numbers</a></h1></br><br />
<br>[2] <a href="http://openwetware.org/images/5/5b/NORgate%2BChemWires_SuppInfo_nature09565-s1.pdf">Tamsir et al. 'Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’: Supplementary Information'. Nature 469, 212–215 (13 January 2011)</a></hi></br><br />
<br>[3] <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a></br><br />
<br>[4] Adjusted from <a href="http://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1330.full">literature</a></hi> to fit our data.</br><br />
<br>[5] <a href="http://parts.igem.org/Part:BBa_K750000">"Part:BBa_K750000" The Registry of Standard Biological Parts </a></hi></br> <br />
<br>[6] <a href="http://www.ncbi.nlm.nih.gov/pubmed/8707053">Cormack et al. 'FACS-optimized mutants of the green fluorescent protein (GFP).' Gene. 1996;173(1 Spec No):33-8.</a></hi></br><br />
</div> <br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/ModelingTeam:UC Davis/Modeling2013-10-28T23:44:01Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
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<img src="https://static.igem.org/mediawiki/2013/c/cf/Modelingbanner_UCDavis.jpg" class="banner" width="967" height="226"><br />
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<div class="floatbox"><br />
<a id="equations"> </a><br />
<h1>Equations</h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/3/35/UCDavis_Testing_modelscheme_n.png" width= 620 height=195></center><br />
<br></br><br />
<br><br />
The equations below model the concentrations of bound transcription factors. That is, they serve to model the concentration of araC bound to pBAD and tetR bound to pTET given the concentrations of the ligands, arabinose and aTc.</br><br>The subsequent equations model the probability of active complex for each element in our circuit. P<sub>BAD</sub> represents the probability that the pBAD promoter will be unbound by araC and thus active. P<sub>TET</sub> represents the probability that the pTET promoter will be unbound by tetR and thus active. P<sub>Riboswitch</sub> expresses the probability that the riboswitch is bound by theophylline, and thus active. For simplicity, it has been modeled here as an activator-controlled promoter. P<sub>Tale Binding Site</sub>, which may be abbreviated to P<sub>TBS</sub> expresses the probability that the TALe binding site is unbound by the TAL repressor, and thus active.</br><br />
<br>The third set of equations are ordinary differential equations modeling the change in concentration over time of the riboswitch-TALe transcript, TAL repressor, GFP mRNA, GFP protein intermediate, and GFP protein. In this model we have taken into account the maturation time of GFP.</br> <br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/9/96/Ucdavismodeling1n.png" width=1019 height=299><br />
<br></br><br />
<img src="https://static.igem.org/mediawiki/2013/4/42/UCDavis_Probofactivecomplex.png" width=1019 height=330><br />
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<img src="https://static.igem.org/mediawiki/2013/c/cc/UCDavis_Testing_odes.png" width=938 height=520><br />
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<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#equations">Equations</a></li><br />
<li><a href="#parameters">Parameters</a></li><br />
<li><a href="#MATLABsim">MATLAB Simulation</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="parameters"></a><br />
<h1>Parameters <a href="#top" class="to_top">^back to top</a></h1><br />
Included here are the parameters used in this model. Please refer to the <a href="#References">References</a> section of this page for the source of each parameter value. <br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4e/Ucdavisparameters1.jpg" class="genpic"></center><br />
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<center><img src="https://static.igem.org/mediawiki/2013/d/de/Ucdavisparameters2.jpg" class="genpic"></center><br />
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<center><img src="https://static.igem.org/mediawiki/2013/a/aa/Ucdavisparameters3.jpg" class="genpic"></center><br />
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<a id="MATLABsim"></a><br />
<h1>MATLAB Simulation<a href="https://static.igem.org/mediawiki/2013/8/8b/UCD2013RiboTALe.m"><i>See the Code!</i></a><a href="#top" class="to_top">^back to top</a></h1><br />
<br />
<h3>TALe Binding Site K<sub>D</sub> As a Source of Tunability</h3><br />
<br>Each state variable in the system of ODEs was given an initial condition of 0. The dynamic response of the system was calculated and plotted over a time span of 10 hours. The results of the model support our data in that the RiboTALe with the larger dissociation constant (RiboTALe 1) is less effective at repressing GFP than RiboTALe 8 under the same induction conditions. The peak seen in the dynamic response of both simulations is a result of the kinematics of the system; there is lag between the initiation of GFP production and when the concentration of active TAL repressors is enough to tip the system.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/0/0a/Ucdavismodel2.png" class="genpic"></center><br />
<br>It should be noted that "less effective" does not mean that RiboTALe 1 is an inferior part, merely that it generates a distinct system response and displays kinematic behavior that may be specifically needed in a future circuit design. We have demonstrated that through their engineerable tunability RiboTALes are capable of achieving a broad range of system responses, a conclusion that is supported by this model. </br><br />
<br></br><br />
<h3>RiboTALe Modulation Through Theophylline Induction Levels</h3><br />
<br>This simulation was carried out under the same conditions defined above, but interrogated only one RiboTALe, RiboTALe 1 with a K<sub>D</sub> of 240 nM. The concentration of theophylline, however, was varied over a range of 1 mM to 10 mM and the results plotted. This simulation also supports our data in that it is clear that the riboswitch is, in fact, responsive to theophylline and that final GFP counts are inversely proportional to the amount of theophylline added.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/2c/Ucdavismodel3.png"class="genpic"></center><br />
<br>Future work with this model may involve the simulation of RiboTALe activity under different non-theophylline riboswitches and an investigation of the orthogonality achievable when using multiple riboswitches in a system.</br><br />
<br></br><br />
<h3>Amplifying System Response Through Transcript Induction</h3> <br />
<br>To investigate the effects of increasing GFP transcript while maintaining constant levels of arabinose and theophylline, the dynamic response of the system under RiboTALe 1 repression was simulated for aTc levels of 0, 25 ng/mL, and 100 ng/mL, where aTC is the inducer of the GFP transcript. The model results show the expected behavior: at higher concentrations of aTc GFP reaches greater peak concentration before repression by the RiboTALe becomes evident. Moreover, this event occurs later in the simulation under conditions of 100 ng/mL of aTc than it does for the other two simulated responses.</br><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/c/c9/Ucdavismodel4.png"></center><br />
<br></br><br />
This model can be further developed to take into account riboswitch leakiness and system stochasticity, and the parameters fine-tuned. It is, however, a useful model in that it provides a mathematical basis that supports the functionality of our RiboTALe devices and shows the wide variety of system responses achievable through the modulation of the engineerable and tunable elements of our construct. We tested combinations of two TAL repressors and two theophylline riboswitches. With this model we will be able to predict the response of a library of RiboTALes, composed a much greater variety of riboswitches and TAL repressors, and perhaps identify with which combination and under what induction conditions a desired system response may be achieved. <br />
</div><br />
<br />
<div class="floatboxwide" id="References"><br />
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<h3>100 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1.16158623970914, 21.6609858203536,, 10.7238390098074, 0.0296612965982997</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1.16158623970914,<br />
1.38324321672162,<br />
1.56787760577646,<br />
3.44344634823164,<br />
28.1550374713881,<br />
1.12263448435245,<br />
1.23446651043791,<br />
1.37349621495727,<br />
2.91488533171903,<br />
21.6609858203536,<br />
0.681669891981254,<br />
1.2548618440377,<br />
1.55449707407215,<br />
4.34727847535614,<br />
19.9321910930205,<br />
0.838126720409999,<br />
0.7232594704811,<br />
1.67616077719149,<br />
6.75843601081123,<br />
21.5221383017144,<br />
0.761406719776734,<br />
0.955255402108518,<br />
0.700049421274729,<br />
10.7238390098074,<br />
0.485471902706462,<br />
0.0196404111837095,<br />
0.0296612965982997,<br />
0.0313199181732694,<br />
0.108325822385663,<br />
0.442737316405862,<br />
<br />
<br />
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25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
1311.329197,4760.869684,27024.29053,1093.892416,4443.585781,38823.82162,<br />
2008.220704,2791.712027,4832.427592,1998.827702,3182.527189,4394.124731--><br />
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5461, 6111, 7360, 2909, 2919, 7100--><br />
</span><br />
<span class='stdevs_3d'><br />
</span><br />
<br />
<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
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1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
<!--2369.383411,2422.018312,2387.445279,2327.26362,2303.398056,2254.056792,<br />
2345.828233,2307.888693,2292.350333,2258.230481,2233.40043,2104.700912,<br />
2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
1551.596136,1338.209982,1205.165033,1125.333333,1165.549228,1147.629325--><br />
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<span class='stdevs_3d'><br />
</span><br />
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<br />
<br />
<div class="floatboxwide" id="References"><br />
<h1>References<a href="#top" class="to_top">^back to top</a></h1><br />
<br>[1] <a href="http://bionumbers.hms.harvard.edu/KeyNumbers.aspx?redirect=false">"Key Numbers for Cell Biologists." Bionumbers: The Database of Useful Biological Numbers</a></h1></br><br />
<br>[2] <a href="http://openwetware.org/images/5/5b/NORgate%2BChemWires_SuppInfo_nature09565-s1.pdf">Tamsir et al. 'Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’: Supplementary Information'. Nature 469, 212–215 (13 January 2011)</a></hi></br><br />
<br>[3] <a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a></br><br />
<br>[4] Adjusted from <a href="http://nar.oxfordjournals.org/content/early/2012/12/25/nar.gks1330.full">literature</a></hi> to fit our data.</br><br />
<br>[5] <a href="http://parts.igem.org/Part:BBa_K750000">"Part:BBa_K750000" The Registry of Standard Biological Parts </a></hi></br> <br />
<br>[6] <a href="http://www.ncbi.nlm.nih.gov/pubmed/8707053">Cormack et al. 'FACS-optimized mutants of the green fluorescent protein (GFP).' Gene. 1996;173(1 Spec No):33-8.</a></hi></br><br />
</div> <br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/DataTeam:UC Davis/Data2013-10-28T23:35:49Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<div class="floatbox"><br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
</div--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p></a><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#graph1">RiboTALe Activity</a></li><br />
<li><a href="#graph2">TALe Tunability</a></li><br />
<li><a href="#graph3">Riboswitch Activation</a></li><br />
<li><a href="#widget">RiboTALe KO3D</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1 id="studies">Proof of Concept: Our Testing Construct</h1><br />
<br>To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Segal Lab and Tagkopoulos Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies <a href="http://www.ncbi.nlm.nih.gov/pubmed/19033367">A flow cytometry-based screen for synthetic riboswitches</a><a href="#ref"> [1]</a>, and <a href="http://aem.asm.org/content/76/23/7881.abstract">Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species</a></hi><a href="#ref"> [2]</a>. The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.</br><br />
<br>We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.</br><br />
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<br>We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.</br><br />
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<h1 id="graph1">Translation is modulated by theophylline concentrations <a href="#top" class="to_top">^back to top</a></h1><br />
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<center><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/UCDavis_graph1c.png/800px-UCDavis_graph1c.png"></center><br />
<br></br><br>We subjected <a href="http://parts.igem.org/Part:BBa_K1212015">our testing construct</a></h1> to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. We also subjected the construct to the induction condition of 0.1% arabinose, which would produce a nominal level of RiboTALe transcript. We varied only the concentration of theophylline, over a range of 0 mM to 10 mM. Thus, difference in fluorescence between induction conditions would be due only to the RiboTALe repression activity. We measured the fluorescence of our construct in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Protocols">Protocols</a></hi> page for details on our culture preparation and Tecan testing parameters. </br><br />
<p id="itworks"><br />
<br />The image above illustrates that GFP fluorescence is inversely related to theophylline concentrations, indicating that the <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor</a></hi> is in fact being translated at rates corresponding to the theophylline induction levels, and effectively binding to its target site. At maximal theophylline concentrations, the expression of GFP is reduced 2.6 fold.<br />
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<p><br />
<br />Next, we investigated what difference in system response we could achieve by altering the binding affinity of the TAL repressor protein.<br />
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<h1 id="graph2">Binding affinities of the TAL repressors provide tunability<a href="#top" class="to_top">^back to top</a></h1> <br />
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<center><img src="https://static.igem.org/mediawiki/2013/thumb/e/ed/UCDavis_graph2dn.png/799px-UCDavis_graph2dn.png"></center><br />
<br></br><br>The <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor protein</a></hi> expressed by our <a href="http://parts.igem.org/Part:BBa_K1212015">RiboTALe device</a></hi> in our initial experiment has a dissociation constant K<sub>D</sub> 1.3 &plusmn; .03 nM. We compared the activity of this RiboTALe to one under the control of the same <a href="http://parts.igem.org/Part:BBa_K1212001">theophylline riboswitch</a></hi>, but that expressed a <a href="http://parts.igem.org/Part:BBa_K1212004">TAL repressor</a></hi> with K<sub>D</sub> 240 &plusmn; 40 nM. We subjected both RiboTALes to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:</br><br />
<br></br><br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
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<br></br> <br>The image above displays the peak fluorescence of two RiboTALe constructs, <a href="http://parts.igem.org/Part:BBa_K1212014">one</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212004">TALe 1</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212015">other</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.</br><br />
<br>It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the <a href="http://parts.igem.org/Part:BBa_K1212001">riboswitch</a></hi> we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.</br><br />
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<h1 id="graph3">Riboswitch leakiness modulates RiboTALe activity<a href="#top" class="to_top">^back to top</a></h1><br />
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<center><img src="https://static.igem.org/mediawiki/2013/thumb/2/2b/UCDavis_graph3en.png/800px-UCDavis_graph3en.png"></center><!--The study <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615613/">A flow cytometry-based screen for synthetic riboswitches</a></hi> by Sean Lynch and Justin Gallivan <a href="#ref">[1]</a> presents a library of theophylline riboswitches with randomized 8 base pair sequences in the Shine-Dalgarno region of the transcript that were screened for riboswitch behavior.--><br></br><br />
