Team:Yale/Project

From 2011.igem.org

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<b>Characterization of function:</b>
<b>Characterization of function:</b>
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<li>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.
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<li><img src="https://static.igem.org/mediawiki/2011/2/2b/Yale-Assays.jpg" style="float:right; margin: 5px; padding-left:10px;" />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.
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<li>RiAFP inhibits ice recrystallization in a concentration-dependent manner. Serial dilutions of purified RiAFP inhibited ice crystal formation in a dose-dependent manner compared to BSA control solutions. Two ice recrystallization inhibition assays, the splat assay and the capillary assay, were used to demonstrate this effect.
<li>RiAFP inhibits ice recrystallization in a concentration-dependent manner. Serial dilutions of purified RiAFP inhibited ice crystal formation in a dose-dependent manner compared to BSA control solutions. Two ice recrystallization inhibition assays, the splat assay and the capillary assay, were used to demonstrate this effect.
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<li>Rat liver cryoprotection: Darren, results are coming soon for this.
<li>Rat liver cryoprotection: Darren, results are coming soon for this.
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<li>To read more about our functional assays, please visit <a href="https://2011.igem.org/Team:Yale/Project/Assays">our BioBricks and proteins page.</a></li>
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Revision as of 13:54, 28 September 2011

iGEM Yale

Cloning:
  • 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.
  • We improved upon the existing TmAFP biobrick in the registry. Team Tokyo Tech 2009 previously submitted a biobrick of the Tenebrio olitor antifreeze protein, TmAFP (BBa_K193209). However, this biobrick contains an internal EcoRI site and is therefore incompatible with BBF RFC10. Additionally, the TmAFP protein in the Tokyo Tech part seems to be truncated; we have characterized and documented these details on their parts page. 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).
  • To read more about our BioBricks, please visit our BioBricks and proteins page.
Expression:
  • 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.
  • To read more about our protein expression, please visit our BioBricks and proteins page.
Purification:
  • 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.
Characterization of function:
  • 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 inhibits ice recrystallization in a concentration-dependent manner. Serial dilutions of purified RiAFP inhibited ice crystal formation in a dose-dependent manner compared to BSA control solutions. Two ice recrystallization inhibition assays, the splat assay and the capillary assay, were used to demonstrate this effect.
  • RiAFP protein was observed absorbed on ice surfaces. The tagging of RiAFP to GFP allowed visualization of adsorbed proteins on ice surfaces. Intensely fluorescent edges are consistent with surface adsorption of the AFP.
  • Rat liver cryoprotection: Darren, results are coming soon for this.
  • To read more about our functional assays, please visit our BioBricks and proteins page.
MAGE: Optimization of antifreeze protein:
  • 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.
  • 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.
X-ray crystallography:
  • 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.
Outreach
  • Team Amsterdam found out during the summer that we were working on antifreeze proteins and requested biobricks of RiAFP, ZeAFP, and TmAFP for them to incorporate in their project. We sent them three parts for them to use in their icE. Coli project. [https://2011.igem.org/Team:Amsterdam/Project/Description] This was done before our parts were submitted to the iGEM registry.
  • We are donating purified RiAFP to the Fikrig Lab at Yale to use as a positive control in their studies on purifying an antifreeze glycoprotein.
  • We donated competent Origami B cells to another group of researchers from Yale.
  • 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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