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Nature’s Antifreeze: Microbial de-icing solutions for ice cream, aircraft and organ transport

Nature’s Antifreeze: Microbial de-icing solutions for ice cream, aircraft and organ transport. Yale iGEM 2011. Challenge:. Achieve large scale, stable extracellular production of highly active antifreeze protein (AFP) using Escherichia coli. Express, characterize, and purify:

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Nature’s Antifreeze: Microbial de-icing solutions for ice cream, aircraft and organ transport

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  1. Nature’s Antifreeze:Microbial de-icing solutions for ice cream, aircraft and organ transport Yale iGEM 2011

  2. Challenge: • Achieve large scale, stable extracellular production of highly active antifreeze protein (AFP) using Escherichia coli. • Express, characterize, and purify: • ZoarceselongatusZeAFP AFP (ZeAFP) • Tenebriomolitor AFP (TmAFP) • A novel, hyperactive Rhagium Inquisitor AFP (RiAFP)

  3. AFPs

  4. Applications

  5. Motivation • Previous iGEM project on eGFP-TmAFP failed to characterize expression • Current AFPs used in industrial applications are far less active than more recently isolated AFPs • Current cost of type I/II/III anti-freeze proteins: • $10,000 / gram • http://www.afprotein.com/products.htm

  6. RiAFP • Cerambycid beetle Rhagium inquisitor isv. cold-tolerant • Hibernates in polar regions • Readily super-cools to -20oC to -30oC • Expresses RiAFP in hemolymph and tissues • Previous studies (Kristiansen et al., 2008 and 2010) have: • isolated protein from hemolymph, • measured anti-freeze activity (TH), • salt enhances anti-freeze activity • determined primary sequence of RiAFP,

  7. Advantages of using RiAFP RiAFP TmAFP SbAFP I SbAFP II Insect AFPs are up to 100x more active than fish AFPs at low temperatures (Tong, et al 2008) Antifreeze activity as a function of concentration of various insect AFPs. (Kristiansen, et al 2010)

  8. Advantages of using RiAFP • Single disulfide bond (less tightly folded compared to other active insect AFPs) • Small protein (136 amino acid; 12kDa) • Novel AFP

  9. Summary of AFPs

  10. Schematic Presentation of Expression Constructs TEV Generously provided by IDT PelB eGFP RiAFP His6 TEV SS eGFP RiAFP His6 SS RiAFP His6 Generously provided by Peter Davies, Queens University PelB ZeAFP His6 eGFP TmAFP His6 Generously provided by Team Tokyo Tech, 2009

  11. Plasmid Construct • All constructs will be in pSB1K3 • High copy, Kan resistance EcoRI Not I XbaI SpeI Not I PstI

  12. Characterization • Basic Characterization: • GFP Fluorescence Assay • SDS/Page (supernatant and pellet) • Western blot (His6 antibody)

  13. Assays – Fikrig Lab • Freeze tolerance survival assay • Induce protein expression in E. coli • Harvest cells, wash in ddH2O, resuspend ddH2O • Plate cells, assay viability, then freeze & thaw tube and assay viability once more • Modified growth assays • Comparison of growth at 4oC induced/uninduced

  14. Assays – Fikrig Lab • Ice recrystallization assay (Graham et al, 2009) Buffer + I. scapularis AGFP Buffer Denatured Buffer + ZeAFP AFP

  15. Assays – Fikrig Lab • Ice morphology assay (DeLuca, et al 1998) • in the absence of protein • in the presence of thioredoxin (10 mg/ml) in 100 mM NH4HCO3 (pH 7.9) • in the presence of maltose-binding protein (20 mg/ml) in 100 mM NH4HCO3 (pH 7.9). Right panel: 100 mM NH4HCO3 (pH 7.9) and mM concentrations: (d) ZeAFP AFP, (e) TRX: ZeAFPAFP, (f) MBP: ZeAFPAFP

  16. (1) Achieve stable, high-level expression of ZeAFP and TmAFP • PCR out ZeAFP from pET20b expression vector clone into iGEM plasmid backbone and submit as biobrick • Induce expression of ZeAFP (on pET20b) in BL21(DE3) • Characterize ZeAFP by SDS/PAGE and/or western • Supernatant/Pellet • Practice survival assays and ice assay • Purification? RBS PelB ZeAFP His6 T7 –IPTG RBS PelB ZeAFP His6 Terminator

  17. (1) Achieve stable, high-level expression of ZeAFP and TmAFP • Transform eGFP-TmAFP-His6 fusion biobrick (Tokyo tech, 2009) into Origami and Bl21 cells • Express at different temperatures • Characterize expression by fluorescence / SDS / Western • Survival Assay • Ice Assay • Purification?

  18. (2) Achieve stable, high-level, extracellular expression of RiAFP • Construct following bricks: TEV RBS PelB eGFP RiAFP His6 Terminator TEV T7 –IPTG RBS PelB eGFP RiAFP His6 Terminator T7 –IPTG RBS PelB RiAFP His6 Terminator TEV Promoter-RBS SS eGFP RiAFP His6 Terminator Promoter-RBS RiAFP His6 Terminator

  19. (2) Achieve stable, high-level, extracellular expression of RiAFP • Characterize expression by fluorescence / SDS / Western • Survival Assay • Ice Assay • Comparison of activity of RiAFP versus eGFP-RiAFP fusion protein – cooperativity? • Purification?

