"
Team:Washington/LightSensing
From 2013.igem.org
(→Our System) |
(→Our System) |
||
| Line 122: | Line 122: | ||
| <p dir="ltr">Output inverter</p> | | <p dir="ltr">Output inverter</p> | ||
|} | |} | ||
| + | <html> | ||
</div> | </div> | ||
</div> | </div> | ||
Revision as of 04:45, 25 September 2013
Contents |
Background
Recently, techniques have been developed that afford researchers the ability to control gene expression with light. To date, sensors that respond to red, green, and blue wavelengths of light have been reported. Light induced expression of genes has a number of advantages over chemical induction methods. For example, light induced expression is cheaper than chemical methods, can be used to finely tune expression levels through modulations of intensity, and can be rapidly removed -- a feature lacking in chemical induction systems. Furthermore, many light expression systems are reversible, depending on the wavelength of light used. Finally, the ability to control the expression levels of multiple genes of interest simultaneously could have far reaching implications for tuning biosynthetic pathways. The use of chemical inducers for this application would likely prove to be difficult and cost prohibitive, but multichromatic gene induction represents a potential solution to this problem. To this end, the 2013 University of Washington iGEM team chose to continue a project we began in 2012 that is aimed at the development and characterization of a set of tools that bring multichromatic gene expression into the realm of possibility for synthetic biologists.
In 2012, the University of Washington iGEM Team developed a freely available app for the Android operating system that affords spatiotemporal control over light wavelengths and intensities. When installed on an appropriate tablet (see XXXXX section) the app could be useful for carrying out complex light induced expressions. In 2012, we were unable to fully characterize the app’s ability to control expression of light inducible genes. Thus, the 2013 iGEM Team’s goals include fully characterizing the app’s ability to control gene expression with light, and the development of a toolkit of light induced expression biobricks that are confirmed to work with the tablet app.
Our System
The light induced expression systems we used both require multiple protein components. In general, a small molecule chromophore is synthesized in the cell, and acts as a substrate for a transmembrane light sensing protein. When the protein sensor bound-chromophore-bound is exposed to light of the correct wavelength, a conformational change is induced in the sensor protein which in turn causes the phosphorylation of a transcription factor. The active form of the transcription factor then modulates the expression of any gene downstream of its cognate promoter. We worked with a red and green light inducible gene expression system this year. Both systems utilize the same chromophore, phycocyanobilin, but differ in the transmembrane light sensing proteins that bind the chromophore, and the downstream regulator proteins. The systems are described graphically in Figure 1.
Figure 1: Generalized Light-Induced Expression System
The genes required to construct a functional light induced protein expression system were generously provided by Jeff Tabor at Rice University. Table 1.1 lists the genes required for each system and their respective functions. In order to construct a functional light responsive system, a multiple genes are transformed into E. coli. Regardless of the system in question, the minimal components required are: 1) genes utilized in the synthesis of the chromophore, 2) a transmembrane light sensing protein, and 3) response regulators which are activated by from the light sensitive proteins, and ultimately function as transcription factors.
| Source | Component | Function |
| Chromophore: | Plac/ara-HO1-PcyA | PCB chromophore expression plasmid |
| pPLPCB | Plac/ara | Hybrid lactose and arabinose promoter |
| pPLPCB | HO1 | Heme oxygenase |
| pPLPCB | PcyA | Phycocyanobillin ferridoxin oxidoreductase |
| Green light sensor: | [PCpcG2-RBS-sfGFP-CcaS](rev)-CcaR | Sense green light with GFP response |
| pJT119b | CcaS | Green light receptor |
| pJT119b | CcaR | Response regulator |
| pJT119b | PcpcG2 | Synechocystis phycocyanoybillin promoter |
| pJT119b | BBa_B0034 | Strong Elowitz RBS |
| pJT119b | sfGFP | Superfolder GFP reporter |
| Red light sensor: | | Sense red light |
| pCPH8 | PLTetO1+CPH8 | |
| pCPH8 | PLTetO1 | Tet repressible promoter constuitive in JT2 |
| pCPH8 | CPH8 | Red light receptor EnvZ fusion |
| pEO100c | POmpC-BBa_B0034-sfGFP | Red light inactivated GFP output |
| pEO100c | POmpC | OmpC promoter |
| pEO100c | BBa_B0034 | Strong Elowitz RBS |
| pEO100c | sfGFP | Superfolder GFP reporter |
| pJT106b | PLambda-RBS-LacZ-T1-POmpC-CI-T1 | Red light activated GFP output |
| pJT106b | Plambda-CI-Term | Output inverter |