We proceeded to investigate the differences achievable in RiboTALe system response by varying the riboswitch controlling the translation of the TAL repressor. To this end we tested two RiboTALe devices, both of which expressed <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>. <a href="http://parts.igem.org/Part:BBa_K1212015">One</a></hi> of these RiboTALes was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212012">other</a> was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a></hi>. We subjected both RiboTALes to the induction condition of 100 ng/uL aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:<br />
<br></br><br />
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<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
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The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios<a href="#ref">[1,2]</a>. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is active. Under identical induction conditions, the RiboTALe under riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a> exhibits no repression activity. The fluorescence measured was in fact <i>higher</i> than the baseline for reasons not understood. From the data we conclude that <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is leakier and yet stronger than <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a>, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability. <br />
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<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
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<h3>100 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
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<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
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89341.08341, 88566.62349, 89727.21455, 58754.65109, 55356.67872, 59500.96347,<br />
71372.62047, 72710.06759, 74182.52136, 39969.45281, 41701.09012, 58907.74009,<br />
22868.50882, 26697.24765, 31385.92672, 12146.13773, 13292.34931, 29857.02244<br />
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25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
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<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
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<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
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1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
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2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[2] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/DataTeam:UC Davis/Data2013-10-28T23:31:19Z<p>Arneckelmann: </p>
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
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<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
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<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#graph1">RiboTALe Activity</a></li><br />
<li><a href="#graph2">TALe Tunability</a></li><br />
<li><a href="#graph3">Riboswitch Activation</a></li><br />
<li><a href="#widget">RiboTALe KO3D</a></li><br />
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<h1 id="studies">Proof of Concept: Our Testing Construct</h1><br />
<br>To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Segal Lab and Tagkopoulos Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies <a href="http://www.ncbi.nlm.nih.gov/pubmed/19033367">A flow cytometry-based screen for synthetic riboswitches</a><a href="#ref"> [1]</a>, and <a href="http://aem.asm.org/content/76/23/7881.abstract">Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species</a></hi><a href="#ref"> [2]</a>. The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.</br><br />
<br>We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.</br><br />
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<br>We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.</br><br />
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<h1 id="graph1">Translation is modulated by theophylline concentrations <a href="#top" class="to_top">^back to top</a></h1><br />
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<center><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/UCDavis_graph1c.png/800px-UCDavis_graph1c.png"></center><br />
<br></br><br>We subjected <a href="http://parts.igem.org/Part:BBa_K1212015">our testing construct</a></h1> to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. We also subjected the construct to the induction condition of 0.1% arabinose, which would produce a nominal level of RiboTALe transcript. We varied only the concentration of theophylline, over a range of 0 mM to 10 mM. Thus, difference in fluorescence between induction conditions would be due only to the RiboTALe repression activity. We measured the fluorescence of our construct in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Protocols">Protocols</a></hi> page for details on our culture preparation and Tecan testing parameters. </br><br />
<p id="itworks"><br />
<br />The image above illustrates that GFP fluorescence is inversely related to theophylline concentrations, indicating that the <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor</a></hi> is in fact being translated at rates corresponding to the theophylline induction levels, and effectively binding to its target site. At maximal theophylline concentrations, the expression of GFP is reduced 2.6 fold.<br />
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<p><br />
<br />Next, we investigated what difference in system response we could achieve by altering the binding affinity of the TAL repressor protein.<br />
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<h1 id="graph2">Binding affinities of the TAL repressors provide tunability<a href="#top" class="to_top">^back to top</a></h1> <br />
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<center><img src="https://static.igem.org/mediawiki/2013/thumb/e/ed/UCDavis_graph2dn.png/799px-UCDavis_graph2dn.png"></center><br />
<br></br><br>The <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor protein</a></hi> expressed by our <a href="http://parts.igem.org/Part:BBa_K1212015">RiboTALe device</a></hi> in our initial experiment has a dissociation constant K<sub>D</sub> 1.3 &plusmn; .03 nM. We compared the activity of this RiboTALe to one under the control of the same <a href="http://parts.igem.org/Part:BBa_K1212001">theophylline riboswitch</a></hi>, but that expressed a <a href="http://parts.igem.org/Part:BBa_K1212004">TAL repressor</a></hi> with K<sub>D</sub> 240 &plusmn; 40 nM. We subjected both RiboTALes to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:</br><br />
<br></br><br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
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<br></br> <br>The image above displays the peak fluorescence of two RiboTALe constructs, <a href="http://parts.igem.org/Part:BBa_K1212014">one</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212004">TALe 1</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212015">other</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.</br><br />
<br>It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the <a href="http://parts.igem.org/Part:BBa_K1212001">riboswitch</a></hi> we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.</br><br />
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<h1 id="graph3">Riboswitch leakiness modulates RiboTALe activity<a href="#top" class="to_top">^back to top</a></h1><br />
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<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/2/2b/UCDavis_graph3en.png/800px-UCDavis_graph3en.png"></center><!--The study <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615613/">A flow cytometry-based screen for synthetic riboswitches</a></hi> by Sean Lynch and Justin Gallivan <a href="#ref">[1]</a> presents a library of theophylline riboswitches with randomized 8 base pair sequences in the Shine-Dalgarno region of the transcript that were screened for riboswitch behavior.--><br></br><br />
We proceeded to investigate the differences achievable in RiboTALe system response by varying the riboswitch controlling the translation of the TAL repressor. To this end we tested two RiboTALe devices, both of which expressed <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>. <a href="http://parts.igem.org/Part:BBa_K1212015">One</a></hi> of these RiboTALes was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212012">other</a> was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a></hi>. We subjected both RiboTALes to the induction condition of 100 ng/uL aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:<br />
<br></br><br />
<br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br><br />
The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios<a href="#ref">[1,2]</a>. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is active. Under identical induction conditions, the RiboTALe under riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a> exhibits no repression activity. The fluorescence measured was in fact <i>higher</i> than the baseline for reasons not understood. From the data we conclude that <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is leakier and yet stronger than <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a>, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability. <br />
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<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
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<h3>100 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
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<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
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<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
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<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
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1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
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1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
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<div class="playme"><br />
<p id="play">Play With Me</p><br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
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</td><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[2] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_DavisTeam:UC Davis2013-10-28T23:03:45Z<p>Arneckelmann: </p>
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<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br /><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoter"><img src="https://static.igem.org/mediawiki/2012/e/ee/Svn12_hp_new.png" class="checkmark"></a><p>We also made the Anderson promoter family controllable through induction.</p><br />
</p><br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
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<img src="https://static.igem.org/mediawiki/2013/2/2a/IGEM_2013_Group_Photo.png" width="200" height="300" align="left" class="genpic"><br />
<!--<img src="https://static.igem.org/mediawiki/2013/f/fd/UCDavisMeettheteam.gif" width="200" height="300" align="left" class="genpic">--><br />
Welcome to the 2013 UC Davis iGEM Wiki!<br></br>We have a created a novel class of transcription factors known as RiboTALs. We sought to address the constraints placed on circuit design by the limited number of well characterized promoters at our disposal, and their respective transcription factors. Our device is a hybrid system composed of two parts: Transcriptional Activator-Like (TAL) effectors which can be engineered to bind to and repress any sequence of interest and riboswitches that can respond to any inducer molecule due to their engineerable and modular aptamer binding domains. <br></br> We also designed and implemented a Biobrick characterization database called The Depot to promote sharing and openness in iGEM.</p><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_DavisTeam:UC Davis2013-10-28T22:46:43Z<p>Arneckelmann: </p>
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<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br /><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark"> <p>We also made the Anderson promoter family controllable by induction.</p><br />
</p><br />
</td><br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
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<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
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<!--<img src="https://static.igem.org/mediawiki/2013/f/fd/UCDavisMeettheteam.gif" width="200" height="300" align="left" class="genpic">--><br />
Welcome to the 2013 UC Davis iGEM Wiki!<br></br>We have a created a novel class of transcription factors known as RiboTALs. We sought to address the constraints placed on circuit design by the limited number of well characterized promoters at our disposal, and their respective transcription factors. Our device is a hybrid system composed of two parts: Transcriptional Activator-Like (TAL) effectors which can be engineered to bind to and repress any sequence of interest and riboswitches that can respond to any inducer molecule due to their engineerable and modular aptamer binding domains. <br></br> We also designed and implemented a Biobrick characterization database called The Depot to promote sharing and openness in iGEM.</p><br />
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</html></div>Arneckelmannhttp://2013.igem.org/File:UCD_2013_new_icon.PNGFile:UCD 2013 new icon.PNG2013-10-28T22:30:12Z<p>Arneckelmann: </p>
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<div></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:55:35Z<p>Arneckelmann: </p>
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<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
<div id="sitemapbox" class="floatbox"><br />
</div><br />
<br />
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</body><br />
<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:41:53Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#itworks{font-weight:bolder;<br />
color:#ffff99;}<br />
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left:-400px;<br />
top:110px;<br />
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visibility:hidden; }<br />
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</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
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$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");<br />
</script></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:40:06Z<p>Arneckelmann: </p>
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<div>{{Team:UC_Davis/Head}}<br />
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<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
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<div class="floatbox"><br />
<table class = "showbox"><br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /> <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
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<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
</body><br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/DataTeam:UC Davis/Data2013-10-28T00:29:54Z<p>Arneckelmann: </p>
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{{Team:UC_Davis/KO3D}}<br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
</div--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p></a><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#graph1">RiboTALe Activity</a></li><br />
<li><a href="#graph2">TALe Tunability</a></li><br />
<li><a href="#graph3">Riboswitch Activation</a></li><br />
<li><a href="#widget">RiboTALe KO3D</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1 id="studies">Proof of Concept: Our Testing Construct</h1><br />
<br>To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Segal Lab and Tagkopoulos Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies <a href="http://www.ncbi.nlm.nih.gov/pubmed/19033367">A flow cytometry-based screen for synthetic riboswitches</a><a href="#ref"> [1]</a>, and <a href="http://aem.asm.org/content/76/23/7881.abstract">Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species</a></hi><a href="#ref"> [2]</a>. The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.</br><br />
<br>We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.</br><br />
<br></br> <br />
<center><img src="https://static.igem.org/mediawiki/2013/6/61/UCDAVIStestTOP.gif" class="genpic" width=345px height=360></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/6/6d/Ucdavisplusarabinose.png" class="genpic" width=488 height=160></center><br />
