Difference between revisions of "Team:Paris Bettencourt/Transmembrane Proteins"
Aliciagraham (Talk | contribs) |
Aliciagraham (Talk | contribs) |
||
| Line 57: | Line 57: | ||
</div> | </div> | ||
<div class=text6> These two light sensors were used to activate two repressors: phIF by blue light and CI by the red light that then act on components that complete the T7 RNAP core, which in turn act to allow production of the genes of interest. 4 plasmids were used to express the system: | <div class=text6> These two light sensors were used to activate two repressors: phIF by blue light and CI by the red light that then act on components that complete the T7 RNAP core, which in turn act to allow production of the genes of interest. 4 plasmids were used to express the system: | ||
| − | + | <table> | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
| − | <table | + | |
<tr> | <tr> | ||
| − | <th | + | <th></th> |
| − | <th | + | <th>Role in circuit</th> |
| − | <th | + | <th>Components</th> |
| − | <th | + | <th>Backbone (resistance, ori)</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td | + | <td>Plasmid One</td> |
| − | <td | + | <td> Photoreceptors: YF1/FixL and Cph8</td> |
| − | <td | + | <td>YF1/FixL and Cph8</td> |
| − | <td | + | <td>CamR, colE1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td | + | <td>Plasmid Two</td> |
| − | <td | + | <td> Repressor activated by YF1/FixL: phIF</td> |
| − | <td | + | <td>phIF repressorin front of FixJ promotor.</td> |
| − | <td | + | <td>SpecR, p15A</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td | + | <td>Plasmid Three</td> |
| − | <td | + | <td>Repressor activated by Cph8: CI</td> |
| − | <td | + | <td>CI repressor in front of OmpR promotor.<br>photobilins: ho1 and pcyA</td> |
| − | <td | + | <td>TrimR, incW</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
| − | <td | + | <td>Plasmid Four</td> |
| − | <td | + | <td>Genes of interest (Biomaterials),</td> |
| − | <td | + | <td>PhaA and PhaB under K1F T7 RNAP promoterm and PhaC under T3 Promoter</td> |
| − | <td | + | <td>AmpR, pSC101</td> |
</tr> | </tr> | ||
</table> | </table> | ||
| + | |||
Revision as of 22:48, 1 November 2017
MEMBRANE PHOTORECEPTOR
Introduction
Light is one of the crucial determinants of the environment and as such, many naturally occuring organisms, including microorganisms , have evolved mechanisms to respond to light. This is done through photoreceptor proteins embedded in the cell membrane, that either change conformity or energy state when exposed to specific wavelengths to then induce a singalling cascade within the cell.
With the advent of genetic engineering, more specifically synthetic biology, scientists have been able to harness the power of these membrane photosensors and create powerful optogenetic tools that are used in a range of applications. Additionally, protein engineering have increased the sensitivity and portability of these enzymes, resulting in accurate modular systems.
State of the art and inspiration
These optogenetic tools have become vital in Synthetic biology, and have been used to construct genetic circuits, used for both fundamental and applicative research(Ref). Most recently, these proteins have been used to create logical circuits, allowing for control of expression of cells.
The Chris Voigt Lab published a paper where they incorporate photoreceptor proteins into a circuit which when exposed to different light wavelengths produce a specific pigments (ref).Their system was used to express, red (gusA), blue (lacZ) and green ((bFMO) , proteins in response to red blue and green light respectively. (figure 1)
Aims
In our design, we aim to activate the production of a biomaterial through an AND gate. For our design we used the multi-enzyme pathway formation of PHB.
- Create and AND gate using two different light wavelengths
- See production of a biomaterial when activated at the specific location where two lights are shone.
- Create a modular desing that can be used with different biomaterials.
Design
We used two wavelengths of light to activate gene expression: red and blue. The photosensors we used are previously characterised parts from the iGEM registry.
SENSING RED LIGHT
The Cph8 photoreceptor system was used as a red light sensing module. This system contains the Cph8 fusion protein created with the photoreceptor Cph1, originating from Cyanobacteria, and the EnvZ histidine kinase native in E.coli. The light sensing unit is connected to transcription via the EnvZ-OmpR signalling pathway, with phosphorylated OmpR acting as a transcription factor. When exposed to Red light, the protein is inactivated and the signal cascade is repressed.
SENSING BLUE LIGHT
The YF1/FixJ photo-sensing system was used as a blue light sensing module. This system contains a fusion protein consisting of the YF1 protein is a fusion protein consisting of the LOV blue light sensor domain of Bacillus subtilis and the heme-binding PAS sensor domain of FixL from Bradyrhizobium japonicum. The light sensing domain is connected to transcription via the FixL/FixJ signalling pathway, with phosphorylated FixJ acting as a transcription factor. When exposed to blue light, the protein is inactivated and the signal cascade is repressed.
These two light sensors were used to activate two repressors: phIF by blue light and CI by the red light that then act on components that complete the T7 RNAP core, which in turn act to allow production of the genes of interest. 4 plasmids were used to express the system:
| Role in circuit | Components | Backbone (resistance, ori) | |
|---|---|---|---|
| Plasmid One | Photoreceptors: YF1/FixL and Cph8 | YF1/FixL and Cph8 | CamR, colE1 |
| Plasmid Two | Repressor activated by YF1/FixL: phIF | phIF repressorin front of FixJ promotor. | SpecR, p15A |
| Plasmid Three | Repressor activated by Cph8: CI | CI repressor in front of OmpR promotor. photobilins: ho1 and pcyA |
TrimR, incW |
| Plasmid Four | Genes of interest (Biomaterials), | PhaA and PhaB under K1F T7 RNAP promoterm and PhaC under T3 Promoter | AmpR, pSC101 |
Sub-title5
Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
bettencourt.igem2017@gmail.com
bettencourt.igem2017@gmail.com 
