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Team:UC Davis/AndersonPromoters
From 2013.igem.org
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<h2>Quick Links<h2> | <h2>Quick Links<h2> | ||
<ul> | <ul> | ||
| + | <li><a href="#section1">Anderson Promoters</a></li> | ||
| + | <li><a href="#graph1">Inducible Repression</a></li> | ||
| + | <li><a href="#mutantwidget">KO3D</a></li> | ||
</ul> | </ul> | ||
</div> | </div> | ||
<div class="floatbox"> | <div class="floatbox"> | ||
| - | <h1>Targeting the Anderson Promoters</h1> | + | <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 /> | ||
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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. | 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> | ||
| + | </div> | ||
| + | |||
| + | <div class="floatboxwide"> | ||
| + | <h1 id="graph1">RiboTALs induce repression of constitutive promoters</h1> | ||
| + | <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. | ||
</div> | </div> | ||
Revision as of 17:46, 28 October 2013
Testing ConstructsCheck out our initial experiments with our testing constructs that served as a proof of concept for RiboTAL function. |
 Anderson PromotersFind out how we controlled the Anderson family of promoters through induction. |
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 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].
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
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.
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.
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, 26.97401505,5.834718515,34.98550127,4.245354108,1113.394961,39.7836588J23101
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, 59.56731494,383.2462543,143.1029041,203.0060701,284.1131969,78.24576474J23105
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, 28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289J23106
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, 11.17418373,4.27155103,21.04377116,16.94729334,23.00187831,19.99511793J23109
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, 15.68057096,17.49085027,0.78231349,5.867255805,7.709058,15.41543204Play With Me
