Detection of Environmental Coliphage through Alpha-Complementation of Beta-Galactosidase in a Fast-Acting Bacterial-Based Biosensor. Team: IvyTech_SouthBend_IN . A . K . A “The Beta-Data-Quantatata!”
Water Serves Many Purposes: It is Essential to Life imgkid.com/ biology.kenyon.edu ww.bbc.co.uk www.portlavaca.org natioanlgeographic.com www.glogster.com www.jigzone.com
Global Problem: Human Waste Contamination of Water in Untreated Sewage How does it occur? ● Direct Dumping ● Sanitary Sewer Overflow (SSO). stock-clip.com www.who.org geograph.org.uk
Problem of Sewage for People Impacts Drinking restrictions ● Bathing or swimming restrictions ● Spread of illness through pathogens ● Typical Pathogens Ailments Health Costs (U.S.A.) Polio virus Acute fever over $500 ● ● Hepatitis viruses Gastroenteritis ● ● million Norovirus Hepatitis ● ● Treatment Cholera Dysentery ● ● ● Emergency care Cryptosporidiosis ● ● Hospitalization ●
Worldwide Response International Water Management Institute - non profit working in many countries World Health Organization - guidelines for maintenance of clean water
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 ● Potentiostatic cell ● Paper analytical device (PAD) ( PDMS:Polydimethylsiloxane)
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 ● 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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