Biofilms and Crushing Ice Crystals Our Team 9/27/2015 WPI iGEM 2 - - PowerPoint PPT Presentation

Antifreeze Proteins: Busting Biofilms and Crushing Ice Crystals

Our Team

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Roadmap

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Background

• Antifreeze Proteins
• Biofilms

Our Project

• Goals
• Freeze Survival Assay
• Biofilm Formation Assay
• Future Directions

Policy & Practices

• Collaboration & Interlab Study
• Educational Engagement

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.

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• 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)

AFPs in iGEM

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http://2011.igem.org/Team:Yale/Project/Assays

Yale 2011 Freeze Survival

{

20-40% Increase

• 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 (Groningen iGEM 2015!)

Kostakioti, M., M. Hadjifrangiskou, and S. J. Hultgren. "Bacterial Biofilms: Development, Dispersal, and Therapeutic Strategies in the Dawn of the Postantibiotic Era." Cold Spring Harbor Perspectives in Medicine 3, no. 4 (2013). Accessed September 25, 2015. doi:10.1101/cshperspect.a010306. Donlan, R. M., and J. W. Costerton. "Biofilms: Survival Mechanisms of Clinically Relevant September 25, 2015. doi:10.1128/C Microorganisms." Clinical Microbiology Reviews 15, no. 2 (2002): 167-93. Accessed MR.15.2.167–193.2002. Akers, Kevin S., Anthony P. Cardile, Joseph C. Wenke, and Clinton K. Murray. "Biofilm Formation by Clinical Isolates and Its Relevance to Clinical Infections." Advances in Experimental Medicine and Biology Biofilm- based Healthcare associated Infections 830 (2014): 1-28. Accessed September 25, 2015. doi:10.1007/978-3-319-11038-7_1.

Biofilms

9/27/2015 WPI iGEM 6 https://upload.wikimedia.org/wikipedia/commons/e/e 4/Staphylococcus_aureus_biofilm_01.jpg

Abdallah, Marwan, Corinne Benoliel, Djamel Drider, Pascal Dhulster, and Nour-Eddine Chihib. "Biofilm Formation and Persistence on Abiotic Surfaces in the Context of Food and Medical Environments." Archives

• f Microbiology, 2014, 453-72.

doi:10.1007/s00203-014-0983-1.

Biofilm Disruption by IAFGP

• IAFGP (a tick antifreeze

protein) inhibits the formation of biofilms by Staphylococcus aureus (Heisig et al., 2014).

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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.

Roadmap

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Background

• Antifreeze Proteins
• Biofilms

Our Project

• Goals
• Freeze Survival Assay
• Biofilm Formation Assay
• Future Directions

Policy & Practices

• Collaboration & Interlab Study
• Educational Engagement

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.

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Cloning

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• Untagged inserts synthesized by IDT
• Cloned into pSB1C3 via Standard Assembly
• Untagged AFPs either cytoplasmic or secreted
• BclA-tag PCRed from WPI 2014’s BclA-YFP Biobrick
• AFP + Double Terminator PCRed from our untagged inserts
• BclA-tagged AFPs

Surface Localized

Freeze Survival Assay

11

• 20 C

Adapted from Wang et al., 2010 protocol

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Freeze Survival Results

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• 50
• 40
• 30
• 20
• 10

10 20 30 40 50

Percent change in freeze survival at -20oC against empty vector control

Percent Change from Control

Biofilm Assay

Adapted from Merritt et al., 2011 protocol

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Biofilm Results

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Structural Analysis

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TiAFP CfAFP

Biofilm Inhibitors

ZeAFP IAFGP

Biofilm Enhancers

Future Directions

• Phage therapy

– Packaging AFP into phages, delivering to endotoxic or lytic bacterial infections

• Coating biomaterials
• Biofilm filters
• Additive properties
• Data driven synthetic

AFP

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http://i.ytimg.com/vi/UjVcn5Mmz-U/maxresdefault.jpg http://www.biofilm.montana.edu/files/CBE/images/bi

• PretreatWtrSM.jpg

Direct Application: Circuit

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• Switchable circuit to induce/inhibit biofilm

formation

Roadmap

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Background

• Antifreeze Proteins
• Biofilms

Our Project

• Goals
• Freeze Survival Assay
• Biofilm Formation Assay
• Future Directions

Policy & Practices

• Collaboration & Interlab Study
• Educational Engagement

Harvard’s Data

Collaborations

• Collaborated with Harvard Biodesign
• Determined whether or not our AFPs worked in a

different biofilm-forming strain of E. coli.

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WPI’s Data:

Interlab Study

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Public Engagement

• Touch Tomorrow

– One-day public festival at WPI – Ran a synthetic biology workshop

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Public Engagement

• Women in Science Day Camp

– Middle school girls interested in STEM – Quantitative analysis and measurement of DNA

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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

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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!

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Thank you! Questions?

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