Team:UC Davis/AndersonPromoters2

From 2013.igem.org

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       <h2>Quick Links<h2>
       <h2>Quick Links<h2>
       <ul>
       <ul>
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      <li><a href="#graph1">J23106 Activity</a></li>
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      <li><a href="#table1">Altered Strength</a></li>
       <li><a href="#widget">KO3D</a></li>
       <li><a href="#widget">KO3D</a></li>
       </ul>
       </ul>
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<div class="floatbox">
<div class="floatbox">
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<h1 id="section1">Targeting the Anderson Promoters</h1>
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<h1 id="section1">Targeting the Anderson Promoters (Secondary Data)</h1>
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<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 />
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<p>  
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We inserted five different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>,  
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We inserted four different Anderson promoters (<a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>,  
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<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>,  
<a href="http://parts.igem.org/Part:BBa_J23101">J23101</a>,  
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<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>, and  
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<a href="http://parts.igem.org/Part:BBa_J23105">J23105</a>,
 +
<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>.
<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 />
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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 />
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We expected to see GFP expression vary with promoter strength, with a larger relative strength overall showing 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 strengths of the promoters we used. This was generally true for most of our constructs.<br /><br />
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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.  
+
However, our J23106 construct did not perform as expected with much lower expression levels of fluorescence than its relative strength would predict. This can be seen in the data below, along with how the addition of the TAL binding sequence may have affected promoter strength.
</p>
</p>
 +
</div>
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<div class="floatboxwide">
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</center>
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<h1 id="graph1">J23106 activity under RiboTAL control compared to J23109</h1>
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<p>The constructs 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>
 +
<img src="https://static.igem.org/mediawiki/2013/6/65/J23106_9graph_UCDavis.jpg" class="centerimg" width=800 height=525 />
 +
<p>As seen in the graph above, J23106 showed fluorescence levels barely above those of the theoretically much weaker promoter J23109. There are many possible reasons why this result may have occurred, such as mutations in the sequence, environmental variables that differ between labs or the addition of the TAL binding site altering promoter strength. Below is our investigation of how the TAL binding site may have affected the relative strengths of the Anderson promoters.
</div>
</div>
<div class="floatboxwide">
<div class="floatboxwide">
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<h1>TAL Binding Site alters Anderson Promoter Activity<a href="#top" class="to_top">Return to Top</a></h1>
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<h1 id="table1">TAL Binding Site alters Anderson Promoter Activity<a href="#top" class="to_top">Return to Top</a></h1>
<center>
<center>
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<table class="showbox1">
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<table class="showbox1" width=400px;>
<tr>
<tr>
<td> Promoter </td>
<td> Promoter </td>
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<td> Relative Strength  </td>
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<td> Expected Relative Strength  </td>
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<td> Fluorescence/OD </td>
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<td> Measured Fluorescence/OD </td>
<td> Expected Fluorescence/OD </td>
<td> Expected Fluorescence/OD </td>
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<td>
 
</tr>
</tr>
<tr>
<tr>
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</table>
</table>
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</center>
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<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>
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<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. Both J23105 and J23106 exhibit different promoter activities with the presence of the TAL Binding Site. In earlier characterizations, J23106 is a stronger promoter than J23105, but with the inclusion of the TAL binding site, the opposite is true. These findings demonstrate the limitations in composability of J23106.  Furthermore, the general discrepancy between expected fluorescence values and actual fluorescences values for the weaker promoters may be due to the presence of a degradation tag on GFP.</p>
</div>
</div>
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<div class="floatboxwide">
<div class="floatboxwide">
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1>
<h1 id="widget">3D RiboTALe Data Plot<a href="#top" class="to_top">Return to Top</a></h1>
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<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.
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<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>
 +
<p><br />This plot shows a smaller scale with the max Fluorescence/OD of 7000, so the plots for J23105, J23106 and J23109 can be seen more easily.</p>
</p>
</p>
<div id="mutantwidget" class="floatbox">
<div id="mutantwidget" class="floatbox">
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<span class="dataMax">80000</span>
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<span class="dataMax">7000</span>
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<h3>J23100</h3>
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<span class="xdata">0, 1, 2, 5, 10</span> <!--0.1% arabinose-->
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-
<span class="ydata">74318.64217, 55342.66284, 54073.96075, 49394.76169, 1400.261385</span>
+
-
<span class="stdevs">1562.749688,3248.697529,965.2085914,2193.314194,34.98550127</span>
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-
<span class='xdata_3d'>0, .01, .1, .25, .5, 1</span>
+
-
<span class='ydata_3d'>0, 1, 2, 5, 10</span>
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-
<span class='zdata_3d'>
+
-
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,
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66589.08966, 56376.24302, 49394.76169, 34927.02648, 17220.67242, 63658.22289,
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1447.535492, 1266.048078, 1400.261385, 1144.055496, 1275.615995, 1337.135852
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-
</span>
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-
<span class='stdevs_3d'>
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-
1933.487778,931.7141933,1562.749688,1786.454908,2695.794862,1693.280045,
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-
4423.195561,640.7031028,3248.697529,370.5129918,969.3078718,12068.86966,
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-
1349.034681,326.3302848,965.2085914,2425.375001,10608.92141,2611.189652,
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-
10931.22028,1498.98258,2193.314194,3811.555031,728.0709108,2629.400873,
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-
26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588
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-
</span>
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-
 
