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Team:UCL PG/Project
From 2013.igem.org
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| - | In order to turn the dual light sensitive chimera to full usability, optimization of the protein structure is necessary. Rational approach is both time consuming and challenging. Alternatively, directed evolution is tedious but they always work and it is where most of the time significant "surprises" are be discovered. An elegant solution of it is | + | In order to turn the dual light sensitive chimera to full usability, optimization of the protein structure is necessary. To achieve that, there are generally two ways: Rational Design or Directed Evolution. Rational approach is both time consuming and challenging unless the target protein had been well studied. Alternatively, directed evolution is tedious but they always work and it is where most of the time significant "surprises" are be discovered. In our case where we are optimizing a chimera, rational approach is beyond interpretation. Hence, we setup a journey to engineer an evolution circuit.<br> |
| + | An elegant solution of it is to introduce a GFP reporter with Omp promoter to respond to level of phosphorylated OmpR. Omp promoter capture the signal and output it as GFP expression which correspond to the strength of response to blue or red light. In short, the better is the mutant, the greener is the cell. | ||
<img style="border-radius:25px;border-width:10px;border-style:solid;border-color:#636363;" src="https://dl.dropboxusercontent.com/u/45600819/SpectraWiki/HomeTest/bigpicturecircuit.jpg"> | <img style="border-radius:25px;border-width:10px;border-style:solid;border-color:#636363;" src="https://dl.dropboxusercontent.com/u/45600819/SpectraWiki/HomeTest/bigpicturecircuit.jpg"> | ||
Revision as of 00:21, 4 October 2013
Live had been on earth for 3.6 billion years. Over time living-beings evolved, merged, devoured and mutated. Cells are having ever massive networks and interactions that is far beyond foreseeable. And now we are only slowly discovering the secret of life. Through this sea of proteins, fluorescent and bioluminescent proteins are like light houses that allows scientist to hold on with faith. As the saying goes, "Seeing is believing". Therefore, we decided to dedicate our iGEM project to this wonderful technology.
We focus on producing a dual light sensitive circuit in order to evolve a red fluorescent protein, mKeima. At the same time we wish to establish a noble concept of "Evolution circuit" and associated high-throughput evolution protocols.
The architecture of dual light sensor revolves around the trick of overlapping fluorescent spectra. In this project, we improvised the overlapping of mKeima emission and Cph8 excitation wavelength. They are fused together, becoming Cph8-mKeima chimera to bring them to close proximity. Cph8 is a fusion protein which was firstly used for bacterial photography.It is able to relay signal to E.coli via phosphorylation of OmpR.
In order to turn the dual light sensitive chimera to full usability, optimization of the protein structure is necessary. To achieve that, there are generally two ways: Rational Design or Directed Evolution. Rational approach is both time consuming and challenging unless the target protein had been well studied. Alternatively, directed evolution is tedious but they always work and it is where most of the time significant "surprises" are be discovered. In our case where we are optimizing a chimera, rational approach is beyond interpretation. Hence, we setup a journey to engineer an evolution circuit.
An elegant solution of it is to introduce a GFP reporter with Omp promoter to respond to level of phosphorylated OmpR. Omp promoter capture the signal and output it as GFP expression which correspond to the strength of response to blue or red light. In short, the better is the mutant, the greener is the cell.
There are three compartments we are building: PCB generator ()
Part I
Results
Part II
Results
Part III
Results
