Team:UC Davis/AndersonPromoters

From 2013.igem.org

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<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">^back to top</a></h1>
<h1 id="graph1">RiboTALs induce repression of constitutive promoters<a href="#top" class="to_top">^back to top</a></h1>
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<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 RiboTAL 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.  
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<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.  
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<center><img src="https://static.igem.org/mediawiki/2013/0/09/UCDavis_andBarchart_n.png" height=525 width=800></center>
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The data show that the GFP transcript is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are sensitive to repression by the RiboTALe. The data also show that the promoters maintain, qualitatively, their relative strengths.  
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The data show that the GFP transcript is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are sensitive to repression by the RiboTALe. The data also show that the promoters maintain, qualitatively, their relative strengths. 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.
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<h1>Repression of GFP Fluorescence is Modulated by Theophylline</h1>
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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>
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<center><img src="https://static.igem.org/mediawiki/2013/8/8c/UCDavis_andTheophyllinedep_n.png" height=525 width=800></center>
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Revision as of 20:06, 28 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.

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].

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 promoters^back 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.



The data show that the GFP transcript is repressible by the synthetic transcription factor. In other words, the once constitutive promoters are sensitive to repression by the RiboTALe. The data also show that the promoters maintain, qualitatively, their relative strengths. 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 of GFP Fluorescence is Modulated by Theophylline

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.

3D RiboTALe Data Plot^back 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.

80000

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

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

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