Team:UC Davis/AndersonPromoters

From 2013.igem.org

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                     <!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src=" " class="blur"></a-->
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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>
                     <a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a>
                     <a href="https://2013.igem.org/Team:UC_Davis/Data"><h3>Testing Constructs</h3></a>
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                     <p>Check out our initial experiments with our testing constructs.
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                     <p>Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.
                     </p>
                     </p>
               </td>               
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               <!--a href="https://2013.igem.org/Team:UC_Davis/Data"><img src=" " class="blur"></a-->
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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>
               <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a>
               <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters"><h3>Anderson Promoters</h3></a>
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                     <p>In progress.  
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                     <p>See how we engineered the constitutive Anderson promoters into a family of inducible promoters with RiboTALs. <br />
 +
                        Also, see the secondary data page, <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">here</a>.
                     </p>
                     </p>
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       <h2>Quick Links<h2>
       <h2>Quick Links<h2>
       <ul>
       <ul>
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      <li><a href="#section1">Anderson Promoters</a></li>
 +
      <li><a href="#graph1">Inducible Repression</a></li>
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      <li><a href="#graph2">Promoter Strength</a></li>
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      <li><a href="#graph3">RiboTAL Control</a></li>
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      <li><a href="#widget">KO3D</a></li>
       </ul>
       </ul>
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</div>
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<h1>Targeting the Anderson Promoters</h1>
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<h1 id="section1">Targeting the Anderson Promoters</h1>
<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 />
<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 />
-
We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>,  
+
We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>,  
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>,  
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>,  
-
<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>,  
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<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and  
-
<a href="http://parts.igem.org/Part:BBa_J23106">J23106</a> and  
+
<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 />
-
<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>.
+
(See the <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">secondary data</a> page for data on an additional construct involving J23106.)
-
<br /> <br /> <br /> <br />
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<br /> <br />
 +
<img src="https://static.igem.org/mediawiki/2013/f/fb/UCDavis_And_modelscheme.png" width= 620 height=195 class="centerimg" />
-
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 />
+
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.  
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.  
</p>
</p>
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<div class="floatboxwide">
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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>
 +
<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.
 +
</p><br>
 +
<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center>
 +
<br><p>
 +
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.
 +
</p>
 +
</div>
 +
<div class="floatboxwide">
 +
<h1 id="graph2">Repression is modulated by theophylline and promoter strength<a href="#top" class="to_top">Return to Top</a></h1><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>
 +
<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center>
 +
<br></br><p>
 +
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.
 +
</p>
 +
</div>
 +
<div class="floatboxwide">
 +
<h1 id="graph3">Control of RiboTALe  transcript levels reliably alters system response<a href="#top" class="to_top">Return to Top</a></h1>
 +
<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>
 +
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.
 +
</p>
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<div class="floatboxwide">
<div class="floatboxwide">
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<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">^back to top</a></h1>
+
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1>
-
<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.
+
<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 /><br />
 +
(See the <a href="https://2013.igem.org/Team:UC_Davis/AndersonPromoters2">secondary data</a> page for data on an additional construct involving J23106 and a smaller scale 3D plot for a better view of the J23105 and J23109 data.)
</p>
</p>
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<h3>J23100</h3>
<h3>J23100</h3>
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<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose-->
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<h3>J23105</h3><!--0.1% arabinose-->
<h3>J23105</h3><!--0.1% arabinose-->
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<span class="stdevs">84.42209965,49.33864245,131.2734258,86.18750714,50.51754508</span>
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<!--5031.489167,3510.447702,3345.751695,3188.787952,3046.043038-->
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<span class="stdevs">474.3233961,235.1842546,264.0535921,144.8641036,56.24994637
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131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229,
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237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174,
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</span>
-
<h3>J23106</h3><!--0.1% arabinose-->
 
-
<span class="xdata">0, 1, 2, 5, 10</span>
 
-
<span class="ydata">1143.671555,1113.875803,1095.652133,1014.904465,1011.776987</span>
 
-
<span class="stdevs">6.122578322,52.76486731,56.15251207,23.41746144,21.04377116</span>
 
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<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span>
 
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<span class='ydata_3d'>0, 1, 2, 5, 10</span>
 
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<span class='zdata_3d'>
 
-
1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952,
 
-
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-
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-
1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415,
 
-
1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572
 
-
</span>
 
-
<span class='stdevs_3d'>
 
-
29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653,
 
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21.78491439,32.91522832,56.15251207,12.42786451,3.952972368,8.013145325,
 
-
11.85982218,23.14421652,23.41746144,155.4244472,21.62208768,5.821220612,
 
-
11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793
 
-
</span>
 
<h3>J23109</h3><!--0.1% arabinose-->
<h3>J23109</h3><!--0.1% arabinose-->
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Latest revision as of 03:47, 29 October 2013

Testing Constructs

Check out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function.

