Difference between revisions of "Part:BBa K2510018"

 
 
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This promoter is a dually repressible promoter that is part of our team's part collection.
 
This promoter is a dually repressible promoter that is part of our team's part collection.
  
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===Usage and Biology===
 
===Usage and Biology===
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<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.
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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.
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<h3><i>In vivo</i>Testing</h3>
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Promoters were tested in vivo in 3 different strains of <i>E. coli</i>.
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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.
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<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.
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<h3>Cell-free testing</h3>
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<P> This synthetic operator was part of the library that we used to test the activity of repressors caged with Dronpa </p>
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<img src="https://static.igem.org/mediawiki/2017/b/b1/96_conditions.png">
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                            <b>Figure 1 : </b> the experiment conducted with the repressors caged with Dronpa
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                                    </span>
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                                      <img src="https://static.igem.org/mediawiki/2017/7/7d/Repression_strength_of_closed_state.png ">
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                                      <b>Figure 2 : </b> An over view of the results of the cell-free experiment. Each
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                                        promoter was tested with its cognate repressors. Our results show that in 90% of our
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                                        constructs caging the repressors with Dronpa has increased the repression strength.
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                                        We have also obtained a wide linear range of repression strength-indicated by the red
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                                        slope-.
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                                    </span>
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                                    <img src="https://static.igem.org/mediawiki/2017/2/22/TetR_dronpa.png">
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                                    <img src="https://static.igem.org/mediawiki/2017/e/ed/P22c2_dronpa.png">
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                                    <img src="https://static.igem.org/mediawiki/2017/5/5e/HKCI_caged_dronpa.png">
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                                    <span  class="image-span text-center">
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                                            <b>Figure 3:</b> detailed Results of the cell-free experiment. Each promoter was
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                                            tested with its cognate repressors. <b>Top</b>: Testing with TetR caged with
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                                            either wt-Dronpa (BBa_K2510108) or a mutated version(BBa_K2510109)<b>Middle</b>:
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                                            Testing with P22c2 caged with either wt-Dronpa (BBa_K2510112) or a mutated
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                                            version(BBa_K2510113).<b>Bottom</b>: Testing with HKcI caged with either wt-
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                                            Dronpa (BBa_K2510110) or a mutated version(BBa_K2510111)          </span>
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                                  <img src="https://static.igem.org/mediawiki/2017/5/5f/Wtvs_mut_repressors.png" >
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                                    <b>Figure 4:</b> the wtDronpa has a better control of HKC1 and C2P22 repressors while the mutDronpa has a better control of TetR repressors </span>
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Latest revision as of 03:50, 16 December 2017


T7 - O-HKcI - O-P22c2 promoter

This promoter is a dually repressible promoter that is part of our team's part collection.

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

This synthetic operator was part of the library that we used to test the activity of repressors caged with Dronpa

Figure 1 : the experiment conducted with the repressors caged with Dronpa Figure 2 : An over view of the results of the cell-free experiment. Each promoter was tested with its cognate repressors. Our results show that in 90% of our constructs caging the repressors with Dronpa has increased the repression strength. We have also obtained a wide linear range of repression strength-indicated by the red slope-. Figure 3: detailed 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) Figure 4: the wtDronpa has a better control of HKC1 and C2P22 repressors while the mutDronpa has a better control of TetR repressors Sequence and Features BBa_K2510018 SequenceAndFeatures