<br></br><center><img src="https://static.igem.org/mediawiki/2013/7/73/MIDDLE.gif" class="genpic" width=345px height=360></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4a/Ucdavisplustheophylline.png" class="genpic" width=488 height=160></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/2/22/Ucdavis2NDTOBOTTOM.gif" class="genpic" width=345px height=360></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/b/bd/Ucdavistalearrrow.png" class="genpic" width=488 height=160></center><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/5/5d/UCDavisBOTTOM.gif" class="genpic" width=345px height=360></center><br />
<br></br><br />
<br />
<br />
<br>We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.</br><br />
</div><br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="graph1">Translation is modulated by theophylline concentrations <a href="#top" class="to_top">^back to top</a></h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/UCDavis_graph1c.png/800px-UCDavis_graph1c.png"></center><br />
<br></br><br>We subjected <a href="http://parts.igem.org/Part:BBa_K1212015">our testing construct</a></h1> to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. We also subjected the construct to the induction condition of 0.1% arabinose, which would produce a nominal level of RiboTALe transcript. We varied only the concentration of theophylline, over a range of 0 mM to 10 mM. Thus, difference in fluorescence between induction conditions would be due only to the RiboTALe repression activity. We measured the fluorescence of our construct in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Protocols">Protocols</a></hi> page for details on our culture preparation and Tecan testing parameters. </br><br />
<p id="itworks"><br />
<br />The image above illustrates that GFP fluorescence is inversely related to theophylline concentrations, indicating that the <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor</a></hi> is in fact being translated at rates corresponding to the theophylline induction levels, and effectively binding to its target site. At maximal theophylline concentrations, the expression of GFP is reduced 2.6 fold.<br />
</p><br />
<p><br />
<br />Next, we investigated what difference in system response we could achieve by altering the binding affinity of the TAL repressor protein.<br />
<br /><br />
</p><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1 id="graph2">Binding affinities of the TAL repressors provide tunability<a href="#top" class="to_top">^back to top</a></h1> <br />
<br></br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/e/ed/UCDavis_graph2dn.png/799px-UCDavis_graph2dn.png"></center><br />
<br></br><br>The <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor protein</a></hi> expressed by our <a href="http://parts.igem.org/Part:BBa_K1212015">RiboTALe device</a></hi> in our initial experiment has a dissociation constant K<sub>D</sub> 1.3 &plusmn; .03 nM. We compared the activity of this RiboTALe to one under the control of the same <a href="http://parts.igem.org/Part:BBa_K1212001">theophylline riboswitch</a></hi>, but that expressed a <a href="http://parts.igem.org/Part:BBa_K1212004">TAL repressor</a></hi> with K<sub>D</sub> 240 &plusmn; 40 nM. We subjected both RiboTALes to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:</br><br />
<br></br><br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br />
<br></br> <br>The image above displays the peak fluorescence of two RiboTALe constructs, <a href="http://parts.igem.org/Part:BBa_K1212014">one</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212004">TALe 1</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212015">other</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.</br><br />
<br>It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the <a href="http://parts.igem.org/Part:BBa_K1212001">riboswitch</a></hi> we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.</br><br />
<br></br><br />
</div><br />
<div class="floatboxwide"><br />
<h1 id="graph3">Riboswitch leakiness modulates RiboTALe activity<a href="#top" class="to_top">^back to top</a></h1><br />
<br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/2/2b/UCDavis_graph3en.png/800px-UCDavis_graph3en.png"></center><!--The study <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615613/">A flow cytometry-based screen for synthetic riboswitches</a></hi> by Sean Lynch and Justin Gallivan <a href="#ref">[1]</a> presents a library of theophylline riboswitches with randomized 8 base pair sequences in the Shine-Dalgarno region of the transcript that were screened for riboswitch behavior.--><br></br><br />
We proceeded to investigate the differences achievable in RiboTALe system response by varying the riboswitch controlling the translation of the TAL repressor. To this end we tested two RiboTALe devices, both of which expressed <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>. <a href="http://parts.igem.org/Part:BBa_K1212015">One</a></hi> of these RiboTALes was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212012">other</a> was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a></hi>. We subjected both RiboTALes to the induction condition of 100 ng/uL aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:<br />
<br></br><br />
<br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br><br />
The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios<a href="#ref">[1,2]</a>. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is active. Under identical induction conditions, the RiboTALe under riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a> exhibits no repression activity. The fluorescence measured was in fact <i>higher</i> than the baseline for reasons not understood. From the data we conclude that <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is leakier and yet stronger than <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a>, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability. <br />
<br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
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<h3>100 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">81896.13, 78666.88, 74182.52, 31385.93</span><br />
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<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
81399.99914, 82312.01989, 81896.13437, 70965.09262, 71753.41851, 52082.65688,<br />
82598.77679, 90191.2318, 78666.87521, 59658.53566, 53189.6567, 43608.73416,<br />
89341.08341, 88566.62349, 89727.21455, 58754.65109, 55356.67872, 59500.96347,<br />
71372.62047, 72710.06759, 74182.52136, 39969.45281, 41701.09012, 58907.74009,<br />
22868.50882, 26697.24765, 31385.92672, 12146.13773, 13292.34931, 29857.02244<br />
<br />
<!--106247.4518,104406.975,107697.0566,87605.53496,113026.3159,116238.2595,<br />
97390.65405,102272.0717,105314.5467,36495.57538,102836.5068,117957.4598,<br />
25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
1311.329197,4760.869684,27024.29053,1093.892416,4443.585781,38823.82162,<br />
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5461, 6111, 7360, 2909, 2919, 7100--><br />
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<span class='stdevs_3d'><br />
</span><br />
<br />
<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
<!--2369.383411,2422.018312,2387.445279,2327.26362,2303.398056,2254.056792,<br />
2345.828233,2307.888693,2292.350333,2258.230481,2233.40043,2104.700912,<br />
2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
1551.596136,1338.209982,1205.165033,1125.333333,1165.549228,1147.629325--><br />
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<div class="playme"><br />
<p id="play">Play With Me</p><br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
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<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
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<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[2] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
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$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");</script><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/DataTeam:UC Davis/Data2013-10-28T00:27:51Z<p>Arneckelmann: </p>
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<!--img src="https://static.igem.org/mediawiki/2013/b/b8/UCD_2013_Data_Banner.PNG" class="banner" width=967px height=226/--><br />
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<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><br />
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</div--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p></a><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>Find out how we controlled the Anderson family of promoters through induction. <br />
</p><br />
</td> <br />
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<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#graph1">RiboTALe Activity</a></li><br />
<li><a href="#graph2">TALe Tunability</a></li><br />
<li><a href="#graph3">Riboswitch Activation</a></li><br />
<li><a href="#widget">RiboTALe KO3D</a></li><br />
</ul><br />
</div><br />
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<div class="floatbox"><br />
<h1 id="studies">Proof of Concept: Our Testing Construct</h1><br />
<br>To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Segal Lab and Tagkopoulos Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies <a href="http://www.ncbi.nlm.nih.gov/pubmed/19033367">A flow cytometry-based screen for synthetic riboswitches</a><a href="#ref"> [1]</a>, and <a href="http://aem.asm.org/content/76/23/7881.abstract">Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species</a></hi><a href="#ref"> [2]</a>. The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.</br><br />
<br>We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.</br><br />
<br></br> <br />
<center><img src="https://static.igem.org/mediawiki/2013/6/61/UCDAVIStestTOP.gif" class="genpic" width=345px height=360></center><br />
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<center><img src="https://static.igem.org/mediawiki/2013/6/6d/Ucdavisplusarabinose.png" class="genpic" width=488 height=160></center><br />
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<center><img src="https://static.igem.org/mediawiki/2013/4/4a/Ucdavisplustheophylline.png" class="genpic" width=488 height=160></center><br />
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<br />
<br />
<br>We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.</br><br />
</div><br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="graph1">Translation is modulated by theophylline concentrations <a href="#top" class="to_top">^back to top</a></h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/UCDavis_graph1c.png/800px-UCDavis_graph1c.png"></center><br />
<br></br><br>We subjected <a href="http://parts.igem.org/Part:BBa_K1212015">our testing construct</a></h1> to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. We also subjected the construct to the induction condition of 0.1% arabinose, which would produce a nominal level of RiboTALe transcript. We varied only the concentration of theophylline, over a range of 0 mM to 10 mM. Thus, difference in fluorescence between induction conditions would be due only to the RiboTALe repression activity. We measured the fluorescence of our construct in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Protocols">Protocols</a></hi> page for details on our culture preparation and Tecan testing parameters. </br><br />
<p id="itworks"><br />
<br />The image above illustrates that GFP fluorescence is inversely related to theophylline concentrations, indicating that the <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor</a></hi> is in fact being translated at rates corresponding to the theophylline induction levels, and effectively binding to its target site. At maximal theophylline concentrations, the expression of GFP is reduced 2.6 fold.<br />
</p><br />
<p><br />
<br />Next, we investigated what difference in system response we could achieve by altering the binding affinity of the TAL repressor protein.<br />
<br /><br />
</p><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1 id="graph2">Binding affinities of the TAL repressors provide tunability<a href="#top" class="to_top">^back to top</a></h1> <br />
<br></br><br />
<br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/e/ed/UCDavis_graph2dn.png/799px-UCDavis_graph2dn.png"></center><br />
<br></br><br>The <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor protein</a></hi> expressed by our <a href="http://parts.igem.org/Part:BBa_K1212015">RiboTALe device</a></hi> in our initial experiment has a dissociation constant K<sub>D</sub> 1.3 &plusmn; .03 nM. We compared the activity of this RiboTALe to one under the control of the same <a href="http://parts.igem.org/Part:BBa_K1212001">theophylline riboswitch</a></hi>, but that expressed a <a href="http://parts.igem.org/Part:BBa_K1212004">TAL repressor</a></hi> with K<sub>D</sub> 240 &plusmn; 40 nM. We subjected both RiboTALes to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:</br><br />
<br></br><br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br />
<br></br> <br>The image above displays the peak fluorescence of two RiboTALe constructs, <a href="http://parts.igem.org/Part:BBa_K1212014">one</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212004">TALe 1</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212015">other</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.</br><br />
<br>It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the <a href="http://parts.igem.org/Part:BBa_K1212001">riboswitch</a></hi> we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.</br><br />
<br></br><br />
</div><br />
<div class="floatboxwide"><br />
<h1 id="graph3">Riboswitch leakiness modulates RiboTALe activity<a href="#top" class="to_top">^back to top</a></h1><br />
<br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/2/2b/UCDavis_graph3en.png/800px-UCDavis_graph3en.png"></center><!--The study <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615613/">A flow cytometry-based screen for synthetic riboswitches</a></hi> by Sean Lynch and Justin Gallivan <a href="#ref">[1]</a> presents a library of theophylline riboswitches with randomized 8 base pair sequences in the Shine-Dalgarno region of the transcript that were screened for riboswitch behavior.--><br></br><br />
We proceeded to investigate the differences achievable in RiboTALe system response by varying the riboswitch controlling the translation of the TAL repressor. To this end we tested two RiboTALe devices, both of which expressed <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>. <a href="http://parts.igem.org/Part:BBa_K1212015">One</a></hi> of these RiboTALes was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212012">other</a> was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a></hi>. We subjected both RiboTALes to the induction condition of 100 ng/uL aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:<br />
<br></br><br />
<br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br><br />
The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios<a href="#ref">[1,2]</a>. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is active. Under identical induction conditions, the RiboTALe under riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a> exhibits no repression activity. The fluorescence measured was in fact <i>higher</i> than the baseline for reasons not understood. From the data we conclude that <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is leakier and yet stronger than <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a>, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability. <br />
<br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">90000</span><br />
<h3>100 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">81896.13, 78666.88, 74182.52, 31385.93</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
81399.99914, 82312.01989, 81896.13437, 70965.09262, 71753.41851, 52082.65688,<br />
82598.77679, 90191.2318, 78666.87521, 59658.53566, 53189.6567, 43608.73416,<br />
89341.08341, 88566.62349, 89727.21455, 58754.65109, 55356.67872, 59500.96347,<br />
71372.62047, 72710.06759, 74182.52136, 39969.45281, 41701.09012, 58907.74009,<br />
22868.50882, 26697.24765, 31385.92672, 12146.13773, 13292.34931, 29857.02244<br />
<br />
<!--106247.4518,104406.975,107697.0566,87605.53496,113026.3159,116238.2595,<br />
97390.65405,102272.0717,105314.5467,36495.57538,102836.5068,117957.4598,<br />
25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
1311.329197,4760.869684,27024.29053,1093.892416,4443.585781,38823.82162,<br />
