Team:UC Davis/AndersonPromoters2

From 2013.igem.org

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<h1 id="section1">Targeting the Anderson Promoters (Secondary Data)</h1>
<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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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>,
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<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>.
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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.  
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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.
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Revision as of 02:48, 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.

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.

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.

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