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Team:Yale/Project
From 2011.igem.org
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<li>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. </li> | <li>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. </li> | ||
| + | <li> (Right) Figure modified from Wang et al., 2009. </li> | ||
<li>To read more about our MAGE experiments, please visit <a href="https://2011.igem.org/Team:Yale/Project/MAGE">our MAGE page.</a></li> | <li>To read more about our MAGE experiments, please visit <a href="https://2011.igem.org/Team:Yale/Project/MAGE">our MAGE page.</a></li> | ||
</ul> | </ul> | ||
Revision as of 03:03, 29 September 2011
Fourteen new biobricks were successfully cloned. Four of which were cloned into in the pSB1C3 vector and submitted to the registry. The type III Zoarces elongatus antifreeze protein (ZeAFP) and Tenebrio molitor antifreeze protein (TmAFP) have been previously characterized in the literature and are now in the registry for other teams to use. Our project primarily focused on extensively characterizing our novel Rhagium inquisitor antifreeze protein (RiAFP) biobrick, as very little is currently known about the structure or function of this protein, besides its hyperactive thermal hystersis property. All parts were verified by fully sequencing them by our team using Yale's Keck Biotechnology center.
Large scale production and subsequent purification of 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 the TmAFP BioBrick by SDS-PAGE, Western blotting, and flourimetry. This characterization was not previously done by Tokyo Tech, a team that had previously worked with TmAFP. 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.
Purification of RiAFP was achieved in high quantities. We tried several methods of purification, including cobalt, loose-beaded and pre-packed Ni-NTA metal affinity chromatography to purify RiAFP, which had a C-terminal 6-His tag. To further ensure purity, size exclusion chromatography via FPLC was completed. 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. A novel purification method to take advantage of the active ice-binding sites of antifreeze protein in using ice-affinity chromatography, as a facile, inexpensive method to purify any AFP fusion constructs, was also explored.
E. coli expressing RiAFP exhibit increased survival post-freezing. Relative freezing tolerance was calculated by determining number of viable cells before and after freezing from three biological replicates. Survival rates for cells expressing RiAFP increased by up to 60% relative to the vector control (p < 0.05). Uninduced transgenic cells also exhibited increased survivability, likely due to leaky expression in the BL21*(DE3) strain.
RiAFP was successfully integrated into the genome of the EcNR2 strain. In vivo genomic engineering was performed using the lambda-red recombination system. The RiAFP gene was linked to kanamycin by crossover PCR. dsDNA recombination efficiency data from Conjugative Assembly Genome Engineering experiments (Isaacs, et al 2011) was used to identify 3 highly recombinogenic sites in the EcNR2 genome.
As a recently discovered hyperactive antifreeze protein, RiAFP has thus far only had its primary protein structure, with no predicted homologies/models for its secondary structure. Thus, we have begun protein crystallography to determine the structure of RiAFP and subsequent MAGE-generated variants to understand the source of its potent antifreeze activity. Preliminary and promising “fuzzy-ball” crystal hits for RiAFP have been obtained thus far. 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.
