Team:Yale/Project

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iGEM Yale

Project Overview Cloning: o Fourteen new biobricks were successfully cloned. Four of which were cloned into in the pSB1C3 vector and submitted to the registry. The type III ZeAFP protein and TmAFP 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 RiAFP biobrick, as very little is currently known about the structure or function of this protein. All parts were verified by fully sequencing them by our team. 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). - 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. - 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. - Characterization of function: o 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 35% 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. o 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. o 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. o Rat liver cryoprotection: Darren, results are coming soon for this. - MAGE: Optimization of antifreeze protein: o 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. 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. - 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. - Outreach o Team Amsterdam contacted us during the summer requesting biobricks of RiAFP, ZeAFP, and TmAFP to incorporate in their project. We sent them three parts for them to use in their icE. Coli project. o 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. o We donated competent Origami B cells to another group of researchers from Yale. 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.

>> go to more about antifreeze protein

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+        <p><b>Project Overview</b>
-            A number of different proteins have evolved to interfere with ice crystal growth. These are known as antifreeze proteins (AFPs), thermal hysteresis proteins, ice structuring proteins, or ice binding proteins. Antifreeze proteins inhibit ice recrystallization and nucleation, modify ice morphology, and display thermal hysteresis – the depression of the freezing point of water without altering the melting point (Bar et al., 2006). The activity of antifreeze proteins varies from about 1oC depression of freezing temperature in fish to 5-7oC depression in the hemolymph of many overwintering insects (Strom et al., 2006). Antifreeze proteins have a variety of applications in cryopreservation of food, cells, and organs, as well as in cryosurgery, agriculture, and as non-polluting de-icing agents. These compounds are more than 300 times more effective in preventing freezing than conventional chemical antifreezes at the same concentrations (Graham, 1997).
+              Cloning:
-       </p>
-<p>
-While moderately active fish AFPs are already being used in industry (Unilever and Breyers, for example incorporate fish AFPs into some of their American ice creams to allow for production of very creamy, dense, reduced fat ice cream with fewer additives), to the best of our knowledge, hyperactive AFPs have not been utilized for applications outside of basic science. This may seem surprising at first, since the Tenebrio molitor antifreeze protein has up to 100 times the specific activity of fish antifreeze proteins (Graham, 1997). One of the main reasons for the lack of use of hyperactive antifreeze proteins in industry is that production of these proteins is prohibitively expensive and currently inefficient compared to moderate fish AFPs. Recombinant expression of most hyperactive antifreeze proteins results in inclusion bodies of largely inactive material and requires laborious and expensive refolding protocols.
-</p>
-<p>
+o   Fourteen new biobricks were successfully cloned. Four of which were cloned into in the pSB1C3 vector and submitted to the registry. The type III ZeAFP protein and TmAFP 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 RiAFP biobrick, as very little is currently known about the structure or function of this protein. All parts were verified by fully sequencing them by our team.
-Recent studies have isolated a novel, hyperactive antifreeze protein from the hemolymph of the cold tolerant Siberian cerambycid beetle, Rhagium inquisitor (RiAFP) (Kristiansen, 2011). The only reported details of this protein thus far are its primary sequence, and its thermal hysteresis activity. Thermal hysteresis measurements indicate that RiAFP is the most active AFP isolated thus far. The protein seems to have significantly fewer disulfide bonds and fewer repeated sequences compared to other active insect AFPs. Whereas the Tenebrio molitor antifreeze protein has a cysteine content of 19% and a total of eight disulfide bonds in its core, the Rhagium Inquisitor AFP has a cysteine content of <1% and has only one disulfide bond (Kristiansen, 2011). These features, together with its small size (12kDa), lead our team to hypothesize that RiAFP would make a good candidate for recombinant expression. Our hope is to demonstrate that RiAFP is an attractive reagent for applications requiring freeze resistance or the control of ice growth and morphology. Importantly, we provide some of the first functional characterizations of this protein, since little is known about its structure or activity other than its thermal hysteresis properties.
-</p>
-<p>
+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).
-Recently, multiplex automated genome engineering (MAGE) was developed for large-scale programming and evolution of cells (Wang, 2009). MAGE allows rapid generation of sequence diversity across a large population of cells through oligo-mediated allelic replacement (Wang, 2009). Synthetic oligonucleotides are repeatedly and continuously introduced to a population of cells and are incorporated at the lagging strand of the replication fork during DNA replication. This approach was previously used to optimize the DXP biosynthesis pathway in E coli to overproduce the industrially important isoprenoid lycopene.
-</p>
-<p>
+-          Expression:
-We designed degenerate oligos to target several sites of the hypothesized ice-binding pocket of the protein. The hypothesized binding site of RiAFP contains stretches riches in alternating Thr and Ala residues, similar to nonhomolgous Thr-rich AFPs. The spacing between ice-binding Thr residues is a close match to the spacing of oxygen atoms on several planes of ice. Twenty two sets of degenerate oligos were designed to insert additional Tx repeats, delete Tx repeats, delete entire TxT segments, and replace regions with degenerate TxTxTxT repeats. After incorporating RiAFP into the genome of the EcNR2 strain (using labda-red recombination) MAGE was used to generate a diverse population of mutants for the antifreeze protein sequence. Multiple freeze thaw cycles were applied as a selective pressure for enhanced survivability after each MAGE cycle.
-</p>
+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.
+-          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.
+-          Characterization of function:
+o   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 35% 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.
+o   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.
+o   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.
+o   Rat liver cryoprotection: Darren, results are coming soon for this.
+-          MAGE: Optimization of antifreeze protein:
+o   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.
+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.
+-          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.
+-          Outreach
+o   Team Amsterdam contacted us during the summer requesting biobricks of RiAFP, ZeAFP, and TmAFP to incorporate in their project. We sent them three parts for them to use in their icE. Coli project.
+o   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.
+o   We donated competent Origami B cells to another group of researchers from Yale.
+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.
+</p>
 <p>
 >> go to more about antifreeze protein</p>
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