2008.220704,2791.712027,4832.427592,1998.827702,3182.527189,4394.124731--><br />
<!--27676, 27805, 27124, 20736, 22057, 19281,<br />
25713, 27355, 25079, 18345, 15425, 14779,<br />
25355, 26322, 26972, 17362, 17072, 18481,<br />
18771, 19210, 21053, 10468, 11105, 16665,<br />
5461, 6111, 7360, 2909, 2919, 7100--><br />
</span><br />
<span class='stdevs_3d'><br />
</span><br />
<br />
<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
<!--2369.383411,2422.018312,2387.445279,2327.26362,2303.398056,2254.056792,<br />
2345.828233,2307.888693,2292.350333,2258.230481,2233.40043,2104.700912,<br />
2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
1551.596136,1338.209982,1205.165033,1125.333333,1165.549228,1147.629325--><br />
</span><br />
<span class='stdevs_3d'><br />
</span><br />
</div><br />
<br />
<div class="playme"><br />
<p id="play">Play With Me</p><br />
</div><br />
</div><br />
<br />
<br />
<!--Begin Showbox--><br />
<div class="floatbox"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
</tr><br />
</table><br />
<br />
</div><br />
<!--End Showbox--><br />
<br />
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<div class="floatboxwide"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[2] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
</p><br />
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</div><br />
<br />
</body><br />
<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");</script><br />
<br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:19:34Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#itworks{font-weight:bolder;<br />
color:#ffff99;}<br />
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#play {position:relative;<br />
left:-400px;<br />
top:110px;<br />
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}<br />
#mutantwidget:hover + .playme{ <br />
visibility:hidden; }<br />
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</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /> <br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
</body><br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:16:06Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#itworks{font-weight:bolder;<br />
color:#ffff99;}<br />
<br />
#play {position:relative;<br />
left:-400px;<br />
top:110px;<br />
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}<br />
#mutantwidget:hover + .playme{ <br />
visibility:hidden; }<br />
<br />
</style><br />
</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src=" " class="blur"></a--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/6/64/UCD_RiboTAL_Icon_v2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /> <br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
</body><br />
</html></div>Arneckelmannhttp://2013.igem.org/File:UCD_RiboTAL_Icon_v2.PNGFile:UCD RiboTAL Icon v2.PNG2013-10-28T00:15:17Z<p>Arneckelmann: </p>
<hr />
<div></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:11:10Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#itworks{font-weight:bolder;<br />
color:#ffff99;}<br />
<br />
#play {position:relative;<br />
left:-400px;<br />
top:110px;<br />
font-size:30pt;<br />
}<br />
#mutantwidget:hover + .playme{ <br />
visibility:hidden; }<br />
<br />
</style><br />
</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src=" " class="blur"></a--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/da/AndersonRiboTAL_Iconv2.PNG" class="blur"></a></div><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /> <br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
</body><br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-28T00:09:43Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#itworks{font-weight:bolder;<br />
color:#ffff99;}<br />
<br />
#play {position:relative;<br />
left:-400px;<br />
top:110px;<br />
font-size:30pt;<br />
}<br />
#mutantwidget:hover + .playme{ <br />
visibility:hidden; }<br />
<br />
</style><br />
</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src=" " class="blur"></a--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/da/AndersonRiboTAL_Iconv2.PNG" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /> <br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
</body><br />
</html></div>Arneckelmannhttp://2013.igem.org/File:AndersonRiboTAL_Iconv2.PNGFile:AndersonRiboTAL Iconv2.PNG2013-10-28T00:07:35Z<p>Arneckelmann: </p>
<hr />
<div></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/AndersonPromotersTeam:UC Davis/AndersonPromoters2013-10-27T19:58:44Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
{{Team:UC_Davis/KO3D}}<br />
<html><br />
<head><br />
<style type="text/css"><br />
#itworks{font-weight:bolder;<br />
color:#ffff99;}<br />
<br />
#play {position:relative;<br />
left:-400px;<br />
top:110px;<br />
font-size:30pt;<br />
}<br />
#mutantwidget:hover + .playme{ <br />
visibility:hidden; }<br />
<br />
</style><br />
</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/a/a8/UCDavis_databanner.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatbox"><br />
<table class = "showbox"><br />
<tr><br />
<td><br />
<!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src=" " class="blur"></a--><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a><br />
<p>Check out our initial experiments with our testing constructs.<br />
</p><br />
</td> <br />
<td><br />
<!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/c/c0/AndersonRiboTAL_Icon.PNG" class="blur"></a--><br />
<a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a><br />
<p>In progress. <br />
</p><br />
</td> <br />
</tr><br />
</table><br />
</div><br />
<br />
<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
</ul><br />
</div><br />
<br />
<div class="floatbox"><br />
<h1>Targeting the Anderson Promoters</h1><br />
<p> After proving that our <a href="https://2013.igem.org/Team:UC_Davis/Data#graph1">RiboTALs worked</a> as transcription factors for an already inducible expression system with pTet upstream of our TALe binding sites corresponding to the TAL repressors used in our characterization experiments and a reporter, GFP, we decided to next target constitutive promoters that have no other form of inducible control. We are targeting the well characterized Anderson Promoter Family. With their known relative activities, we hope we can achieve predictable system responses from these promoters when placed upstream of GFP and under the control of our RiboTALs. <br /> <br /><br />
<br />
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, <br />
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our <a href="http://parts.igem.org/Part:BBa_K1212012">construct</a> containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD <a href="#ref">[1]</a>.<br />
<br /> <br /> <br /> <br /><br />
<br />
Similarly to our initial testing constructs, we tested our Anderson promoter and RiboTAL constructs by subjecting the pBAD promoter and the theophylline riboswitch to a range of induction levels with arabinose and theophylline, respectively. It was expected that at low levels of arabinose and theophylline, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter and the theophylline riboswitch. <br /><br />
Unlike our initial testing constructs, we expected to see GFP expression vary with promoter strength. A promoter with a larger relative strength should overall show greater fluorescence levels than one with a smaller relative strength. We used the table of Variant RFP (au) values from the Anderson promoter pages as our measure for the relative strengthes of the promoters we used. <br />
</p><br />
</div><br />
<br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
<div id="mutantwidget" class="floatbox"><br />
<span class="dataMax">80000</span><br />
<h3>J23100</h3><br />
<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose--><br />
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span><br />
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556,<br />
71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523,<br />
66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922,<br />
66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,<br />
1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852<br />
</span><br />
<span class='stdevs_3d'><br />
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,<br />
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,<br />
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,<br />
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,<br />
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588<br />
</span><br />
<br />
<h3>J23101</h3> <!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span><br />
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,<br />
56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732,<br />
47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869,<br />
21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331,<br />
2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202<br />
</span><br />
<span class='stdevs_3d'><br />
174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805,<br />
1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162,<br />
938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525,<br />
733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417,<br />
59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474<br />
</span><br />
<br />
<h3>J23105</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">7533.047431,6370.331682,5990.223684,2922.509781,2186.773466<br />
<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038--><br />
</span><br />
<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637<br />
<!--84.42209965,49.33864245,131.2734258,86.18750714,50.51754508--></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113,<br />
7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523,<br />
6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175,<br />
3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258,<br />
2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702<br />
<br />
<!--5718.361606,5169.127949,5031.489167,4634.991328,4083.204797,2655.861704,<br />
3718.355779,3580.669712,3510.447702,3335.670901,2910.032655,2826.228369,<br />
3460.931882,3449.533885,3345.751695,3216.095915,2763.955863,2704.578296,<br />
3381.493036,3249.669019,3188.787952,3043.687531,2651.760386,2787.069081,<br />
2998.026717,2779.749183,3046.043038,2811.939279,3205.590598,2961.0219--><br />
</span><br />
<span class='stdevs_3d'><br />
901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122,<br />
173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492,<br />
131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,<br />
237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,<br />
28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289<br />
<br />
<!--151.4713363,49.99710014,84.42209965,210.3718956,341.3775726,251.1386117,<br />
250.4062843,38.58019825,49.33864245,195.9502595,127.0445151,438.3061591,<br />
174.2089269,26.34361885,131.2734258,13.80571526,120.6585221,68.21636888,<br />
34.72855284,87.29251749,86.18750714,37.88522959,230.3528339,90.41846107,<br />
34.72644874,14.18299119,50.51754508,56.20927247,84.87382334,126.8705022--><br />
</span><br />
<br />
<h3>J23106</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span><br />
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,<br />
1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909,<br />
1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199,<br />
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,<br />
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572<br />
</span><br />
<span class='stdevs_3d'><br />
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,<br />
35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047,<br />
21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,<br />
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,<br />
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793<br />
</span><br />
<br />
<h3>J23109</h3><!--0.1% arabinose--><br />
<span class="xdata">0, 1, 2, 5, 10</span><br />
<span class="ydata">1037.815719,1003.632865,997.6508013,912.070168,664.1533463</span><br />
<span class="stdevs">6.533826992,7.610089852,12.17300425,16.37293291,0.78231349</span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544,<br />
861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299,<br />
599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452,<br />
1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409,<br />
655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495<br />
</span><br />
<span class='stdevs_3d'><br />
6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733,<br />
12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657,<br />
334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358,<br />
204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375,<br />
15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204<br />
</span><br />
<br />
</div><br />
</div><br />
<br />
<div class="floatbox"><br />
<a id="ref"></a><h3>References</h3><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
</p><br />
</div><br />
</body><br />
</html></div>Arneckelmannhttp://2013.igem.org/File:AndersonRiboTAL_Icon.PNGFile:AndersonRiboTAL Icon.PNG2013-10-27T19:57:44Z<p>Arneckelmann: </p>
<hr />
<div></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/PartsTeam:UC Davis/Parts2013-10-27T06:12:49Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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font-family: verdana;<br />
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font-style:normal;<br />
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<div><br />
<img src="https://static.igem.org/mediawiki/2013/2/2b/Partsbanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1>Parts</h1><br />
The following parts were assembled throughout the development of this project:<br />
<br></br><br />
<center>Well-Characterized Parts</center><br />
<table id="myTable" class=" "><br />
<br />
<thead><br />
<tr><br />
<th>&nbsp;Name</th><br />
<th>&nbsp;Type</th><br />
<th>&nbsp;Description</th><br />
<th>&nbsp;Designer</th><br />
<th>&nbsp;Length&nbsp; </th><br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212011">&nbsp;BBa_K1212011</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+TAL1</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2612</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212012">&nbsp;BBa_K1212012</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+TAL8</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2616</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212013">&nbsp;BBa_K1212013</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pTet+TBS 1+RBS+GFP+T</td><br />
<td>&nbsp;Aura Ferreiro</td><br />
<td>&nbsp;1003</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212014">&nbsp;BBa_K1212014</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 1+TAL 1</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2616</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212015">&nbsp;BBa_K1212015</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 1+TAL 8</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2620</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212016">&nbsp;BBa_K1212016</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pTet+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1003</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212021">&nbsp;BBa_K1212021</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23100+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="darkColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212022">&nbsp;BBa_K1212022</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23101+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;974</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212023">&nbsp;BBa_K1212023</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23105+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;974</td><br />
</tr><br />
<br />
<tr class="darkColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212024">&nbsp;BBa_K1212024</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23106+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212025">&nbsp;BBa_K1212025</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23109+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;974</td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br></br><br />
<center>Other Parts</center><br />
<table id="myTable" class=" "><br />
<br />
<thead><br />
<tr><br />
<th>&nbsp;Name</th><br />
<th>&nbsp;Type</th><br />
<th>&nbsp;Description</th><br />
<th>&nbsp;Designer</th><br />
<th>&nbsp;Length&nbsp; </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212002">&nbsp;BBa_K1212002&nbsp;</a></td><br />
<td>&nbsp;Plasmid_Backbone&nbsp;</td><br />
<td>&nbsp;Golden Gate Compatible pSB3K3&nbsp;</td><br />
