IvyTech SouthBend IN Championship Presentation

Detection of Environmental Coliphage through
Alpha-Complementation of Beta-Galactosidase in a
Fast-Acting Bacterial-Based Biosensor.
Team: IvyTech_SouthBend_IN
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“The Beta-Data-Quantatata!”
Water Serves Many Purposes: It is Essential to Life
ww.bbc.co.uk
biology.kenyon.edu
imgkid.com/
www.glogster.com
www.jigzone.com
www.portlavaca.org
natioanlgeographic.com
Global Problem: Human Waste Contamination of Water in Untreated Sewage
How does it occur?
● Direct Dumping
● Sanitary Sewer Overflow (SSO).
stock-clip.com
geograph.org.uk
www.who.org
Problem of Sewage for People
Impacts
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Drinking restrictions
Bathing or swimming restrictions
Spread of illness through pathogens
Typical Pathogens
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Polio virus
Hepatitis viruses
Norovirus
Cholera
Cryptosporidiosis
Ailments
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Acute fever
Gastroenteritis
Hepatitis
Dysentery
Health Costs (U.S.A.)
over $500
million
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Treatment
Emergency care
Hospitalization
Worldwide Response
International Water Management Institute
non profit working in many countries
World Health Organization guidelines for maintenance of clean water
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Surveillance: How do You Detect “Dangerous Water?”
Standard Method Includes Detection of Coliforms:
● Concentration of microbes in the water by centrifugation or filtration
● Culture on selective media
● A colorimetric test
● Takes a minimum of 24 hours
Limitations of this Test:
● Indirect test (not detecting human pathogens)
● Duration of incubation required
● Interference by antagonistic organisms
● Lack of specificity
● Poor detection of slow-growing “viable but non-culturable” microbes (VBNC)
● Environmental conditions can cause the quality of water to change in less than 24 hours
Need for a Reliable, Faster-acting Test
Enzyme β-galactosidase Activity as an Indirect Indicator of Bacteria Presence
● Colorimetric assay is more sensitive
● Still lacks specificity
Coliphage (Enteric Bacteriophage Detection) (Ijzerman)
Advantages over Coliform Detection
● Increased specificity and sensitivity
● Correlates with presence of human enteric pathogens
● Not dependent upon coliform viability
● Increased environmental stability over bacteria
Disadvantage
● Conventional plaque assay requires 24 hours
Bacteriophage Release of β-galactosidase (Stanek)
Advantages of Bacteriophage over Plaque Assay
● Reduces assay time to 4-6 hours
Disadvantage
● Incompatible with a field test: Requires laboratory technical skill and centrifugation
Alpha Complementation of the Beta-Galactosidase Enzyme
Proposed Field Biosensor For Coliphage
Construction of a LacZ α Polypeptide Generator:
BBa_K1477014
E. coli-C and Top 10 Cells are Equivalent
Permissive Hosts for Coliphage Replication
Design of an Colihage Immediate Lysis Cassette
Typical Phage Replication Cycle
Short circuit by immediate lysis cassette
Development of Fast Lysis: Building BBa_K1477030
Activation of the “Immediate Lysis Device”
by Coliphage T7
Microfluidic Biosensor Device
1. Mask
3. Glass Bonded PDMS:
Fluid flowed easily!
2. SU-8 Mold:
Picture taken after PDMS cast was pulled off (you can see some areas
of SU-8 got peeled up). Due to an adhesion issue, the third inlet
channel was lost after development.
4.Other Options for Platform
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( PDMS:Polydimethylsiloxane)
Potentiostatic cell
Paper analytical device (PAD)
Community Outreach
SCIENCE ALIVE
Our team has been committed to helping our local
community and recently helped with the Saint Joseph
County Public Library's "Science Alive."
This is an event to help get younger students all over the
city more interested and more excited about science!
IGEM TEAMS AID
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Carnegie Mellon
Cornell
ETH Zurich and other surveys
Academic Outreach:
Poster Presentations
Community College Undergaduate Research Initiative - Spring Poster Session
Montgomery Community College, Philadelphia, PA May 20, 2014
Summer Undergraduate Research Symposium
University of Notre Dame, Notre Dame, IN August 3, 2014
Community College Undergraduate Research Initiative - National Meeting
Hart Senate Building, Washington D.C. September 29, 2014
Summary
● Submitted and characterized two BioBricks to the
Registry: BBa_K1477014 and BBa_K1477030.
● Assessed Top10 E.coli cells as a replacement for the
standard E. coli C for water quality screening
● Demonstrated extracellular alpha-complementation of
Bgal as a result of coliphage lysis of target E.coli
● Designed and developing a microfluidic chamber to
house device
● Performed local community outreach
● Supported the IGEM community
Thanks & Attributions
For BioBricks from the Registry:
● Berkeley Teams for the Constitutive Promoter the Endolysin Cassette.
● USTC Team for the LacZ α fragments
● “Antiquity Group” for the RBSs and Terminators.
Special thanks to
● George Twaddle
● Christina Arisio
● Shaunasee Kosen (F-Cubed, LLC)
Financial Support
● Ivy Tech Foundation
● Ivy Tech Office of Student Life
● Community College Undergraduate Research Initiative
● The Steel Warehouse Company

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