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Team:Washington/LightSensing
From 2013.igem.org
Contents |
Background
The optimization of bacterial compound expression has its roots in metabolic pathways. Often, the production pathways of multiple precursor compounds require tuning before a synthetic system can work at full efficiency. As a result, the use of different light wavelengths to manipulate expression levels was of interest to the 2012 iGEM Team. Light expression is cheap relative to chemical methods. Moreover, light expression systems can undergo fine manipulation through changes in intensity, and can undergo rapid removal of inputs in a simple, straightforward manner. Finally, many light expression systems are reversible, depending on the wavelength of light used. The 2012 iGEM Team attempted to modulate expression of beta-galactosidase using a green light sensor and red light sensor. The final results were unsatisfactory; therefore, the 2013 iGEM Team sought to improve and refine compound expression through the use of the green and red light sensors.
Methods
Our planned light expression systems revolved around multiple gene inserts. PLPCB expresses the chromophore PCB. When PCB is expressed alongside CcaS, green light at a wavelength of 535nm causes increased autophosphorylation of CcaS, which phosphorylates response regulator CcaR. CcaR binds to Pcpcg2, expressing GFP. For the red light systems, PCB is used alongside pcph8, a protein that is phosphorylated by 705nm light and dephosphorylated by 650nm light. When phosphorylated, pcph8 phosphorylates OmpR, which activates transcription from PompC. In conjunction with a genetic inverter, this system was used to produce beta-galactosidase or GFP as an output.
Cloning
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Our System
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