IGEM:Caltech/2007/Project/cisTransResults: Difference between revisions
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So far, trans activation has been tested for cis3 and cis4 (with their trans combinations 1 and 2), as well as trans3 with cis8, making the total count: <br> | |||
* cis3trans1 | |||
* cis3trans2 | |||
* cis4trans1 | |||
* cis4trans2 | |||
* cis8trans3 | |||
<br> | |||
The repressions and activation levels of viral protein was tested by replacing the viral protein gene with the reported molecule YFP, with fluorescence now an indicator of host cell protein concentration. Cells were prepared according to the [[IGEM:Caltech/2007/Protocols/Flow_Cytometry |flow cytometry]] protocol, and YFP fluorescence was measured with Quanta, with results below. | |||
The fluorescence levels of D1210, YFP (the YFP construct with no cis repressor), and YFP + aTc serve as a control (as in the case of D1210, to show the fluorescence levels of background cells), as a guideline for minimum activation needed (as titering results show that N and Q levels in the (no-cis) construct are sufficient to cause lysis), and as maximum protein production possible (100% level set by the YFP + aTc results). Thus, the YFP fluorescence levels provide a estimate of target trans activation levels. Arabinose is added to the trans samples to induce trans production, and aTc added to increase production of the cis transcript.<br> | |||
[[Image:20071020_cis3.png|right]] | |||
<br> | |||
<b>cis3 and accompanying trans</b> | |||
Geometric mean was found for each cell type using FloJo: | |||
* cis3trans2 + ARAB + aTc : 163.67 | |||
* cis3trans2 + ARAB : 11.63 | |||
* cis3trans1 + ARAB + aTc : 159.38 | |||
* cis3trans1 + ARAB : 12.05 | |||
* cis3 + aTc : 32.75 | |||
* cis3 : 4.05 | |||
* YFP + aTc : 1308.11 | |||
* YFP : 345.19 | |||
* D1210 : 9.78 | |||
<br> | |||
When activated by both arabinose and aTc, the geometric mean shows that cis3trans1 and cis3trans2 fluorescence levels reach almost half of the tetR repressed (no aTc) system. When compared to the 100% fluorescence value (set by the mean of the YFP + aTc culture), trans activation for these two combinations reach more than 10%, while cis repression (cis3 + aTc) is approximately 2%. | |||
[[Image:20071020_cis4.png|cis4]] | |||
[[Image:20071020_cis8.png]] | |||
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Revision as of 11:37, 26 October 2007
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So far, trans activation has been tested for cis3 and cis4 (with their trans combinations 1 and 2), as well as trans3 with cis8, making the total count:
The fluorescence levels of D1210, YFP (the YFP construct with no cis repressor), and YFP + aTc serve as a control (as in the case of D1210, to show the fluorescence levels of background cells), as a guideline for minimum activation needed (as titering results show that N and Q levels in the (no-cis) construct are sufficient to cause lysis), and as maximum protein production possible (100% level set by the YFP + aTc results). Thus, the YFP fluorescence levels provide a estimate of target trans activation levels. Arabinose is added to the trans samples to induce trans production, and aTc added to increase production of the cis transcript. 
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