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<h3>J23101</h3> <!--0.1% arabinose-->
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-
<span class="xdata">0, 1, 2, 5, 10</span>
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-
<span class="ydata">65225.42561,52589.99271,42644.87763,14935.51109,2712.895963</span>
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-
<span class="stdevs">537.8232768,1838.324601,1703.320859,211.5468525,143.1029041</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'>
+
-
65469.43353,64888.68512,65225.42561,64941.19503,64873.03094,64461.38353,
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-
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
+
-
</span>
+
-
<span class='stdevs_3d'>
+
-
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
+
-
</span>
+
<h3>J23105</h3><!--0.1% arabinose-->
<h3>J23105</h3><!--0.1% arabinose-->

Latest revision as of 03:54, 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

Find out how we controlled the Anderson family of promoters through induction.
Also, see the secondary data page, here.

Quick Links

Targeting the Anderson Promoters (Secondary Data)

We inserted five different Anderson promoters (J23100, J23101, J23105, J23106, 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].

We expected to see GFP expression vary with promoter strength, with a larger relative strength overall showing 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 strengths of the promoters we used. This was generally true for most of our constructs.

However, our J23106 construct did not perform as expected with much lower expression levels of fluorescence than its relative strength would predict. This can be seen in the data below, along with how the addition of the TAL binding sequence may have affected promoter strength.

J23106 activity under RiboTAL control compared to J23109

The constructs 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.

As seen in the graph above, J23106 showed fluorescence levels barely above those of the theoretically much weaker promoter J23109. There are many possible reasons why this result may have occurred, such as mutations in the sequence, environmental variables that differ between labs or the addition of the TAL binding site altering promoter strength. Below is our investigation of how the TAL binding site may have affected the relative strengths of the Anderson promoters.

TAL Binding Site alters Anderson Promoter ActivityReturn to Top

Promoter Expected Relative Strength Measured Fluorescence/OD Expected Fluorescence/OD
J23100 1 76048.39 76048.39
J23101 0.7 65469.43 53233.88
J23105 0.24 8812.306 18251.61
J23106 0.47 1341.74 35742.74
J23109 0.04 925.0912 3041.936

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. Both J23105 and J23106 exhibit different promoter activities with the presence of the TAL Binding Site. In earlier characterizations, J23106 is a stronger promoter than J23105, but with the inclusion of the TAL binding site, the opposite is true. These findings demonstrate the limitations in composability of J23106. Furthermore, the general discrepancy between expected fluorescence values and actual fluorescences values for the weaker promoters may be due to the presence of a degradation tag on GFP.

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.


This plot shows a smaller scale with the max Fluorescence/OD of 7000, so the plots for J23105, J23106 and J23109 can be seen more easily.

7000

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

J23106

0, 1, 2, 5, 10 1143.671555,1113.875803,1095.652133,1014.904465,1011.776987 6.122578322,52.76486731,56.15251207,23.41746144,21.04377116 0, .01, .1, .25, .5, 1 0, 1, 2, 5, 10 1341.739873,1253.722707,1143.671555,1426.316189,1197.883339,1194.643952, 1245.694646,1192.559043,1113.875803,1365.114347,1091.300427,1140.861909, 1260.370064,1137.689575,1095.652133,1318.814458,1094.357512,1138.75199, 1100.642767,982.5552421,1014.904465,1081.786216,954.6730307,985.045415, 1009.218344,977.0919833,1011.776987,985.080509,1005.811758,1033.441572 29.72707759,19.18243567,6.122578322,20.73434182,55.93695971,37.54780653, 35.19508365,11.5287082,52.76486731,14.77007333,26.52770527,8.296078047, 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

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

Play With Me

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.

Retrieved from "http://2013.igem.org/Team:UC_Davis/AndersonPromoters2"