  20. Schematic representation of cooperativity and non-cooperativity • Previous studies suggest that ZeAFP and TmAFP function independently (no cooperativity) • Fusion proteins were slightly more active than the free ZeAFP AFPs (DeLuca, et al 1998) • Other studies on AFGP and type I, ZeAFP AFP from fish have displayed cooperativity (Ben, et al 2006)

  21. Timeline • What we have accomplished thus far: • Received BioBrick library, stabs of ZeAFP AFP and eGFP-TmTHP • Ordered RiAFP gene from IDT DNA (many thanks!) • Minipreps of eGFP-TmAFP, ZeAFP antifreeze, promoter bricks, GFP bricks, terminator bricks, RBS bricks, OmpA, PelB leader sequences • Made Competent DH5-alpha cells • Design of primers to transfer ZeAFP into iGEM standard vector • Practice E. coli survival assay • June: • Preparation of Origami competent cells • PCR out ZeAFP AFP from pET20b vector & ligate into standard iGEM vector • Express and purify eGFP-Tm-THP in Origami and BL21 cells • Fluorescence/SDS/Western • Ice assays for positive controls

  22. Timeline • July: • Clone synthetic R. inquisitor gene into standard iGEM vector • Ligate to various promoters/etc • PCR out RiAFP and clone into standard iGEM vector • Complete ice assays • August: • Spillover • Optimize export quantities using MAGE technique

  23. Important Dates • 6/7/11 Project Defense • 10/8/11 - 10/10/11 iGEM 2011 Regional Jamboree: Americas • 11/5/11 - 11/7/11 iGEM 2011 World Championship Jamboree, MIT

  24. Thank you! • Raymond and Beverly Sackler Institute for Biological, Physical and Engineering Sciences • Yale College Dean’s Office • Professor Farren Isaacs • Professor Nigel Grindley • Graduate Student Advisors • Integrated DNA Technology • W.M. Keck Foundation • Professor Peter Davies • Tokyo Tech 2009 • iGEM

  25. References • Bouvet, V.R., Lorello, G.R. & Ben, R.N. Aggregation of Antifreeze Glycoprotein Fraction 8 and Its Effect on Antifreeze Activity. Biomacromolecules 7, 565-571 (2006). • DeLuca, C.I., Comley, R. & Davies, P.L. Antifreeze proteins bind independently to ice. Biophys J 74, 1502-1508 (1998). • Garnham, C.P. et al. Compound Ice-Binding Site of an Antifreeze Protein Revealed by Mutagenesis and Fluorescent Tagging. Biochemistry 49, 9063-9071 (2010). • 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). • Kristiansen, E., Pedersen, S.A. & Zachariassen, K.E. Salt-induced enhancement of antifreeze protein activity: A salting-out effect. Cryobiology 57, 122-129 (2008). • Kristiansen, E. et al. Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor. Insect Biochemistry and Molecular Biology 41, 109-117 (2011).

  26. Questions?

  27. Extra slides

  28. AFPs depress the freezing T of fish blood several hundred times more efficiently than regular salts

  29. Structure/Mechanism of AFPs • Structures have evolved independently • Activity varies from about 1 °C in blood of polar to 5–7 °C in the hemolymph of many overwintering insects • Various mechanisms have been proposed to explain the activity/interaction between AFPs and ice. • Adsorption-inhibition hypothesis : Gibbs –Thompson effect • Ice binding surfaces are relatively hydrophobic

  30. Previous Studies • Structural characteristics of a novel antifreeze protein from the longhorn beetle Rhagium inquisitor, Insect Biochemistry and Molecular Biology, 2010 • Report on primary structure of a 12.8 kDa AFP from this beetle (RiAFP). • It has a high capacity to evoke antifreeze activity as compared to other known insect AFPs and it is structurally unique in several aspects. • Six internal repeat segments of a thirteen residue repeat pattern is irregularly spaced apart throughout its sequence. The central part of these repeat segments is preserved as TxTxTxT

  31. Previous Studies • Salt-induced enhancement of antifreeze protein activity: a salting-out effect, Cryobiology, 2008 • Report on salt-induced enhancement of the antifreeze activity of an antifreeze protein from the longhorn beetle • Activity is predetermined by a steady-state distribution of the AFP between the solution and the ice surface region • Isolation and characterization of hemolymph antifreeze proteins from larvae of the longhorn beetle Rhagium inquisitor (L), Comp. Biochem. Physiol, 2005 • Procedure to isolate AFPs from hemolymph of larvae and presents some characteristics of structure • 22% ala, 20% glyc, 34% threonine, 17% serine, 9% Glyx • Mass, pH stability (3-12)

  32. Justification for fusion constructs • Signal sequence: • More easily purified from culture media • Greatly facilitates downstream processing • Continuous cultures for large-scale production • Potential toxicity for cell? • “Sec  SRP  TAT” (disulfide bond, Sec or SRP; PelB common) • eGFP: • Flourescence measurements • Improved solubility • Improved staining with Comassie brilliant blue • TEV cleavage: • Easily purified; co-operativity analysis. • His6 Tag

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