Difference between revisions of "Team:Paris Bettencourt/Logic Circuit"
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<b>A</b> - Only one copy of each repressor is present downstream of the promoter core. | <b>A</b> - Only one copy of each repressor is present downstream of the promoter core. | ||
B - A copy of one of the two different operators is present upstream of the promoter core to possibly enable a better control of gene expression. | B - A copy of one of the two different operators is present upstream of the promoter core to possibly enable a better control of gene expression. | ||
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<div class=text2right>Based on our <a href=”https://2017.igem.org/Team:Paris_Bettencourt/Model#thirdmodel”>modeling</a> results, we designed decided to work of three specific repressors due to their interesting parameter values. For each pair of repressors, four different arrangements of the operators were characterized experimentally. Firstly, the impact of the way the two operators are ordered downstream of the promoter core was studied (Figure 2A). Secondly, we studied the impact of adding a second operator upstream of the promoter core on promoter activity (Figure 2B). This gave us a total of twelve promoters to test (Table 1). Different input combinations were applied to the system, i.e. repressor concentrations were varied. In order to control concentration, the repressors were put under the control of well-known inducible promoters : p<sub>lacI</sub> and p<sub>ara</sub>. Since it is difficult to track the concentration of each repressor in real time, it was approximated by the fluorescence emitted by a fluorescent protein - eyfp or ecfp - regulated by the same inducible promoters as the repressors. The output was measured by the mRFP1 fluorescence emitted. All florescences were measured using a flow cytometer.</div> | <div class=text2right>Based on our <a href=”https://2017.igem.org/Team:Paris_Bettencourt/Model#thirdmodel”>modeling</a> results, we designed decided to work of three specific repressors due to their interesting parameter values. For each pair of repressors, four different arrangements of the operators were characterized experimentally. Firstly, the impact of the way the two operators are ordered downstream of the promoter core was studied (Figure 2A). Secondly, we studied the impact of adding a second operator upstream of the promoter core on promoter activity (Figure 2B). This gave us a total of twelve promoters to test (Table 1). Different input combinations were applied to the system, i.e. repressor concentrations were varied. In order to control concentration, the repressors were put under the control of well-known inducible promoters : p<sub>lacI</sub> and p<sub>ara</sub>. Since it is difficult to track the concentration of each repressor in real time, it was approximated by the fluorescence emitted by a fluorescent protein - eyfp or ecfp - regulated by the same inducible promoters as the repressors. The output was measured by the mRFP1 fluorescence emitted. All florescences were measured using a flow cytometer.</div> | ||
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<tr> <td><a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2510028">BBa_K2510028</a></td><td class=partnamecell>O-TetR - T7 - O-TetR - O-HKcI promoter</td><td class=sequencecell>GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCTGAACCATAAGTTCAGCTCTG</td><td>115</td></tr> | <tr> <td><a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2510028">BBa_K2510028</a></td><td class=partnamecell>O-TetR - T7 - O-TetR - O-HKcI promoter</td><td class=sequencecell>GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCTGAACCATAAGTTCAGCTCTG</td><td>115</td></tr> | ||
<tr> <td><a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2510029">BBa_K2510029</a></td><td class=partnamecell>O-TetR - T7 - O-TetR - O-P22C2 promoter</td><td class=sequencecell>GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG</td><td>125</td></tr></table></div></div> | <tr> <td><a target="_blank" href="http://parts.igem.org/wiki/index.php?title=Part:BBa_K2510029">BBa_K2510029</a></td><td class=partnamecell>O-TetR - T7 - O-TetR - O-P22C2 promoter</td><td class=sequencecell>GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG</td><td>125</td></tr></table></div></div> | ||
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| + | <divclass=text2right><img src="https://static.igem.org/mediawiki/2017/f/f2/Fig2_LC_PB.png"> <span><b>Figure 2</b>: Design of repressor expression device and reporter. <b>A</b> - First repressor and reporter regulated by P<sub>lacI</sub><b>B</b> - Second reporter and repressor regulated by P<sub>Bad</sub></span></div></div> | ||
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<div class=text2><div class="text2left">Testing of the promoters was performed <i>in vivo</i> in <i>E. coli</i> BL21 (AI) and <i>E. coli</i> BL21 (DE3). Both strains were tested to counter the effect of the T7 polymerase and at least one of the repressors being regulated by the same promoter. Analysis of fluorescence using a flow cytometer, testing four conditions for each strain: no inducer, each inducer indivudually - IPTG and arabinose - and both inducers together. The testing was performed on strains containing or not the repressors gene. | <div class=text2><div class="text2left">Testing of the promoters was performed <i>in vivo</i> in <i>E. coli</i> BL21 (AI) and <i>E. coli</i> BL21 (DE3). Both strains were tested to counter the effect of the T7 polymerase and at least one of the repressors being regulated by the same promoter. Analysis of fluorescence using a flow cytometer, testing four conditions for each strain: no inducer, each inducer indivudually - IPTG and arabinose - and both inducers together. The testing was performed on strains containing or not the repressors gene. | ||
</div> | </div> | ||
| − | <div class="text2right" | + | <div class="text2right"></div>Table of all the different conditions.</div> |
<img src=""> | <img src=""> | ||
Revision as of 14:34, 1 November 2017
LOGIC CIRCUIT
Introduction & Background
For our main system to work, where production of a biomaterial, only occurs when two specific lights intersect, a logic-gate needed to be developed. To create a NOR-gate at the promoter level, we aimed at creating dually repressed promoters. Most of NOR-gate promoter designs using tandem repressible promoters (1, 2, 3) have unpredictable properties and leaky expression. The main challenge to create a clean design of NOR-gate containing only one transcription starting point is the lack of standard transcriptional elements smaller than repressible promoters. Recent work on transcription elements showed that assembling insulated synthetic operator upstream and downstream of an insulated T7 promoter core allowed for a more diverse control of gene expression and a more specific response time (1).