<td>Alex Neckelmann</td><br />
<td>&nbsp;2750&nbsp;</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212006">&nbsp;BBa_K1212006</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Golden Gate Compatible GFP</td><br />
<td>&nbsp;Aura Ferreiro</td><br />
<td>&nbsp;1052</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212009">&nbsp;BBa_K1212009</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+GFP+T</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1086</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212010">&nbsp;BBa_K1212010</a></td><br />
<td>&nbsp;Composite </td><br />
<td>&nbsp;pBAD+Riboswitch 1+GFP+T</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1090</td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="floatboxwide"><br />
<p><br />
Our best characterized parts are our RiboTAL constructs, which consist of parts BBa_K1212011-BBa_K1212016. Among these parts, most of characterization was focused on the riboswitch+TAL constructs (BBa_K1212011, BBa_K1212012, BBa_K1212014, BBa_K1212015), as the other parts were simply pTet+GFP, which are already well characterized parts, with some extra base pairs added for our TAL binding sites (BBa_K1212013, BBa_K1212016). Both our pSB3K3 backbone (BBa_K1212002) and our GFP (BBa_K1212006) with their BsaI sites fixed were used in our RiboTAL constructs and functioned as expected but did not need separate characterization. The pBAD+Riboswitch+GFP parts (BBa_K1212009 and BBa_K1212010) were originally planned for testing our riboswitches in case the RiboTAL constructs were not working. Since the RiboTALs did work as expected, we focused on characterizing the RiboTALs over the riboswitch testing constructs.<br />
</p><br />
</div><br />
<br />
<!--Begin Showbox--><br />
<div class="floatboxwide"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
<br />
<br />
</tr><br />
</table><br />
<br />
</div><br />
<!--End Showbox--><br />
<br />
<br />
<div id="sitemapbox" class="floatboxwide"></div><br />
</div><br />
<br />
</body><br />
<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");</script><br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/PartsTeam:UC Davis/Parts2013-10-27T06:07:22Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<br />
<html><br />
<head><br />
<style><br />
<br />
#footer-box{display:none;}<br />
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#myTable {<br />
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font-size: 9.6px;<br />
font-family: verdana;<br />
font-weight:normal;<br />
font-style:normal;<br />
background: #c0c0c0;<br />
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margin-bottom:10px;<br />
border-radius:4px;<br />
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#myTable tfoot tr {<br />
color:white;<br />
background: #333333;<br />
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background: #f0f0f0;<br />
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color: #002bb8}<br />
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</head><br />
<body><br />
<br />
<div><br />
<img src="https://static.igem.org/mediawiki/2013/2/2b/Partsbanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1>Parts</h1><br />
The following parts were assembled throughout the development of this project:<br />
<br></br><br />
<center>Well-Characterized Parts</center><br />
<table id="myTable" class=" "><br />
<br />
<thead><br />
<tr><br />
<th>&nbsp;Name</th><br />
<th>&nbsp;Type</th><br />
<th>&nbsp;Description</th><br />
<th>&nbsp;Designer</th><br />
<th>&nbsp;Length&nbsp; </th><br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212011">&nbsp;BBa_K1212011</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+TAL1</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2612</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212012">&nbsp;BBa_K1212012</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+TAL8</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2616</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212013">&nbsp;BBa_K1212013</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pTet+TBS 1+RBS+GFP+T</td><br />
<td>&nbsp;Aura Ferreiro</td><br />
<td>&nbsp;1003</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212014">&nbsp;BBa_K1212014</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 1+TAL 1</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2616</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212015">&nbsp;BBa_K1212015</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 1+TAL 8</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2620</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212016">&nbsp;BBa_K1212016</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pTet+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1003</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212021">&nbsp;BBa_K1212021</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23100+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="darkColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212022">&nbsp;BBa_K1212022</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23101+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;974</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212023">&nbsp;BBa_K1212023</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23105+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="darkColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212024">&nbsp;BBa_K1212024</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23106+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212025">&nbsp;BBa_K1212025</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23109+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br></br><br />
<center>Other Parts</center><br />
<table id="myTable" class=" "><br />
<br />
<thead><br />
<tr><br />
<th>&nbsp;Name</th><br />
<th>&nbsp;Type</th><br />
<th>&nbsp;Description</th><br />
<th>&nbsp;Designer</th><br />
<th>&nbsp;Length&nbsp; </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212002">&nbsp;BBa_K1212002&nbsp;</a></td><br />
<td>&nbsp;Plasmid_Backbone&nbsp;</td><br />
<td>&nbsp;Golden Gate Compatible pSB3K3&nbsp;</td><br />
<td>Alex Neckelmann</td><br />
<td>&nbsp;2750&nbsp;</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212006">&nbsp;BBa_K1212006</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Golden Gate Compatible GFP</td><br />
<td>&nbsp;Aura Ferreiro</td><br />
<td>&nbsp;1052</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212009">&nbsp;BBa_K1212009</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+GFP+T</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1086</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212010">&nbsp;BBa_K1212010</a></td><br />
<td>&nbsp;Composite </td><br />
<td>&nbsp;pBAD+Riboswitch 1+GFP+T</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1090</td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="floatboxwide"><br />
<p><br />
Our best characterized parts are our RiboTAL constructs, which consist of parts BBa_K1212011-BBa_K1212016. Among these parts, most of characterization was focused on the riboswitch+TAL constructs (BBa_K1212011, BBa_K1212012, BBa_K1212014, BBa_K1212015), as the other parts were simply pTet+GFP, which are already well characterized parts, with some extra base pairs added for our TAL binding sites (BBa_K1212013, BBa_K1212016). Both our pSB3K3 backbone (BBa_K1212002) and our GFP (BBa_K1212006) with their BsaI sites fixed were used in our RiboTAL constructs and functioned as expected but did not need separate characterization. The pBAD+Riboswitch+GFP parts (BBa_K1212009 and BBa_K1212010) were originally planned for testing our riboswitches in case the RiboTAL constructs were not working. Since the RiboTALs did work as expected, we focused on characterizing the RiboTALs over the riboswitch testing constructs.<br />
</p><br />
</div><br />
<br />
<!--Begin Showbox--><br />
<div class="floatboxwide"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
<br />
<br />
</tr><br />
</table><br />
<br />
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<br />
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</div><br />
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</body><br />
<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");</script><br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/PartsTeam:UC Davis/Parts2013-10-27T05:59:48Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
<br />
<html><br />
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<div><br />
<img src="https://static.igem.org/mediawiki/2013/2/2b/Partsbanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1>Parts</h1><br />
The following parts were assembled throughout the development of this project:<br />
<br></br><br />
<center>Well-Characterized Parts</center><br />
<table id="myTable" class=" "><br />
<br />
<thead><br />
<tr><br />
<th>&nbsp;Name</th><br />
<th>&nbsp;Type</th><br />
<th>&nbsp;Description</th><br />
<th>&nbsp;Designer</th><br />
<th>&nbsp;Length&nbsp; </th><br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212011">&nbsp;BBa_K1212011</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+TAL1</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2612</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212012">&nbsp;BBa_K1212012</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+TAL8</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2616</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212013">&nbsp;BBa_K1212013</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pTet+TBS 1+RBS+GFP+T</td><br />
<td>&nbsp;Aura Ferreiro</td><br />
<td>&nbsp;1003</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212014">&nbsp;BBa_K1212014</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 1+TAL 1</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2616</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212015">&nbsp;BBa_K1212015</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 1+TAL 8</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;2620</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212016">&nbsp;BBa_K1212016</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pTet+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1003</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212021">&nbsp;BBa_K1212021</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23100+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="darkColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212022">&nbsp;BBa_K1212022</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23101+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212023">&nbsp;BBa_K1212023</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23105+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="darkColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212024">&nbsp;BBa_K1212024</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23106+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
<tr class="lightColor"><br />
<td><a target="new" href="http://parts.igem.org/Part:BBa_K1212025">&nbsp;BBa_K1212025</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;J23109+TBS 2+RBS+GFP</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;988</td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br></br><br />
<center>Other Parts</center><br />
<table id="myTable" class=" "><br />
<br />
<thead><br />
<tr><br />
<th>&nbsp;Name</th><br />
<th>&nbsp;Type</th><br />
<th>&nbsp;Description</th><br />
<th>&nbsp;Designer</th><br />
<th>&nbsp;Length&nbsp; </th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212002">&nbsp;BBa_K1212002&nbsp;</a></td><br />
<td>&nbsp;Plasmid_Backbone&nbsp;</td><br />
<td>&nbsp;Golden Gate Compatible pSB3K3&nbsp;</td><br />
<td>Alex Neckelmann</td><br />
<td>&nbsp;2750&nbsp;</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212006">&nbsp;BBa_K1212006</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Golden Gate Compatible GFP</td><br />
<td>&nbsp;Aura Ferreiro</td><br />
<td>&nbsp;1052</td><br />
</tr><br />
<tr class="lightColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212009">&nbsp;BBa_K1212009</a></td><br />
<td>&nbsp;Composite</td><br />
<td>&nbsp;pBAD+Riboswitch 2+GFP+T</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1086</td><br />
</tr><br />
<tr class="darkColor"><br />
<td><a target="new" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K1212010">&nbsp;BBa_K1212010</a></td><br />
<td>&nbsp;Composite </td><br />
<td>&nbsp;pBAD+Riboswitch 1+GFP+T</td><br />
<td>&nbsp;Amy Soon</td><br />
<td>&nbsp;1090</td><br />
</tr><br />
<br />
</tbody><br />
</table><br />
<br />
</div><br />
</div><br />
<div class="floatboxwide"><br />
<p><br />
Our best characterized parts are our RiboTAL constructs, which consist of parts BBa_K1212011-BBa_K1212016. Among these parts, most of characterization was focused on the riboswitch+TAL constructs (BBa_K1212011, BBa_K1212012, BBa_K1212014, BBa_K1212015), as the other parts were simply pTet+GFP, which are already well characterized parts, with some extra base pairs added for our TAL binding sites (BBa_K1212013, BBa_K1212016). Both our pSB3K3 backbone (BBa_K1212002) and our GFP (BBa_K1212006) with their BsaI sites fixed were used in our RiboTAL constructs and functioned as expected but did not need separate characterization. The pBAD+Riboswitch+GFP parts (BBa_K1212009 and BBa_K1212010) were originally planned for testing our riboswitches in case the RiboTAL constructs were not working. Since the RiboTALs did work as expected, we focused on characterizing the RiboTALs over the riboswitch testing constructs.<br />
</p><br />
</div><br />
<br />
<!--Begin Showbox--><br />
<div class="floatboxwide"><br />
<table class="showbox"><br />
<tr><br />
<td><br />
<br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
<br />
<br />
<br />
</tr><br />
</table><br />
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</div><br />
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<script type="text/javascript"><br />
$("#sitemapbox").load("https://2013.igem.org/Template:Team:UC_Davis/site_map #sitemap1");</script><br />
</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/CriteriaTeam:UC Davis/Criteria2013-10-26T02:12:55Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<h1>Judging Criteria</h1><br />
<p>BRONZE</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Completed <a href="https://igem.org/Team.cgi?id=1212">Team Registration</a></hi><br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Completed <a href="https://igem.org/Team.cgi?id=1212">Judging Form</a></hi></br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Finalized Team Wiki<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Presented at the North American Regional Jamboree </br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Have documented and submitted at least one new part to the registry. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Parts">Parts</a></hi> page <br />
<br></br><br />
<p>SILVER</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Experimentally validated the expected behavior of 4 RiboTALe constructs, parts BBa_K1212011-BBa_K1212016. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Parts">Parts</a></hi> page<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Provided the characterization data for these parts on the 'Main Page' section of those parts' Registry entries</br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Submitted these parts to the iGEM Parts Registry<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">We have discussed any safety risks associated with our project and the steps we took to mitigate those risks on the <a href="https://2013.igem.org/Team:UC_Davis/Safety">Safety</a></hi> page of this wiki</br><br />
<br />
