Team:Yale

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<b>Nature’s Antifreeze: Microbial Expression and Characterization of a Novel Insect Antifreeze Protein for De-icing Solutions</b><br /><br />
<b>Nature’s Antifreeze: Microbial Expression and Characterization of a Novel Insect Antifreeze Protein for De-icing Solutions</b><br /><br />
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             Antifreeze proteins have applications in cryopreservation of food, cells, and organs, as well as in cryosurgery and agriculture. The purpose of this study was to express, purify, and characterize a novel, hyperactive antifreeze protein recently isolated from the Siberian beetle, Rhagium inquisitor (RiAFP). Large scale (150mg/L), stable production of RiAFP and a RiAFP-GFP fusion protein was achieved in E. coli. Proteins were purified using Ni-NTA affinity chromatography. E. coli expressing RiAFP exhibited increased survival post-freezing. RiAFP inhibited ice recrystallization in both splat and capillary assay. To optimize the activity of the hypothesized RiAFP ice binding site, we are using directed evolution through multiplex automated genome engineering (MAGE). Finally, we are further optimizinge our crystallization conditions for RiAFP to better understand the structure-function relationship, as well as conducting post-freezing survival assays in C. elegans.
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             Antifreeze proteins have applications in cryopreservation of food, cells, and organs, as well as in cryosurgery and agriculture. The purpose of this study was to express, purify, characterize, and optimize a novel, hyperactive antifreeze protein recently isolated from the Siberian beetle, Rhagium inquisitor (RiAFP). Large scale (150mg/L), stable production of RiAFP and a RiAFP-GFP fusion protein was achieved in E. coli. Proteins were purified by Ni-NTA affinity chromatography. E. coli expressing RiAFP exhibited increased survival post-freezing. RiAFP inhibited ice recrystallization in a dose-dependent manner. RiAFP also improved tissue morphology of rat livers post-freezing. Preliminary results indicate that RiAFP may have a cryoprotective effect in C. elegans. To optimize the activity of the hypothesized RiAFP binding site, we used directed evolution through multiplex automated genome engineering and are currently screening for mutants with enhanced properties. Finally, to better understand the structure-function relationship, we have generated promising crystals of RiAFP for x-ray crystallography and are now optimizing crystallization conditions.
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                 <p>We expressed, purified, characterized and have begun optimizing the novel, hyperactive <i>Rhagium inquisitor</i> antifreeze protein using a variety of synthetic and molecular biology techniques.</p>
                 <p>We expressed, purified, characterized and have begun optimizing the novel, hyperactive <i>Rhagium inquisitor</i> antifreeze protein using a variety of synthetic and molecular biology techniques.</p>
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                 <div id="center"><a href="https://2011.igem.org/Team:Yale/Notebook/Week1"><img src="https://static.igem.org/mediawiki/2011/a/ac/YaleBut2.png" /></a></div>
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                 <div id="center"><a href="https://2011.igem.org/Team:Yale/Protocols"><img src="https://static.igem.org/mediawiki/2011/a/ac/YaleBut2.png" /></a></div>
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Latest revision as of 00:53, 29 September 2011

iGEM Yale


Nature’s Antifreeze: Microbial Expression and Characterization of a Novel Insect Antifreeze Protein for De-icing Solutions

Antifreeze proteins have applications in cryopreservation of food, cells, and organs, as well as in cryosurgery and agriculture. The purpose of this study was to express, purify, characterize, and optimize a novel, hyperactive antifreeze protein recently isolated from the Siberian beetle, Rhagium inquisitor (RiAFP). Large scale (150mg/L), stable production of RiAFP and a RiAFP-GFP fusion protein was achieved in E. coli. Proteins were purified by Ni-NTA affinity chromatography. E. coli expressing RiAFP exhibited increased survival post-freezing. RiAFP inhibited ice recrystallization in a dose-dependent manner. RiAFP also improved tissue morphology of rat livers post-freezing. Preliminary results indicate that RiAFP may have a cryoprotective effect in C. elegans. To optimize the activity of the hypothesized RiAFP binding site, we used directed evolution through multiplex automated genome engineering and are currently screening for mutants with enhanced properties. Finally, to better understand the structure-function relationship, we have generated promising crystals of RiAFP for x-ray crystallography and are now optimizing crystallization conditions.

We demonstrate that the Rhagium inquisitor antifreeze protein possesses great potential for applications requiring freeze resistance or the control of ice growth and morphology.

We expressed, purified, characterized and have begun optimizing the novel, hyperactive Rhagium inquisitor antifreeze protein using a variety of synthetic and molecular biology techniques.

A second year entrant to the iGEM competition, the Yale iGEM team is comprised of students from a wide range of backgrounds connected by a common passion for synthetic biology.

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