Anderson Promoters

See how we engineered the constitutive Anderson promoters into a family of inducible promoters with RiboTALs.
Also, see the secondary data page, here.

Quick Links

Targeting the Anderson Promoters

After proving that our RiboTALs worked 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.

We inserted four different Anderson promoters (J23100, J23101, J23105, and J23109) upstream of TALe binding site 2 corresponding to TAL repressor 8 and a reporter, GFP. These constructs were then cotransformed with our construct containing TAL repressor 8 under the control of theophylline riboswitch 8.1* and pBAD [1].
(See the secondary data page for data on an additional construct involving J23106.)

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.

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.

RiboTALs induce repression of constitutive promotersReturn to Top


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.



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.

Repression is modulated by theophylline and promoter strengthReturn to Top


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.



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.

Control of RiboTALe transcript levels reliably alters system responseReturn to Top

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.




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.

3D RiboTALe Data PlotReturn to Top

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.

(See the secondary data page for data on an additional construct involving J23106 and a smaller scale 3D plot for a better view of the J23105 and J23109 data.)

3

J23100

0, 1, 2, 5, 10 74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385 1562.749688,3248.697529,965.2085914,2193.314194,34.98550127 0, .01, .1, .25, .5, 1 0, 1, 2, 5, 10 76048.39311, 77931.58406, 74318.64217, 73627.4482, 75515.86198, 72846.27556, 71772.10022, 66033.21127, 55342.66284, 57974.14724, 44462.20883, 65993.17523, 66291.3375, 64928.82588, 54073.96075, 54428.95081, 32730.77267, 58216.82922, 66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289, 1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852 1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045, 4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966, 1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652, 10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873, 26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588

J23101

0, 1, 2, 5, 10 65225.42561,52589.99271,42644.87763,14935.51109,2712.895963 537.8232768,1838.324601,1703.320859,211.5468525,143.1029041 0, .01, .1, .25, .5, 1 0, 1, 2, 5, 10 65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353, 56339.75509,58140.17603,52589.99271,45254.53728,36563.0243,47208.36732, 47400.79321,48076.73334,42644.87763,34732.24646,24599.22385,36528.04869, 21381.9066,18986.22297,14935.51109,9852.736573,5936.14123,11775.73331, 2682.143899,2676.431614,2712.895963,2619.453617,2981.708074,3020.156202 174.0755085,828.3347761,537.8232768,288.9683597,841.4751661,1102.760805, 1627.453069,1672.025902,1838.324601,944.8314199,1669.466473,1844.206162, 938.7552643,1246.809219,1703.320859,407.3806125,726.2248181,979.5962525, 733.3128496,1561.599886,211.5468525,193.9365247,591.6545979,227.8345417, 59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474

J23105

0, 1, 2, 5, 10 7533.047431,6370.331682,5990.223684,2922.509781,2186.773466 474.3233961,235.1842546,264.0535921,144.8641036,56.24994637 0, .01, .1, .25, .5, 1 0, 1, 2, 5, 10 8812.305599,7892.957239,7533.047431,7579.954392,7888.945451,6805.733113, 7563.486294,6640.472268,6370.331682,6253.6198,5534.528131,5860.052523, 6553.583112,5385.001711,5990.223684,5436.21014,4856.993296,5548.690175, 3109.416405,2875.640374,2922.509781,2660.378131,2699.499797,2876.530258, 2097.690672,2117.866102,2186.773466,2066.628283,2354.494675,2207.135702 901.9688537,352.754544,474.3233961,274.8370082,964.5829582,131.6683122, 173.4047574,83.7298617,235.1842546,87.45576228,147.7822028,45.53135492, 131.1607144,704.5895154,264.0535921,122.033044,96.90805522,113.3800229, 237.4232758,73.51396005,144.8641036,48.61349215,88.64882984,38.65762174, 28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289

J23109

0, 1, 2, 5, 10 1037.815719,1003.632865,997.6508013,912.070168,664.1533463 6.533826992,7.610089852,12.17300425,16.37293291,0.78231349 0, .01, .1, .25, .5, 1 0, 1, 2, 5, 10 925.0911877,960.1962864,1037.815719,1058.657115,986.9207353,896.7875544, 861.8894124,916.1772568,1003.632865,1017.699865,1012.165592,889.6819299, 599.2391802,915.5235762,997.6508013,1007.517453,991.7154276,955.7963452, 1041.621487,906.6285868,912.070168,888.3245713,853.4524525,930.5172409, 655.0211174,676.9155936,664.1533463,661.4786804,680.5517376,668.3791495 6.591307623,31.07102163,6.533826992,34.50916728,87.65383543,7.167267733, 12.79379878,35.46215647,7.610089852,14.47083964,11.54059793,1.192383657, 334.893904,11.84526361,12.17300425,8.505818035,7.936113763,5.568196358, 204.2909781,21.33787907,16.37293291,5.798384994,19.66398171,20.33713375, 15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204

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References

[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.

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