<br>GOLD</br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">We have improved two existing parts, the <a href="http://parts.igem.org/Part:pSB3K3">pSB3K3</a></hi> backbone as well as part <a href="http://parts.igem.org/Part:BBa_K750000?title=Part:BBa_K750000">BBa_K750000</a></hi> (GFP under the regulation of pBAD and with an LVA degradation tag) by subjecting them to site directed mutagenesis in order to make them Golden Gate Assembly compatible. That is, we removed internal BsaI restriction sites. We created pages for the new parts, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212002">BBa_K1212002</a></hi> and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212006">BBa_K1212006</a></hi>, respectively, and have submitted them to the Registry. We also altered the expression behavior of five of the constitutive Anderson promoters (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23101">J23101</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23105">BBa_J23105</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23106">BBa_J23106</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23109">BBa_J23109</a>). By placing a TAL binding site downstream of each promoter, we were able to make each constitutive promoter repressible by our TALE in the presence of theophylline and arabinose. See <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212021">BBa_K1212021</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212022">BBa_K1212022</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212023">BBa_K1212023</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212024">BBa_K1212024</a>, and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212025">BBa_K1212025</a> for more details.<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">We hosted a midsummer regional iGEM meetup, which was attended by the teams from <a href="https://2013.igem.org/Team:Berkeley">Berkeley</a></hi>, <a href="https://2013.igem.org/Team:Stanford-Brown">Stanford-Brown</a></hi>, and <a href="https://2013.igem.org/Team:UCSF">UC San Francisco</a></hi>. During this meetup we introduced and held an open discussion about our respective projects, asking key questions, giving feedback, and helping each other understand our projects from a "judge's" perspective in preparation for the regional Jamboree. We exchanged information about resources and helped form, we hope, a stronger sense of community and cooperation between teams. <br /><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark"><br />
We have developed a prototype database to help promote sharing and openness among iGEM teams. Our BioBrick Characterization Data Depot (The Depot) is a resource through which teams can share their raw characterization data. While originally proposed as a way for our team to share the multivariate characterization data for our RiboTALs, The Depot was expanded to promote sharing in iGEM for any BioBrick type. As a test run, we asked teams from around the world to help us populate our database with characterization data of the Anderson Promoter Collection. We hope to expand this so future teams can upload their project data in a standardized and easily accessible format. Please see our page <a href="https://2013.igem.org/Team:UC_Davis/Database">about the Depot</a></hi> for more information!</br><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/CriteriaTeam:UC Davis/Criteria2013-10-26T02:02:06Z<p>Arneckelmann: </p>
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<h1>Judging Criteria</h1><br />
<p>BRONZE</p><br />
<br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Completed <a href="https://igem.org/Team.cgi?id=1212">Team Registration</a></hi><br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Completed <a href="https://igem.org/Team.cgi?id=1212">Judging Form</a></hi></br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Finalized Team Wiki<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Are scheduled to present at the North American Regional Jamboree </br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Have documented and submitted at least one new part to the registry. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Parts">Parts</a></hi> page <br />
<br></br><br />
<p>SILVER</p><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Experimentally validated the expected behavior of 4 RiboTALe constructs, parts BBa_K1212011-BBa_K1212016. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Parts">Parts</a></hi> page<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Provided the characterization data for these parts on the 'Main Page' section of those parts' Registry entries</br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">Submitted these parts to the iGEM Parts Registry<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">We have discussed any safety risks associated with our project and the steps we took to mitigate those risks on the <a href="https://2013.igem.org/Team:UC_Davis/Safety">Safety</a></hi> page of this wiki</br><br />
<br />
<br>GOLD</br><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">We have improved two existing parts, the <a href="http://parts.igem.org/Part:pSB3K3">pSB3K3</a></hi> backbone as well as part <a href="http://parts.igem.org/Part:BBa_K750000?title=Part:BBa_K750000">BBa_K750000</a></hi> (GFP under the regulation of pBAD and with an LVA degradation tag) by subjecting them to site directed mutagenesis in order to make them Golden Gate Assembly compatible. That is, we removed internal BsaI restriction sites. We created pages for the new parts, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212002">BBa_K1212002</a></hi> and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212006">BBa_K1212006</a></hi>, respectively, and have submitted them to the Registry. We also altered the expression behavior of five of the constitutive Anderson promoters (<a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23101">J23101</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23105">BBa_J23105</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23106">BBa_J23106</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_J23109">BBa_J23109</a>). By placing a TAL binding site downstream of each promoter, we were able to make each constitutive promoter repressible by our TALE in the presence of theophylline and arabinose. See <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212021">BBa_K1212021</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212022">BBa_K1212022</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212023">BBa_K1212023</a>, <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212024">BBa_K1212024</a>, and <a href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K1212025">BBa_K1212025</a> for more details.<br />
<br><img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark">We hosted a midsummer regional iGEM meetup, which was attended by the teams from <a href="https://2013.igem.org/Team:Berkeley">Berkeley</a></hi>, <a href="https://2013.igem.org/Team:Stanford-Brown">Stanford-Brown</a></hi>, and <a href="https://2013.igem.org/Team:UCSF">UC San Francisco</a></hi>. During this meetup we introduced and held an open discussion about our respective projects, asking key questions, giving feedback, and helping each other understand our projects from a "judge's" perspective in preparation for the regional Jamboree. We exchanged information about resources and helped form, we hope, a stronger sense of community and cooperation between teams. <br /><br />
<img src="https://static.igem.org/mediawiki/2013/b/b9/Judgingcheckoff_UCDavis.png" class="checkmark"><br />
We have developed a prototype database to help promote sharing and openness among iGEM teams. Our BioBrick Characterization Data Depot (The Depot) is a resource through which teams can share their raw characterization data. While originally proposed as a way for our team to share the multivariate characterization data for our RiboTALs, The Depot was expanded to promote sharing in iGEM for any BioBrick type. As a test run, we asked teams from around the world to help us populate our database with characterization data of the Anderson Promoter Collection. We hope to expand this so future teams can upload their project data in a standardized and easily accessible format. Please see our page <a href="https://2013.igem.org/Team:UC_Davis/Database">about the Depot</a></hi> for more information!</br><br />
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<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/Notebook/Week_15Team:UC Davis/Notebook/Week 152013-10-25T16:50:42Z<p>Arneckelmann: </p>
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<h1 class="title">Week 15 Pre-Regionals</h1><br />
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<br />9/28/13-10/3/13<br />
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<p>With the wiki frozen and the North American Regional Jamboree looming, we focused on getting all of our data together for the presentation and poster. Refinement of our presentation and poster were our top priorities before heading off to Toronto.</p> <br />
<br /><br />
<br/ >10/4/13-10/6/13 <br /><br />
<p>Time for the North American Regional Jamboree! We were glad to have our presentation early on Saturday, so we could relax and enjoy the other presentations. Everyone did an incredible job, and Toronto was a blast. We are thrilled to have advanced to the World Championship Jamboree and cannot wait to go to MIT.</p><br />
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<h1 class="title">Week 16 Post-Regionals</h1><br />
<br />10/7/13-10/12/13 <br /><br />
<p>After taking a brief break to catch up on some sleep and schoolwork, we met with our advisors to discuss what we plan to accomplish before the World Championships. We decided on expanding our RiboTALs to targeting GFP under the control of the well characterized Anderson Promoter Collection. Since the Anderson Promoters are a family of constitutive promoters that have measured relative strengths, we believe they can give us predictable system responses. We plan to use 5 of the Anderson Promoters: <br />
<a href="http://parts.igem.org/Part:BBa_J23100" >J23100</a>,<br />
<a href="http://parts.igem.org/Part:BBa_J23101"> J23101</a>,<br />
<a href="http://parts.igem.org/Part:BBa_J23105"> J23105</a>,<br />
<a href="http://parts.igem.org/Part:BBa_J23106"> J23106</a> and <br />
<a href="http://parts.igem.org/Part:BBa_J23109">J23109</a>, which have a wide range of activity and use our model to predict the response of the other Anderson Promoters. We will also continue to improve and populate the Depot.<br />
</p><br /> <br /><br />
<h1 class="title">Week 17</h1><br />
<br />10/13/13-10/19/13<br /><br />
<p>We're back to doing more construction as we try to replace the <a href="http://parts.igem.org/Part:BBa_R0040">pTet promoter</a> in front of<br />
<a href="http://parts.igem.org/Part:BBa_K1212016">TBS2+GFP</a> with our chosen Anderson Promoters. After a few issues, we managed to transform the promoters from the iGEM distribution kits and use Standard Assembly to assemble them together with <a href="http://parts.igem.org/Part:BBa_K1212016">TBS2+GFP</a> which we PCR amplified out of our previous constructs. Our Standard Assemblies were successful, so we did contransformations of the Anderson Promoters+TBS2+GFP in <a href="http://parts.igem.org/Part:pSB1A2">pSB1A2</a> and our <a href="http://parts.igem.org/Part:BBa_K1212012">Riboswitch2+TAL8 RiboTAL</a> in <a href="http://parts.igem.org/Part:BBa_K1212002">pSB3K3</a>. We had hoped to start testing our new constructs in the Tecan on Thursday, but due to the lack of/slow growing colonies for our cotransformations testing will have to be held off. Tecan testing will start next week, due to other unforeseen circumstances.</p><br />
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<h1 class="title">Week 18</h1><br />
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<h1 class="title">Project Motivation</h1><br />
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<p><br />
In synthetic biology, every circuit or device contains, at its core, at least one promoter and one protein coding region. While there are a countless number of proteins we could use, circuit design is limited by the small number of well characterized inducible promoters at our disposal, and their respective transcription factors. TetR, LacI, AraC, LuxR, and cI...do these sound familiar? <br />
</p><br />
<img src="https://static.igem.org/mediawiki/2013/a/ac/ProjMotive_UCDavis.png" class="centerimg"/><br></br><br />
<center><h3>We need more available inputs for transcriptional and translational control.</h3></center><br />
<p>What if ... <br />
<ul><br />
<li>we had transcriptional regulators that could be used in any strain or any chassis?</li> <br />
<li>we could directly engineer repressors for target sequences, instead of having to assemble parts to place them under the control of an inducible promoter? </li><br />
<li>we could control repression with our molecule of choice?</li><br />
<li>we could increase the degrees of freedom that we as researchers have over the control of gene expression?</li><br />
</ul><br />
We would have the ability to host multiple, orthogonal systems within the same chassis. A large part of synthetic biology is, ultimately, designing constructs that generate a response to an input stimulus. A construct that is entirely flexible both at its inputs and outputs is the ideal tool to facilitate the engineering of synthetic biology devices. If we decoupled transcription and translation of a repressor device, maintaining fine-tuned control of both processes, and characterized the behavior of all the parts involved, the dynamic range achievable would be stupendous.<br />
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<h1>The Solution</h1><br />
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<p>Transcription activator-like effectors (TALEs) are proteins secreted by the bacterial plant pathogen <i>Xanthomonas</i>. TALEs contain sequence specific DNA binding domains and can act as transcriptional repressors or activators <a href="#ref">[1]</a>. This binding occurs through hydrogen bonds and van der Waals interactions and is stabilized by the protein's secondary structure. The DNA binding domains are sequence specific due to consecutive protein repeats, which are composed to correspond to a certain base preference <a href="#ref">[2]</a>. TAL repressors can therefore be engineered to bind to any DNA sequence of interest, following now well-understood rules for TAL-DNA binding <a href="#ref">[3,4]</a>. TALEs are thus a powerful and modular tool for the control of gene expression in genetic circuits. Current efforts to quantify and predict TALE binding affinities and functionalities are being made in order to create libraries of TALE systems that will serve to streamline research and the development of genetic devices <a href="#ref">[2]</a>.</p><br />
<br />
<p><br />Riboswitches, on the other hand, are regulatory structures in the 5’-UTR of mRNA that undergo a conformational change in the presence of a specific ligand that binds to the aptamer domain of the structure <a href="#ref">[5]</a>. This conformational change can regulate the initiation of translation by sequestering the ribosome binding site of the mRNA sequence, making it unavailable for binding <a href="#ref">[5]</a>. Riboswitches have been shown to work in diverse bacterial species and many natural examples have been found for riboswitches that turn off translation in the presence of the target ligand as well <a href="#ref">[6,7]</a>. Riboswitches have also been well characterized, are dose-dependent and have been engineered to respond to non-natural ligands thus providing an orthogonal control system <a href="#ref">[8]</a>. These RNA-based devices, like the TALE proteins, are also modular and powerful tools for the control of gene expression.</p><br />
<br />
<p><br />The fusion of these two devices--placing the TAL repressors under the control of riboswitches--offers a means by which to control the expression of any gene of interest, using wider variety of inducers than there are currently available. As our understanding of riboswitches and our ability to engineer aptamer binding domains develop, it will be possible to develop a fully orthogonal, highly versatile systems for control of gene expression. There is a potential for multiplexing, as riboswitches designed to respond to different molecules and fused to different TAL repressors can be used in parallel within a single chassis, or can be induced in a temporally sequential manner for applications such as developmental research. We have to demonstrated that RiboTALes function as synthetic transcription factors that are orthogonal to the natural biochemistry of the cell and increase the degrees of freedom available to us in the control of genetic circuits.</p><br />
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<h2>TALE binding to DNA <a href="#ref">[1]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" width=195 height=195 class="genpic"></img><br />
<h2>Riboswitch with inducer bound <a href="#ref">[9]</a></h2><br />
<img src="https://static.igem.org/mediawiki/2013/7/7e/Riboswitchpic_UCDavis.jpg" width=195 height=195 class="genpic"><img/><br />
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<td><br />
<br />
<div><a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><br />
<img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
</div><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project_Overview"><h3>Project Overview</h3></a><br />
<p>Learn about how we combine riboswitches and TALs into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
<br />
</td><br />
<br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs. <br />
</p><br />
</td><br />
<br />
