Difference between revisions of "Part:BBa K2510024"

Revision as of 03:36, 2 November 2017

O-HKcI - T7 - O-HKcI - O-P22c2 promoter

This promoter is a dually repressible promoter

Usage and Biology

This dually repressible promoter allows for the creation of a logic-gate, specifically a NOR-Gate. Indeed, gene expression will occur only when the two repressor are either expressed but non-functioning or absent from the system altogether. To design such a promoter, we combined insulated promoter that had been previously tested to form repressible promoter to create new dually repressible promoters that can be used to form a logic gate. The promoter was tested both in vivo and in a cell-free environment using repressors that were caged with a photoswitchable fluorescent protein.

In vivoTesting

Promoters were tested in vivo in 3 different strains of E. coli. The results show that the system is not as efficient as it could be, and insularity of promoter elements is still a long way off.

Figure 5: Data obtained from flow cytometry experiments. The number showed is the mean of medians for 3 different experiments.

Cell-free testing

We expected to observe no red fluorescence after exposure to violet light and fluorescence after exposure to cyan light, as we thought the system would work similarly to a non-caged repressor. However, it appears that the fluorescence levels are increased over 1000-fold when the repressor is not caged.

One explanation for this behavior could be that the dimerization of Dronpa, instead of preventing repressor binding to its operator, actually makes its binding more permanent, thus leading to the lower fluorescence level.

Figure 4: Results of the cell-free experiment. Each promoter was tested with its cognate repressors. Top: Testing with TetR caged with either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)Middle: Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated version(BBa_K2510113).Bottom: Testing with HKcI caged with either wt-Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111)

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 This promoter is a dually repressible promoter
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+<!-- Add more about the biology of this part here-->
 ===Usage and Biology===
+<html>This dually repressible promoter allows for the creation of a logic-gate, specifically a NOR-Gate. Indeed, gene expression will occur only when the two repressor are either expressed but non-functioning or absent from the system altogether. To design such a promoter, we combined insulated promoter that had been previously tested to form repressible promoter to create new dually repressible promoters that can be used to form a logic gate.
+The promoter was tested both <i>in vivo</i> and in a cell-free environment using repressors that were caged with a photoswitchable fluorescent protein.
+<h3><i>In vivo</i>Testing</h3>
+Promoters were tested in vivo in 3 different strains of <i>E. coli</i>.
+The results show that the system is not as efficient as it could be, and insularity of promoter elements is still a long way off.
+<img src="https://static.igem.org/mediawiki/2017/f/fd/FACS_analysis_PB.png"><span><b>Figure 5</b>: Data obtained from flow cytometry experiments. The number showed is the mean of medians for 3 different experiments.
+<h3>Cell-free testing</h3>
+We expected to observe no red fluorescence after exposure to violet light and fluorescence after exposure to cyan light, as we thought the system would work similarly to a non-caged repressor. However, it appears that the fluorescence levels are increased over 1000-fold when the repressor is not caged.</br></br>
+One explanation for this behavior could be that the dimerization of Dronpa, instead of preventing repressor binding to its operator, actually makes its binding more permanent, thus leading to the lower fluorescence level.
+<img src="https://static.igem.org/mediawiki/2017/e/e2/Logic_tetR_PB.png"></br><img src="https://static.igem.org/mediawiki/2017/8/8e/Logic_P22c2_PB.png"></br><img src="https://static.igem.org/mediawiki/2017/1/19/Logic_HKcIfigure_PB.png"></br><b>Figure 4</b>: Results of the cell-free experiment. Each promoter was tested with its cognate repressors. <b>Top</b>: Testing with TetR caged with either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)<b>Middle</b>: Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated version(BBa_K2510113).<b>Bottom</b>: Testing with HKcI caged with either wt-Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111)</div></html>
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