Difference between revisions of "Part:BBa K2510025"

Latest revision as of 03:54, 16 December 2017

O-HKcI - T7 - O-HKcI - O-TetR promoter

This part 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

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_K2510025 SequenceAndFeatures

@@ Line 18: / Line 18: @@
 <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>
+<P> This synthetic operator was part of the library that we used to test the activity of repressors caged with Dronpa </p>
-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/b/b1/96_conditions.png">
-<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>
+                             <b>Figure 1 : </b> the experiment conducted with the repressors caged with Dronpa
+                                    </span>
+                                      <img src="https://static.igem.org/mediawiki/2017/7/7d/Repression_strength_of_closed_state.png ">
+                                       <b>Figure 2 : </b> 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-.
+                                    </span>
+                                    <img src="https://static.igem.org/mediawiki/2017/2/22/TetR_dronpa.png">
+                                    <img src="https://static.igem.org/mediawiki/2017/e/ed/P22c2_dronpa.png">
+                                     <img src="https://static.igem.org/mediawiki/2017/5/5e/HKCI_caged_dronpa.png">
+                                    <span  class="image-span text-center">
+                                            <b>Figure 3:</b> detailed 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)           </span>
+                                   <img src="https://static.igem.org/mediawiki/2017/5/5f/Wtvs_mut_repressors.png" >
+                                    <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>