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Antifreeze Proteins: Busting
Biofilms and Crushing Ice
Crystals
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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
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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
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• 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
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https://upload.wikimedia.org/wikipedia/commons/e/e4/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 of Microbiology, 2014, 453-72.
doi:10.1007/s00203-014-0983-1.
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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.
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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
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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
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Freeze Survival Assay
11
-20 CAdapted from Wang et al., 2010 protocol
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Freeze Survival Results
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-50
-40
-30
-20
-10
0
10
20
30
40
50
Percent change in freeze survival at -20oC
against empty vector control
Perc
ent
Ch
ange
fro
m C
on
tro
l
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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
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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.jpghttp://www.biofilm.montana.edu/files/CBE/images/bioPretreatWtrSM.jpg
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Direct Application: Circuit
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• Switchable circuit to induce/inhibit biofilm formation
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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
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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:
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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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AttributionsA 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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