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 />
             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.
             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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Revision as of 03:15, 26 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, 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.

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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