Design
Based on our modeling results, we designed decided to work of three specific repressors due to their interesting parameter values. For each pair of repressors, four different arrangements of the operators were characterized experimentally. Firstly, the impact of the way the two operators are ordered downstream of the promoter core was studied (Figure 2A). Secondly, we studied the impact of adding a second operator upstream of the promoter core on promoter activity (Figure 2B). This gave us a total of twelve promoters to test (Table 1). Different input combinations were applied to the system, i.e. repressor concentrations were varied. In order to control concentration, the repressors were put under the control of well-known inducible promoters : placI and para. Since it is difficult to track the concentration of each repressor in real time, it was approximated by the fluorescence emitted by a fluorescent protein - eyfp or ecfp - regulated by the same inducible promoters as the repressors. The output was measured by the mRFP1 fluorescence emitted. All florescences were measured using a flow cytometer.
| Part number | Part Name | Sequence | Size |
|---|---|---|---|
| BBa_K2510018 | T7 - O-HKcI - O-P22c2 promoter | GTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGATTTAAGTGTTCTTTAATCGCTGTTCCGCTG | 74 |
| BBa_K2510019 | T7 - O-HKcI - O-TetR promoter | GTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGTCCCTATCAGTGATAGAGATCACACTCCTTC | 74 |
| BBa_K2510020 | T7 - O-P22c2 - O-HKcI promoter | GTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTGAACCATAAGTTCAGCTCTG | 74 |
| BBa_K2510021 | T7 - O-P22c2 - O-TetR promoter | GTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTCCCTATCAGTGATAGAGATCACACTCCTTC | 84 |
| BBa_K2510022 | T7- O-TetR - O-HKcI promoter | GTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATCACACTCCTTCTGAACCATAAGTTCAGCTCTG | 74 |
| BBa_K2510023 | T7 - O-TetR - O-P22c2 promoter | GTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATCACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG | 84 |
| Part number | Part Name | Sequence | Size |
|---|---|---|---|
| BBa_K2510024 | O-HKcI - T7 - O-HKcI - O-P22c2 promoter | GTGAACCATAAGTTCAGCTATGTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGATTTAAGTGTTCTTTAATCG CTGTTCCGCTG | 95 |
| BBa_K2510025 | O-HKcI - T7 - O-HKcI - O-TetR promoter | GTGAACCATAAGTTCAGCTATGTAATACGACTCACTATAGGGGTGAACCATAAGTTCAGCTCTGTCCCTATCAGTGATAGAGAT CACACTCCTTC | 95 |
| BBa_K2510026 | O-P22C2 - T7 - O-P22C2 - O-HKcI promoter | GTCATTTAAGTGTTCTTTAATGAGCATCTGCTATGTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCGCTGTGAACCATAAGTTCAGCTCTG | 107 |
| BBa_K2510027 | O-P22c2 - T7 - O-P22C2 - O-TetR promoter | GTCATTTAAGTGTTCTTTAATGAGCATCTGCTATGTAATACGACTCACTATAGGGGATTTAAGTGTTCTTTAATCGCTGTTCCG CTGTCCCTATCAGTGATAGAGATCACACTCCTTC | 118 |
| BBa_K2510028 | O-TetR - T7 - O-TetR - O-HKcI promoter | GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCTGAACCATAAGTTCAGCTCTG | 115 |
| BBa_K2510029 | O-TetR - T7 - O-TetR - O-P22C2 promoter | GTCTCCCTATCAGTGATAGAGATCACACTCCTTCAACCTATGTAATACGACTCACTATAGGGGTCCCTATCAGTGATAGAGATC ACACTCCTTCATTTAAGTGTTCTTTAATCGCTGTTCCGCTG | 125 |
Figure 2: Design of repressor expression device and reporter. A - First repressor and reporter regulated by PlacIB - Second reporter and repressor regulated by PBadIn vivo testing
Testing of the promoters was performed in vivo in E. coli BL21 (AI) and E. coli BL21 (DE3). Both strains were tested to counter the effect of the T7 polymerase and at least one of the repressors being regulated by the same promoter. Analysis of fluorescence using a flow cytometer, testing four conditions for each strain: no inducer, each inducer indivudually - IPTG and arabinose - and both inducers together. The testing was performed on strains containing or not the repressors gene.
Table of all the different conditions.As is shown in figure 3, Red fluorescence is lower when either of the repressors are present in the system as well as when both are present (ttest,p<0.5). However, we can see that there is no cummulative effect of the presence of both repressors as the difference between the populations is not significant (ttest,p>0.5).
Cell-free testing using Photocaged repressors
We wanted to combine two aspects of our project: our designed logic gate and the photocaged repressors. We chose to test them in a cell-free environment for both practicality reasons and to show that every aspect of our project was compatible with a cell-free environment.
