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                    <li class='selected'        ><a href="https://2012.igem.org/Team:UC_Davis">Page</a></li>

 

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<h1> Protein Engineering </h1>

Beyond the general circuit outline for our degradation of PET, there are also two different protein engineering projects we performed. In the first project we constructed a more effective and higher yield form of the LC-Cutinase protein. In a second less related project, we mutated the lac-repressor in order to change its ligand specificity. We changed its specificity to bind a harmful chemical called diuron instead of the natural IPTG in order to create a unique biosensor system.  

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<h1>LC-Cutinase Engineering:</h1>

We had two goals with the engineering of cutinase: replicate mutations made in the paper by Sulaiman et. al., which consisted of three residues mutated to alanines, as well as to generate our own chosen mutations. Before we decided to replicate Sulaiman’s mutations we initially wanted to validate if we could expect the same results in our protein with the same mutations. In order to do this we wanted to compare best fit models (proteins with known structure that best fit an input sequence) generated for LC-Cutinase’s sequence and Sulaiman’s Tfu_0883’s sequence. Replicating Sulaiman we used Swiss-Model to generate a best fit model for LC-Cutinase, resulting in the protein 3visB with 61% homology. In Sulaiman’s paper they received 1jfr, a model that was second best in LC-Cutinase’s output with 55% homology. This similarity between generated models and high homology rate gave support to the expectation of similar results from the same mutations.

Further validation was found using a multiple sequence alignment between: LC-Cutinase, 3visB, Tfu_0883 and 1jfr (show pic). The alignment showed strong homology in all active site residues as well as between two of the three residues targeted by Sulaiman et. al. for mutation. The third targeted residue, though significantly different in Tfu_0883 compared to in LC-Cutinase, was expected to result in similar protein activity gains upon mutation to alanine. This was found by loading both models for 3visB and 1jfr in Pymol and assessing that the residues held similar placement above the active site. Their placement, as well as the placement of the other two targeted residues, showed that mutation to a smaller residue, alanine, would result in better binding ability of the active site.  

Mutation results:

Diuron biosensor:

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<hp>References</hp>

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