Antifreeze Proteins: Busting Biofilms and Crushing Ice Crystals
Our Team 9/27/2015 WPI iGEM 2
Roadmap • Antifreeze Proteins • Biofilms Background • Goals • Freeze Survival Assay • Biofilm Formation Assay Our Project • Future Directions • Collaboration & Interlab Study • Educational Engagement Policy & Practices 9/27/2015 WPI iGEM 3
Antifreeze Proteins • Produced by a large variety of species that inhabit a wide range of habitats • Protect cells from the fatal effects of freezing by: – surrounding ice crystals as they form – inhibiting the growth of large, sharp crystals that can rupture the cell. 9/27/2015 WPI iGEM 4
AFPs in iGEM Yale 2011 Freeze Survival • Few past teams using AFPs – Only three ever used in iGEM: RiAFP, ZeAFP, TmAFP { – Best characterized so far: ZeAFP (Arctic Fish) – Yale 2011 characterized RiAFP (Siberian beetle) 20-40% Increase http://2011.igem.org/Team:Yale/Project/Assays 9/27/2015 WPI iGEM 5
Biofilms • 90% Extracellular Matrix (Kostakioti et al., 2013) • Antimicrobial Resistance (Donlan & Costerton, 2002) • Problematic in Medical and Food Production industries (Abdallah et al., 2014; Akers et al., 2015) • Use as filters for water and energy generation Abdallah, Marwan, Corinne Benoliel, Djamel Drider, Pascal Dhulster, and Nour-Eddine Chihib. "Biofilm Formation and Persistence on Abiotic (Groningen iGEM 2015!) Surfaces in the Context of Food and Medical Environments." Archives of Microbiology , 2014, 453-72. doi:10.1007/s00203-014-0983-1. Akers, Kevin S., Anthony P. Cardile, Joseph C. Wenke, and Clinton K. Murray. Kostakioti, M., M. Hadjifrangiskou, and S. J. Hultgren. "Bacterial Biofilms: "Biofilm Formation by Clinical Isolates and Its Relevance to Clinical Development, Dispersal, and Therapeutic Strategies in the Dawn of the Donlan, R. M., and J. W. Costerton. "Biofilms: Survival Mechanisms of https://upload.wikimedia.org/wikipedia/commons/e/e Infections." Advances in Experimental Medicine and Biology Biofilm- Postantibiotic Era." Cold Spring Harbor Perspectives in Medicine 3, no. 4 Clinically Relevant September 25, 2015. doi:10.1128/C based Healthcare associated Infections 830 (2014): 1-28. Accessed 4/Staphylococcus_aureus_biofilm_01.jpg (2013). Accessed September 25, 2015. Microorganisms." Clinical Microbiology Reviews 15, no. 2 (2002): 167-93. September 25, 2015. doi:10.1007/978-3-319-11038-7_1. doi:10.1101/cshperspect.a010306. Accessed MR.15.2.167 – 193.2002. 9/27/2015 WPI iGEM 6
Biofilm Disruption by IAFGP • IAFGP (a tick antifreeze protein) inhibits the formation of biofilms by Staphylococcus aureus (Heisig et al., 2014). Coating catheters with P1, a peptide derived from IAFGP, resulted in decreased S. aureus biofilm formation. Heisig, Martin, et al. "Antivirulence Properties of an Antifreeze Protein." Cell Reports 9, no. 2 (2014): 417-24. doi:10.1016/j.celrep.2014.09.034. 9/27/2015 WPI iGEM 7
Roadmap • Antifreeze Proteins • Biofilms Background • Goals • Freeze Survival Assay • Biofilm Formation Assay Our Project • Future Directions • Collaboration & Interlab Study • Educational Engagement Policy & Practices 9/27/2015 WPI iGEM 8
Project Goals and Outcomes 1. Create a comprehensive library of antifreeze proteins that are quantitatively characterized 2. Examine freeze survival properties of antifreeze proteins 3. Examine biofilm-inhibiting properties of antifreeze proteins 4. Temporal modulation of biofilms. 9/27/2015 WPI iGEM 9
Cloning • BclA-tagged AFPs • Untagged inserts synthesized by IDT • Cloned into pSB1C3 via Standard Assembly • Untagged AFPs either cytoplasmic or secreted Surface Localized • BclA-tag PCRed from WPI 2014’s BclA-YFP Biobrick • AFP + Double Terminator PCRed from our untagged inserts 9/27/2015 WPI iGEM 10
Freeze Survival Assay Adapted from Wang et al., 2010 protocol -20 C 11 9/27/2015 WPI iGEM
Freeze Survival Results Percent change in freeze survival at -20 o C against empty vector control 50 40 Percent Change from Control 30 20 10 0 -10 -20 -30 -40 -50 9/27/2015 WPI iGEM 12
Biofilm Assay Adapted from Merritt et al., 2011 protocol 9/27/2015 WPI iGEM 13
Biofilm Results 9/27/2015 WPI iGEM 14
Structural Analysis Biofilm Enhancers Biofilm Inhibitors ZeAFP IAFGP TiAFP CfAFP 9/27/2015 WPI iGEM 15
Future Directions • Phage therapy – Packaging AFP into phages, delivering to endotoxic or lytic bacterial infections • Coating biomaterials • Biofilm filters • Additive properties http://www.biofilm.montana.edu/files/CBE/images/bi http://i.ytimg.com/vi/UjVcn5Mmz-U/maxresdefault.jpg oPretreatWtrSM.jpg • Data driven synthetic AFP 9/27/2015 WPI iGEM 16
Direct Application: Circuit • Switchable circuit to induce/inhibit biofilm formation 9/27/2015 WPI iGEM 17
Roadmap • Antifreeze Proteins • Biofilms Background • Goals • Freeze Survival Assay • Biofilm Formation Assay Our Project • Future Directions • Collaboration & Interlab Study • Educational Engagement Policy & Practices 9/27/2015 WPI iGEM 18
Collaborations Harvard’s Data WPI’s Data: • Collaborated with Harvard Biodesign Determined whether or not our AFPs worked in a • different biofilm-forming strain of E. coli . 9/27/2015 WPI iGEM 19
Interlab Study 9/27/2015 WPI iGEM 20
Public Engagement • Touch Tomorrow – One-day public festival at WPI – Ran a synthetic biology workshop 9/27/2015 WPI iGEM 21
Public Engagement • Women in Science Day Camp – Middle school girls interested in STEM – Quantitative analysis and measurement of DNA 9/27/2015 WPI iGEM 22
Conclusions • Built a library of 16 unique AFPs – Submitted 23 parts to the registry • Found three AFPs that increase freeze survival >20% • Found two AFPs that inhibit biofilm formation • Found two AFPs that promote biofilm formation 9/27/2015 WPI iGEM 23
Attributions A big thanks goes out to… Professor Farny and Professor Duffy WPI Biology Department WPI Dean of Arts and Sciences, Karen Oates IDT NEGEM Collaborators Harvard BioDesign Yale 2011 and WPI 2014 Thank you for making our project great! 9/27/2015 WPI iGEM 24
Thank you! Questions? 9/27/2015 WPI iGEM 25
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