Team:Yale/Project
From 2011.igem.org
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Cloning: | Cloning: | ||
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o We improved upon the existing TmAFP biobrick in the registry. Team Tokyo Tech 2009 previously submitted a biobrick of the Tenebrio Molitor antifreeze protein, TmAFP (BBa_K193209). However, this biobrick contains an internal EcoRI site and is therefore incompatible with BBF RFC 10. Additionally, the TmAFP protein in the Tokyo Tech part seems to be truncated. Our TmAFP part is RFC10 compatible, and includes the full sequence of this protein. Our sequence was obtained from the Fass Lab, and is reported on in the following paper: Bar, M., Bar-Ziv, R., Scherf, T. & Fass, D. Efficient production of a folded and functional, highly disulfide-bonded [beta]-helix antifreeze protein in bacteria. Protein Expression and Purification 48, 243-252 (2006). | o We improved upon the existing TmAFP biobrick in the registry. Team Tokyo Tech 2009 previously submitted a biobrick of the Tenebrio Molitor antifreeze protein, TmAFP (BBa_K193209). However, this biobrick contains an internal EcoRI site and is therefore incompatible with BBF RFC 10. Additionally, the TmAFP protein in the Tokyo Tech part seems to be truncated. Our TmAFP part is RFC10 compatible, and includes the full sequence of this protein. Our sequence was obtained from the Fass Lab, and is reported on in the following paper: Bar, M., Bar-Ziv, R., Scherf, T. & Fass, D. Efficient production of a folded and functional, highly disulfide-bonded [beta]-helix antifreeze protein in bacteria. Protein Expression and Purification 48, 243-252 (2006). | ||
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- Expression: | - Expression: | ||
o Large scale production RiAFP was achieved. This is the first reported recombinant expression of RiAFP. Expression was verified by SDS-PAGE, Western blotting, and observing green pellets. We also verified expression of our TmAFP biobrick by SDS-PAGE, Western blotting, and flourimetry. Importantly, RiAFP was expressed in soluble form in very high quantities (150mg/L), as determined by UV-vis, Protein A280, and a Bradford Assay. Several temperatures, length of induction, and IPTG concentrations were investigated to optimize yields. The high level of expression is significant because expression of other comparably active insect antifreeze proteins, such as TmAFP, results in inclusion bodies of largely inactive material and requires expensive refolding protocols. This has limited the use of hyperactive insect antifreeze proteins in industry thus far. We believe that RiAFP is an attractive potential commercial reagent for applications requiring freeze resistance. | o Large scale production RiAFP was achieved. This is the first reported recombinant expression of RiAFP. Expression was verified by SDS-PAGE, Western blotting, and observing green pellets. We also verified expression of our TmAFP biobrick by SDS-PAGE, Western blotting, and flourimetry. Importantly, RiAFP was expressed in soluble form in very high quantities (150mg/L), as determined by UV-vis, Protein A280, and a Bradford Assay. Several temperatures, length of induction, and IPTG concentrations were investigated to optimize yields. The high level of expression is significant because expression of other comparably active insect antifreeze proteins, such as TmAFP, results in inclusion bodies of largely inactive material and requires expensive refolding protocols. This has limited the use of hyperactive insect antifreeze proteins in industry thus far. We believe that RiAFP is an attractive potential commercial reagent for applications requiring freeze resistance. | ||
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- Purification: | - Purification: | ||
o Purification of RiAFP was achieved in high quantities. We used Ni-NTA affinity chromatography followed by size exclusion chromatography (FPLC) to purify RiAFP. Purity was verified by SDS-PAGE. Since our RiAFP-GFP fusion protein expressed at much higher levels compared to RiAFP by itself (likely because GFP increases the solubility of the protein), we first purified the GFP-TEV-RiAFP fusion protein, exposed pure fractions to TEV protease, and conducted size exclusion chromatography to isolate RiAFP. | o Purification of RiAFP was achieved in high quantities. We used Ni-NTA affinity chromatography followed by size exclusion chromatography (FPLC) to purify RiAFP. Purity was verified by SDS-PAGE. Since our RiAFP-GFP fusion protein expressed at much higher levels compared to RiAFP by itself (likely because GFP increases the solubility of the protein), we first purified the GFP-TEV-RiAFP fusion protein, exposed pure fractions to TEV protease, and conducted size exclusion chromatography to isolate RiAFP. | ||
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- Characterization of function: | - Characterization of function: | ||
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o Rat liver cryoprotection: Darren, results are coming soon for this. | o Rat liver cryoprotection: Darren, results are coming soon for this. | ||
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- MAGE: Optimization of antifreeze protein: | - MAGE: Optimization of antifreeze protein: | ||
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o Four hundred and thirty four million predicted combinatorial genomic variants of the RiAFP gene have been generated thus far. This is a diverse population of insertion, deletion, and mismatch mutants for the hypothesized ice-binding region of RiAFP. Using multiplex automated genome engineering, we were able to generate more potential “biobricks” than currently exist in the iGEM registry! We are in the process of screening mutants for enhanced survivability and performing iterations of the MAGE cycle. | o Four hundred and thirty four million predicted combinatorial genomic variants of the RiAFP gene have been generated thus far. This is a diverse population of insertion, deletion, and mismatch mutants for the hypothesized ice-binding region of RiAFP. Using multiplex automated genome engineering, we were able to generate more potential “biobricks” than currently exist in the iGEM registry! We are in the process of screening mutants for enhanced survivability and performing iterations of the MAGE cycle. | ||
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- X-ray crystallography: | - X-ray crystallography: | ||
o Preliminary and promising “fuzzy-ball” crystal hits for RiAFP have been generated. We are in the process of further optimizing conditions for crystallization. Additionally, we are in the process of using site-directed mutagenesis to replace methionine residues by Se-met in order to solve the crystallographic phase problem. | o Preliminary and promising “fuzzy-ball” crystal hits for RiAFP have been generated. We are in the process of further optimizing conditions for crystallization. Additionally, we are in the process of using site-directed mutagenesis to replace methionine residues by Se-met in order to solve the crystallographic phase problem. | ||
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- Outreach | - Outreach | ||
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o We donated a TmAFP plasmid to a researcher in the Elizabeth Rhoades lab at Yale looking for a highly disulfide bonded protein to use as a control. | o We donated a TmAFP plasmid to a researcher in the Elizabeth Rhoades lab at Yale looking for a highly disulfide bonded protein to use as a control. | ||
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>> go to more about antifreeze protein</p> | >> go to more about antifreeze protein</p> | ||
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Revision as of 15:43, 26 September 2011