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 />
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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 demonstrate that the <i>Rhagium inquisitor</i> antifreeze protein possesses great potential for applications requiring freeze resistance or the control of ice growth and morphology.</p>
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                <div id="center"><a href="https://2011.igem.org/Team:Yale/Project"><img src="https://static.igem.org/mediawiki/2011/2/21/YaleBut1.png" /></a></div>
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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>
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                <p>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. </p>
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                <div id="center"><a href="https://2011.igem.org/Team:Yale/Team"><img src="https://static.igem.org/mediawiki/2011/0/02/YaleBut3.png" /></a></div>
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<li><a href="https://2011.igem.org/Team:Yale">Home</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/Project">Our Project</a></li>
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<li><a href="https://2011.igem.org/Team:Yale/team">Our Team</a></li>
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<h3> Introduction </h3>
 
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''Please read the attached file.''
 
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<br/>
 
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<html><a href="https://static.igem.org/mediawiki/2011/9/95/Yale_iGEM_2011_Outline.ppt">Summary and Outline of 2011 Yale iGEM (PPT)</a></html>
 
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<h3>  Recent Updates </h3>
 
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''In the past month, we have:"
 
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1) Successfully expressed four different antifreeze protein constructs in BL21 and Origami strain E. coli cells (verified by observing green pellets, fluorimetry, SDS/PAGE Gel electrophoresis, and Western blotting)
 
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2) Demonstrated enhanced cold survivability at -20° C in cells expressing antifreeze protein (microbial plating assay)
 
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3) Verified sequences of all antifreeze protein constructs
 
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4) Cloned three antifreeze protein constructs into the standard iGEM BioBrick vector
 
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We have received the synthetic novel Siberian beetle antifreeze protein gene that was sponsored by IDT and have begun cloning and subsequent experiments on this construct.  We are also developing an ice recrystallization inhibition assay and finishing the cloning of several more BioBricks. Soon, we hope to begin optimizing expression of the antifreeze proteins using directed evolution technologies.
 
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<!--- The Mission, Experiments --->
 
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<h3> Active Directory </h3>
 
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{| style="color:#1b2c8a;background-color:#0c6;" cellpadding="3" cellspacing="1" border="1" bordercolor="#fff" width="62%" align="center"
 
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!align="center"|[[Team:Yale|Home]]
 
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!align="center"|[[Team:Yale/Team|Team]]
 
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!align="center"|[https://igem.org/Team.cgi?year=2011&team_name=Yale Official Team Profile]
 
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!align="center"|[[Team:Yale/Project|Project]]
 
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!align="center"|[[Team:Yale/Parts|Parts Submitted to the Registry]]
 
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!align="center"|[[Team:Yale/Modeling|Modeling]]
 
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!align="center"|[[Team:Yale/Notebook|Notebook]]
 
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!align="center"|[[Team:Yale/Safety|Safety]]
 
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!align="center"|[[Team:Yale/Attributions|Attributions]]
 
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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.

Retrieved from "http://2011.igem.org/Team:Yale"