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Team:UC Davis/AndersonPromoters2
From 2013.igem.org
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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. | 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. | ||
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| + | <h1 id="graph1">J23106 activity under RiboTAL control compared to J23109</h1> | ||
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Revision as of 03:19, 29 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 (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
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. 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 also shows the general variance in characterization and experimental conditions between labs.
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
