Team:UC Davis/AndersonPromoters2
From 2013.igem.org
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![]() 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. |
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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.
TAL Binding Site alters Anderson Promoter ActivityReturn to Top
Promoter | Relative Strength | 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. Supposedly, the
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.
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, 28.38574215,40.28067521,56.24994637,20.55443453,40.29182958,69.83170289J23106
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