<br />
</tr><br />
<tr><br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><br />
<img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur" /><br />
<!--img src="https://static.igem.org/mediawiki/2013/9/97/UCD_2013_HO_Button.jpg" class="blur"--></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we promote sharing in iGEM through The Depot, an open BioBrick characterization database.<br /><br />
<a href="http://dilbert.cs.ucdavis.edu/Depot" class="bold">Visit the Depot!</a><br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"</a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
</p><br />
</td><br />
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<a id="ref"><h3>References</h3></a><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Structural+Basis+for+Sequence-Specific+Recognition+of+DNA+by+TAL+Effectors">[1] D. Dong, Y. Chuangye, P. Xiaojing, M. Mahfouz, W. Jiawei, Z. Jian-Kang, et al., "Structural Basis for Sequence-Specific Recognition of DNA by TAL Effectors," Science, vol. 335, pp. 720-723, 10 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Quantitative+analysis+of+TALE-DNA+interactions+suggests+polarity+effects">[2] J. F. Meckler, M. S. Bhakta, M. S. Kim, R. Ovadia, C. H. Habrian, A. Zykovich, et al., "Quantitative analysis of TALE-DNA interactions suggests polarity effects," Nucleic Acids Res, vol. 41, pp. 4118-28, Apr 2013.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Breaking+the+Code+of+DNA+Binding+Specificity+of+TAL-Type+III+Effectors">[3] J. Boch, H. Scholze, S. Schornack, A. Landgraf, S. Hahn, S. Kay, et al., "Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors," Science, vol. 326, pp. 1509-1512, Dec 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+Simple+Cipher+Governs+DNA+Recognition+by+TAL+Effectors">[4] M. J. Moscou and A. J. Bogdanove, "A Simple Cipher Governs DNA Recognition by TAL Effectors," Science, vol. 326, pp. 1501-1501, Dec 11 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Dual-acting+riboswitch+control+of+translation+initiation+and+mRNA+decay">[5] M. P. Caron, L. Bastet, A. Lussier, M. Simoneau-Roy, E. Masse, and D. A. Lafontaine, "Dual-acting riboswitch control of translation initiation and mRNA decay," Proceedings of the National Academy of Sciences of the United States of America, vol. 109, pp. E3444-E3453, Dec 2012.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[6] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Mechanism-Guided+Library+Design+and+Dual+Genetic+Selection+of+Synthetic+OFF+Riboswitches">[7] N. Muranaka, K. Abe, and Y. Yokobayashi, "Mechanism-Guided Library Design and Dual Genetic Selection of Synthetic OFF Riboswitches," Chembiochem, vol. 10, pp. 2375-2381, Sep 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Reengineering+orthogonally+selective+riboswitches">[8] N. Dixon, J. N. Duncan, T. Geerlings, M. S. Dunstan, J. E. G. McCarthy, D. Leys, et al., "Reengineering orthogonally selective riboswitches," Proceedings of the National Academy of Sciences of the United States of America, vol. 107, pp. 2830-2835, Feb 16 2010.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[9] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
</p><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/BiosTeam:UC Davis/Bios2013-10-08T01:27:43Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d5/Teambiobanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<div class="floatboxwide"><br />
<h1>2013 UC Davis iGEM Team</h1><br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/Aurabio_UCDavis.jpg" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3> Aura Ferreiro</h3><br />
<p><br />
I am a 5th year undergraduate majoring in Biological Systems Engineering. I enjoy playing my ukulele and coming up with ways to integrate quinoa and tofu into every meal I cook. I plan to apply for graduate programs in the same field I study now, and look forward to a career in design and modeling of industrial biological systems, or of synthetic biological devices with commercial applications.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/8/82/Ucdavis13_amy_photo.JPG" class="leftpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Amy Soon</h3><br />
<p><br />
I am a senior majoring in Biomedical Engineering and specializing in Systems and Synthetic Biology. Outside of school and the lab, I enjoy fantasy and mystery stories in tv shows, games or novels. I am still in the process of deciding on my future career path and whether I will go to graduate school directly after graduating or take some time off to get work experience first. I hope to be able to continue working with synthetic biology in a career in industry.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e2/Ucdavis13_alex_photo.JPG" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Alex Neckelmann</h3><br />
<p><br />
I am a Senior majoring in Biotechnology with an emphasis in microbiology and awesome. When I’m not at school, I enjoy playing tennis or reading biographies. I plan on graduating and applying to graduate school in the fall. After graduate school, I would really enjoy working on projects involved in bioremediation or optimizing industrial bioprocesses through synthetic biology.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cb/Ucdavis13_david_photo.JPG" class="leftpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>David Hwang</h3><br />
<p><br />
Hi, I'm David Hwang, and I'm entering my fourth year as a Computer Science major this coming fall. My current professional interests are computer science applications in healthcare, education, and clearly, biology. My unprofessional interests are ultimate frisbee (I am on the school's club team where we placed in the top 8 in the nation this past year), and cooking.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d0/Lucasbio_UCDavis.jpg" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Lucas Murray</h3><br />
<p><br />
Hi, I am a third year biomedical engineering major with an emphasis in systems and synthetic biology. When I'm not going back and forth between labs, I enjoy competitive sailing, freediving, and underwater photography. If I had free time, you'd find me doing any of those three. My main background is in instrumentation and chemistry, but my academic focus is on synthetic biology, biophysics, and molecular biology. I plan to apply to graduate school next fall, where I would like to continue to study synthetic biology.<br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatboxwide"><br />
<h1>Advisors:</h1><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/UCD_13_Andrew_profile.jpg" class="leftpic"><br />
<h3>Andrew Yao</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>Wetlab Advisor</li><br />
<li>B.S. in Biomedical Engineering</li><br />
<li>Department: Biomedical Engineering</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/UCD_13_Nick_profile.jpg" class="rightpic"><br />
<h3>Nick Csicsery</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>iGEM Veteran Advisor</li><br />
<li>B.S. in Biological Systems Engineering</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/5/56/UCD_13_photo_Navneet.jpg" class="leftpic"><br />
<h3>Navneet Rai</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>Postdoctoral Research Associate</li><br />
<li>Wetlab Advisor</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h2>Instructors:</h2><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c3/UCD_2013_Marc_Bio.png" class="leftpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Marc Facciotti</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in Biophysics</li><br />
<li>Department: Biomedical Engineering and UC Davis Genome Center</li><br />
<li><a target="new" href=http://www.bme.ucdavis.edu/facciotti>Facciotti Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a3/UCD_13_photo_Justin.jpg" class="rightpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Justin Siegel</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in BioMolecular Structure and Design</li><br />
<li>Department of Biochemistry and Molecular Medicine, School of Medicine</li><br />
<li><a target="new" href=http://sites.google.com/site/ucdsiegellab/>Siegel Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/UCD_13_photo_Ilias.jpg" class="leftpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Ilias Tagkopoulos</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in Electrical Engineering</li><br />
<li>Department: Computer Science and UC Davis Genome Center</li><br />
<li><a target="new" href=http://www.cs.ucdavis.edu/~iliast>Tagkopoulos Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e3/Ucdavisteampic.gif" class="genpic" width=600 height=403></center><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/BiosTeam:UC Davis/Bios2013-10-08T01:27:29Z<p>Arneckelmann: </p>
<hr />
<div>{{Team:UC_Davis/Head}}<br />
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<img src="https://static.igem.org/mediawiki/2013/d/d5/Teambiobanner_UCDavis.jpg" class="banner" width=967 height=226 /><br />
</div><br />
<div class="floatboxwide"><br />
<h1>2013 UC Davis iGEM Team</h1><br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/0/06/Aurabio_UCDavis.jpg" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3> Aura Ferreiro</h3><br />
<p><br />
I am a 5th year undergraduate majoring in Biological Systems Engineering. I enjoy playing my ukulele and coming up with ways to integrate quinoa and tofu into every meal I cook. I plan to apply for graduate programs in the same field I study now, and look forward to a career in design and modeling of industrial biological systems, or of synthetic biological devices with commercial applications.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/8/82/Ucdavis13_amy_photo.JPG" class="leftpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Amy Soon</h3><br />
<p><br />
I am a senior majoring in Biomedical Engineering and specializing in Systems and Synthetic Biology. Outside of school and the lab, I enjoy fantasy and mystery stories in tv shows, games or novels. I am still in the process of deciding on my future career path and whether I will go to graduate school directly after graduating or take some time off to get work experience first. I hope to be able to continue working with synthetic biology in a career in industry.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/e/e2/Ucdavis13_alex_photo.JPG" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Alex Neckelmann</h3><br />
<p>asdf<br />
I am a Senior majoring in Biotechnology with an emphasis in microbiology and awesome. When I’m not at school, I enjoy playing tennis or reading biographies. I plan on graduating and applying to graduate school in the fall. After graduate school, I would really enjoy working on projects involved in bioremediation or optimizing industrial bioprocesses through synthetic biology.<br />
</p><br />
</div><br />
</div><br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/c/cb/Ucdavis13_david_photo.JPG" class="leftpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>David Hwang</h3><br />
<p><br />
Hi, I'm David Hwang, and I'm entering my fourth year as a Computer Science major this coming fall. My current professional interests are computer science applications in healthcare, education, and clearly, biology. My unprofessional interests are ultimate frisbee (I am on the school's club team where we placed in the top 8 in the nation this past year), and cooking.<br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/d/d0/Lucasbio_UCDavis.jpg" class="rightpic" width="150" height="150"><br />
<div class="teampagetext"><br />
<h3>Lucas Murray</h3><br />
<p><br />
Hi, I am a third year biomedical engineering major with an emphasis in systems and synthetic biology. When I'm not going back and forth between labs, I enjoy competitive sailing, freediving, and underwater photography. If I had free time, you'd find me doing any of those three. My main background is in instrumentation and chemistry, but my academic focus is on synthetic biology, biophysics, and molecular biology. I plan to apply to graduate school next fall, where I would like to continue to study synthetic biology.<br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatboxwide"><br />
<h1>Advisors:</h1><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a0/UCD_13_Andrew_profile.jpg" class="leftpic"><br />
<h3>Andrew Yao</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>Wetlab Advisor</li><br />
<li>B.S. in Biomedical Engineering</li><br />
<li>Department: Biomedical Engineering</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/8/89/UCD_13_Nick_profile.jpg" class="rightpic"><br />
<h3>Nick Csicsery</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>iGEM Veteran Advisor</li><br />
<li>B.S. in Biological Systems Engineering</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/5/56/UCD_13_photo_Navneet.jpg" class="leftpic"><br />
<h3>Navneet Rai</h3><br />
<div class="teampagetext"><br />
<p><br />
<ul><br />
<li>Postdoctoral Research Associate</li><br />
<li>Wetlab Advisor</li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<br />
<br />
<div class="floatboxwide"><br />
<h2>Instructors:</h2><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/c/c3/UCD_2013_Marc_Bio.png" class="leftpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Marc Facciotti</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in Biophysics</li><br />
<li>Department: Biomedical Engineering and UC Davis Genome Center</li><br />
<li><a target="new" href=http://www.bme.ucdavis.edu/facciotti>Facciotti Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/a/a3/UCD_13_photo_Justin.jpg" class="rightpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Justin Siegel</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in BioMolecular Structure and Design</li><br />
<li>Department of Biochemistry and Molecular Medicine, School of Medicine</li><br />
<li><a target="new" href=http://sites.google.com/site/ucdsiegellab/>Siegel Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
<br />
<br />
<div class="floatbox1"><br />
<img src="https://static.igem.org/mediawiki/2013/5/5d/UCD_13_photo_Ilias.jpg" class="leftpic"><br />
<div class="teampagetext"><br />
<h3>Dr. Ilias Tagkopoulos</h3><br />
<p><br />
<ul><br />
<li>Ph.D. in Electrical Engineering</li><br />
<li>Department: Computer Science and UC Davis Genome Center</li><br />
<li><a target="new" href=http://www.cs.ucdavis.edu/~iliast>Tagkopoulos Lab Website</a></li><br />
</ul><br />
</p><br />
</div><br />
</div><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<center><img src="https://static.igem.org/mediawiki/2013/e/e3/Ucdavisteampic.gif" class="genpic" width=600 height=403></center><br />
</div><br />
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/OutreachTeam:UC Davis/Outreach2013-09-28T03:46:30Z<p>Arneckelmann: </p>
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<h1>NorCal iGEM Meetup</h1><br />
<p>Over the summer, UC Davis organized an iGEM meetup with UCSF, UC Berkeley, and the Stanford-Brown teams. We started off the day giving a brief talk on each of our projects. After each talk, we had a chance to ask more questions about their project and offer possible constructive criticism. From this, we all received a different perspective on some of the tough problems we were trying to solve with our projects. After the meetup, we all had lunch and got to talk even more about synthetic biology among other things. To change things up, we also went bowling.</p><br />
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<center><img src="https://static.igem.org/mediawiki/2013/4/4a/UCD_2013_Meetup_Picture.png" class="genpic" width=803px height=535px/></center><br />
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<h1>Other Efforts in Human Outreach</h1><br />
<ul><li>Provided DNA extractions for E.coli expressing RFP for the biotechnology class at Davis Senior High School.</li><br />
<br />
<li>Worked with classmate Nicholas Armstrong to create a Synthetic Biology Club at UC Davis<br />
<ul><br />
<li>Team member Alex Neckelmann took the role of Activities Chair</li><br />
<li>Check out the club's Facebook page for more information</li><br />
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</head></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/OutreachTeam:UC Davis/Outreach2013-09-28T03:43:02Z<p>Arneckelmann: </p>
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<div class="floatboxwide"><br />
<br />
<h1>NorCal iGEM Meetup</h1><br />
