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Team:Yale/Project
From 2011.igem.org
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<b>MAGE: Optimization of antifreeze protein:</b> | <b>MAGE: Optimization of antifreeze protein:</b> | ||
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| - | <li>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. | + | <li><img src="https://static.igem.org/mediawiki/2011/b/b4/Yale-MAGE.jpg" style="float:right; margin: 5px; padding-left:10px;" />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. |
</li> | </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>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>To read more about our MAGE experiments, please visit <a href="https://2011.igem.org/Team:Yale/Project/MAGE">our MAGE page.</a></li> | ||
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Revision as of 14:02, 28 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 antifreeze property. All parts were verified by fully sequencing them by our team using Yale's Keck Biotechnology center.
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.
