The Beta-Data-Quantatata! Water Serves Many Purposes: It is - - PowerPoint PPT Presentation

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The Beta-Data-Quantatata! Water Serves Many Purposes: It is - - PowerPoint PPT Presentation

Mar 04, 2023 200 likes •419 views

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

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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!”

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Water Serves Many Purposes: It is Essential to Life

www.glogster.com ww.bbc.co.uk www.jigzone.com imgkid.com/ www.portlavaca.org

natioanlgeographic.com biology.kenyon.edu

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Global Problem: Human Waste Contamination of Water in Untreated Sewage

www.who.org

How does it occur?

  • Direct Dumping
  • Sanitary Sewer Overflow (SSO).

stock-clip.com geograph.org.uk

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Problem of Sewage for People

Ailments

  • Acute fever
  • Gastroenteritis
  • Hepatitis
  • Dysentery

Health Costs (U.S.A.)

  • ver $500

million

  • Treatment
  • Emergency care
  • Hospitalization

Typical Pathogens

  • Polio virus
  • Hepatitis viruses
  • Norovirus
  • Cholera
  • Cryptosporidiosis

Impacts

  • Drinking restrictions
  • Bathing or swimming restrictions
  • Spread of illness through pathogens
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SLIDE 5

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
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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
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Alpha Complementation of the Beta-Galactosidase Enzyme

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Proposed Field Biosensor For Coliphage

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Construction of a LacZ α Polypeptide Generator: BBa_K1477014

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  • E. coli-C and Top 10 Cells are Equivalent

Permissive Hosts for Coliphage Replication

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Typical Phage Replication Cycle Short circuit by immediate lysis cassette

Design of an Colihage Immediate Lysis Cassette

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Development of Fast Lysis: Building BBa_K1477030

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Activation of the “Immediate Lysis Device” by Coliphage T7

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Microfluidic Biosensor Device

  • 3. Glass Bonded PDMS:

Fluid flowed easily! Picture taken after PDMS cast was pulled off (you can see some areas

  • f SU-8 got peeled up). Due to an adhesion issue, the third inlet

channel was lost after development.

  • 2. SU-8 Mold:

( PDMS:Polydimethylsiloxane)

  • 1. Mask

4.Other Options for Platform

  • Potentiostatic cell
  • Paper analytical device (PAD)
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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!

Community Outreach

IGEM TEAMS AID

  • Carnegie Mellon
  • Cornell
  • ETH Zurich and other surveys
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SLIDE 19

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

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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
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SLIDE 21

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