<p>Over the summer, UC Davis organized an iGEM meetup with UCSF, UC Berkeley, and the Stanford-Brown teams. We started off the day giving a brief talk on each of our projects. After each talk, we had a chance to ask more questions about their project and offer possible constructive criticism. From this, we all received a different perspective on some of the tough problems we were trying to solve with our projects. After the meetup, we all had lunch and got to talk even more about synthetic biology among other things. To change things up, we also went bowling.</p><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/4/4a/UCD_2013_Meetup_Picture.png" class="genpic" width=803px height=535px/></center><br />
<br></br><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1>Other Efforts in Human Outreach</h1><br />
<ul><li>Provided DNA extractions for E.coli expressing RFP for the biotechnology class at Davis Senior High School.</li><br />
<br />
<li>Worked with classmate Nicholas Armstrong to create a Synthetic Biology Club at UC Davis<br />
<ul><br />
<li>Team member Alex Neckelmann took the role of Activities Chair</li><br />
<li>Check out the club's <a href="https://www.facebook.com/groups/SyntheticBiologyClub>Facebook page</a> for more information</li><br />
</ul><br />
</li><br />
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</head></div>Arneckelmannhttp://2013.igem.org/File:UCD_2013_Team_Photo.PNGFile:UCD 2013 Team Photo.PNG2013-09-28T03:30:17Z<p>Arneckelmann: </p>
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</html></div>Arneckelmannhttp://2013.igem.org/Team:UC_Davis/DataTeam:UC Davis/Data2013-09-28T03:07:41Z<p>Arneckelmann: </p>
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<h1 id="studies">Proof of Concept: Our Testing Construct</h1><br />
<br>To characterize the behavior of a RiboTALe device, we acquired cells containing the sequences for TAL repressors from the Siegel Lab at UC Davis, with which we have worked closely. We placed the TAL repressors downstream of theophylline-responsive riboswitches, the sequences of which were taken from the studies <a href="http://www.ncbi.nlm.nih.gov/pubmed/19033367">A flow cytometry-based screen for synthetic riboswitches</a><a href="#ref"> [1]</a>, and <a href="http://aem.asm.org/content/76/23/7881.abstract">Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species</a></hi><a href="#ref"> [2]</a>. The riboswitch-TALe sequences were placed under the regulation of a pBAD promoter.</br><br />
<br>We inserted previously engineered TALe binding sites corresponding to the TAL repressors used in our characterization experiments upstream of a reporter, GFP. This target sequence was placed under the regulation of a pTET promoter.</br><br />
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<center><img src="https://static.igem.org/mediawiki/2013/6/61/UCDAVIStestTOP.gif" class="genpic" width=345px height=360></center><br />
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<center><img src="https://static.igem.org/mediawiki/2013/6/6d/Ucdavisplusarabinose.png" class="genpic" width=488 height=160></center><br />
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<br>We tested our construct by subjecting the pBAD promoter, the theophylline riboswitch, and the pTET promoter to a range of induction levels with arabinose, theophylline, and aTc, respectively. It was expected that at low levels of arabinose and theophylline, but at high levels of aTc, GFP expression would be maximal due to the very low production of TAL repressor protein. On the other hand, at high levels of arabinose and theophylline it was expected that fluorescence levels would be greatly reduced due the higher rate of TAL repressor production. We also expected to see many instances of neither total GFP expression or total GFP repression, depending on the relative states of induction of the pBAD promoter, the theophylline riboswitch, and the pTET promoter.</br><br />
</div><br />
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<div class="floatbox2"> <br />
<h2>Quick Links<h2><br />
<ul><br />
<li><a href="#graph1">RiboTALe Activity</a></li><br />
<li><a href="#graph2">TALe Tunability</a></li><br />
<li><a href="#graph3">Riboswitch Activation</a></li><br />
<li><a href="#widget">RiboTALe KO3D</a></li><br />
</ul><br />
</div><br />
<br />
<div class="floatboxwide"><br />
<h1 id="graph1">Fluorescence is modulated by theophylline concentrations</h1><br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/f/f1/UCDavis_graph1c.png/800px-UCDavis_graph1c.png"></center><br />
<br></br><br>We subjected <a href="http://parts.igem.org/Part:BBa_K1212015">our testing construct</a></h1> to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. We also subjected the construct to the induction condition of 0.1% arabinose, which would produce a nominal level of RiboTALe transcript. We varied only the concentration of theophylline, over a range of 0 mM to 10 mM. Thus, difference in fluorescence between induction conditions would be due only to the RiboTALe repression activity. We measured the fluorescence of our construct in E. Coli strain MG1655Z1 over a course of 9-10 hours using the Tecan Infinite 200Pro microplate reader. Please refer to the <a href="https://2013.igem.org/Team:UC_Davis/Protocols">Protocols</a></hi> page for details on our culture preparation and Tecan testing parameters. </br><br />
<p id="itworks"><br />
<br />The image above illustrates that GFP fluorescence is inversely related to theophylline concentrations, indicating that the <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor</a></hi> is in fact being translated at rates corresponding to the theophylline induction levels, and effectively binding to its target site. At maximal theophylline concentrations, the expression of GFP is reduced 2.6 fold.<br />
</p><br />
<p><br />
<br />Next, we investigated what difference in system response we could achieve by altering the binding affinity of the TAL repressor protein.<br />
<br /><br />
</p><br />
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<h1 id="graph2">Binding affinities of the TAL repressors provide tunability</h1> <br />
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<br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/e/ed/UCDavis_graph2dn.png/799px-UCDavis_graph2dn.png"></center><br />
<br></br><br>The <a href="http://parts.igem.org/Part:BBa_K1212007">TAL repressor protein</a></hi> expressed by our <a href="http://parts.igem.org/Part:BBa_K1212015">RiboTALe device</a></hi> in our initial experiment has a dissociation constant K<sub>D</sub> 1.3 &plusmn; .03 nM. We compared the activity of this RiboTALe to one under the control of the same <a href="http://parts.igem.org/Part:BBa_K1212001">theophylline riboswitch</a></hi>, but that expressed a <a href="http://parts.igem.org/Part:BBa_K1212004">TAL repressor</a></hi> with K<sub>D</sub> 240 &plusmn; 40 nM. We subjected both RiboTALes to the induction condition of 100 ng/mL aTc, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:</br><br />
<br></br><br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br />
<br></br> <br>The image above displays the peak fluorescence of two RiboTALe constructs, <a href="http://parts.igem.org/Part:BBa_K1212014">one</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212004">TALe 1</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212015">other</a></hi> expressing <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>, under different induction conditions for arabinose and theophylline. Both RiboTALes exhibit the expected behavior pattern given the induction conditions, but at consistently different levels of fluorescence. We have attributed this to the difference in binding affinities of the two TAL repressors to their respective binding sites.This variable, if well characterized for different TAL repressors, will provide a powerful means to control the tunability of these devices.</br><br />
<br>It is similarly interesting to note that under conditions of 1% arabinose, but no theophylline, there was clearly some reduction in fluorescence. We concluded that the <a href="http://parts.igem.org/Part:BBa_K1212001">riboswitch</a></hi> we used in this experiment had some degree of leakiness. We next investigated the possibility of altering riboswitch leakiness as another means to increase the tunability of our RiboTALe devices.</br><br />
<br></br><br />
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<div class="floatboxwide"><br />
<h1 id="graph3">Riboswitch leakiness modulates RiboTALe activity</h1><br />
<br />
<br></br><br />
<center><img src="https://static.igem.org/mediawiki/2013/thumb/2/2b/UCDavis_graph3en.png/800px-UCDavis_graph3en.png"></center><!--The study <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2615613/">A flow cytometry-based screen for synthetic riboswitches</a></hi> by Sean Lynch and Justin Gallivan <a href="#ref">[1]</a> presents a library of theophylline riboswitches with randomized 8 base pair sequences in the Shine-Dalgarno region of the transcript that were screened for riboswitch behavior.--><br></br><br />
We proceeded to investigate the differences achievable in RiboTALe system response by varying the riboswitch controlling the translation of the TAL repressor. To this end we tested two RiboTALe devices, both of which expressed <a href="http://parts.igem.org/Part:BBa_K1212007">TALe 8</a></hi>. <a href="http://parts.igem.org/Part:BBa_K1212015">One</a></hi> of these RiboTALes was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a></hi> and the <a href="http://parts.igem.org/Part:BBa_K1212012">other</a> was under the control of theophylline riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a></hi>. We subjected both RiboTALes to the induction condition of 100 ng/uL aTC, which would result in constitutive and maximal expression of GFP given no repression. At the same time, we subjected both RiboTALes to the following conditions:<br />
<br></br><br />
<br />
<table class="black"><br />
<tr><th></th><br />
<th>Arabinose Concentration (%)</th><br />
<th>Theophylline Concentration (mM)</th><br />
<th>Expected Result</th><br />
</tr><br />
<tr><th></th><br />
<td>0.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of RiboTALe transcript and thus TAL repressor</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>0.0</td><br />
<td>Full GFP expression due to a lack of riboswitch inducer, and thus translation of the TAL protein. Decreased GFP expression could be attributed to riboswitch leakiness</td><br />
</tr><br />
<tr><th></th><br />
<td>1.0</td><br />
<td>10.0</td><br />
<td>Full GFP repression, due the nominal expression of the RiboTALe transcript and the TAL repressor</td><br />
</tr><br />
</table><br />
<br><br />
The image above displays the fluorescence results for the two RiboTALe devices tested in this experiment. According to the literature both riboswitches have similar reported fold activation ratios<a href="#ref">[1,2]</a>. But it is clear that the two RiboTALe devices, differing only in the riboswitch controlling the translation of the TAL repressor, exhibit consistently different behavior. The data show that at 1% arabinose (the inducer for the RiboTALe transcript), but in the absence of theophylline, the RiboTALe under control of riboswitch <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is active. Under identical induction conditions, the RiboTALe under riboswitch <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a> exhibits no repression activity. The fluorescence measured was in fact <i>higher</i> than the baseline for reasons not understood. From the data we conclude that <a href="http://parts.igem.org/Part:BBa_K1212001">Clone E</a> is leakier and yet stronger than <a href="http://parts.igem.org/Part:BBa_K1212000">Clone 8.1*</a>, generating a 3.68 fold reduction in fluorescence as opposed to a 2.42 fold reduction. These data indicate that differences riboswitch leakiness and strength do impact RiboTALe system behavior, and can be engineered into RiboTALe designs as sources of tunability. <br />
<br />
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<div class="floatboxwide"><br />
<h1 id="widget">3D RiboTALe Data Plot</h1><br />
<p>Here is a graphical representation of some of our RiboTALe characterization data. The graph can be toggled between 2D and 3D plot modes. The data sets plotted can also be turned on or off through the use of the corresponding buttons in the upper right of the graph. Feel free to click the navigation buttons or drag the 3D graph in order to get a better view.<br />
</p><br />
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<br />
<h3>100 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">81896.13, 78666.88, 74182.52, 31385.93</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
81399.99914, 82312.01989, 81896.13437, 70965.09262, 71753.41851, 52082.65688,<br />
82598.77679, 90191.2318, 78666.87521, 59658.53566, 53189.6567, 43608.73416,<br />
89341.08341, 88566.62349, 89727.21455, 58754.65109, 55356.67872, 59500.96347,<br />
71372.62047, 72710.06759, 74182.52136, 39969.45281, 41701.09012, 58907.74009,<br />
22868.50882, 26697.24765, 31385.92672, 12146.13773, 13292.34931, 29857.02244<br />
<br />
<!--106247.4518,104406.975,107697.0566,87605.53496,113026.3159,116238.2595,<br />
97390.65405,102272.0717,105314.5467,36495.57538,102836.5068,117957.4598,<br />
25845.65451,24721.27778,98984.39646,3460.195648,19476.66271,106190.2008,<br />
1311.329197,4760.869684,27024.29053,1093.892416,4443.585781,38823.82162,<br />
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<h3>0 ng/mL aTc</h3><br />
<span class="xdata">0, 1, 5, 10</span><br />
<span class="ydata">1629.559472, 1700.829702, 1701.217487, 1116.65006</span><br />
<span class="stdevs"></span><br />
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span><br />
<span class='ydata_3d'>0, 1, 2, 5, 10</span><br />
<span class='zdata_3d'><br />
1629.559472,1634.441888,1565.606412,1568.030923,1549.936772,1550.608345,<br />
1700.829702,1756.172098,1716.475113,1714.999974,1687.625775,1673.997409,<br />
1792.94241,1759.305223,1757.164411,1746.514631,1719.24679,1750.824618,<br />
1701.217487,1990.586906,2022.536824,2028.438816,2074.652724,2075.872599,<br />
1116.65006,1816.239221,1806.522633,1788.908717,1827.918679,1805.555476<br />
<!--2369.383411,2422.018312,2387.445279,2327.26362,2303.398056,2254.056792,<br />
2345.828233,2307.888693,2292.350333,2258.230481,2233.40043,2104.700912,<br />
2216.924946,2059.606099,1971.680454,1990.172062,1954.929637,1854.387133,<br />
1346.787494,1021.299836,914.3222794,887.978114,880.5460371,789.473705,<br />
1551.596136,1338.209982,1205.165033,1125.333333,1165.549228,1147.629325--><br />
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<!--div class="playme"><br />
<p id="play">Play With Me</p><br />
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<a href="https://2013.igem.org/Team:UC_Davis/Project"><img src="https://static.igem.org/mediawiki/2013/b/bf/TALpic_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Project"><h3>Project Background</h3></a><br />
<p>Learn about how we combine riboswitches and TAL's into robust orthogonal mechanisms for inducible repression. <br />
</p></a><br />
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<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><img src="https://static.igem.org/mediawiki/2013/d/d5/Resultsicon_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Results</h3></a><br />
<p>Check out the cool results of our experiments with RiboTALs.<br />
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<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><img src="https://static.igem.org/mediawiki/2013/3/35/Humanpracbutton2_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/HumanPracticesOverview"><h3>Human Practices</h3></a><br />
<p>Take a look at how we designed a new database for better raw data characterization of Biobricks. <br />
</p><br />
</td> <br />
<td><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><img src="https://static.igem.org/mediawiki/2013/f/f3/Judgingbutton_UCDavis.jpg" class="blur"></a><br />
<a href="https://2013.igem.org/Team:UC_Davis/Criteria"><h3>Judging Criteria</h3></a><br />
<p>Here's the criteria that we met for this year's team. <br />
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<a id="ref"><h3>References</h3></a><br />
<p><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=A+flow+cytometry-based+screen+for+synthetic+riboswitches">[1] S. A. Lynch and J. P. Gallivan, "A flow cytometry-based screen for synthetic riboswitches," Nucleic Acids Research, vol. 37, pp. 184-192, Jan 2009.</a><br />
<br /><br />
<a href="http://www.ncbi.nlm.nih.gov/pubmed/?term=Synthetic+Riboswitches+That+Induce+Gene+Expression+in+Diverse+Bacterial+Species">[2] S. Topp, C. M. K. Reynoso, J. C. Seeliger, I. S. Goldlust, S. K. Desai, D. Murat, et al., "Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species (vol 76, pg 7881, 2010)," Applied and Environmental Microbiology, vol. 77, pp. 2199-2199, Mar 2011.</a><br />
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</html></div>Arneckelmann