[PPT] - iGEM 2014 Giant Jamboree 10/31/14 Our Team 1 2 Worcester PowerPoint Presentation

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iGEM 2014

Giant Jamboree 10/31/14

Ag-goat-ination: Combating Antibiotic Resistance through Diagnostic Agglutination Assays

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Worcester Polytechnic Institute

Our Team

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Worcester Polytechnic Institute

Overall Project Goal

Design a New Method for Disease Diagnostics More Efficient Diagnoses of Infections Reduction of Blanket Antibiotic Treatment Curb Rise of Antibiotic Resistance

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Worcester Polytechnic Institute

Road Map

Background

Our Motivation
Choosing a Pathogen

Our Main Project

Surface Display of Proteins
Agglutination
Project Summary
Future Uses

Subprojects

Beyond the Bench
Practices
Improving characterization of existing BioBrick

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Worcester Polytechnic Institute

According to the 2013 CDC Report: Antibiotic Resistance Threats in the United States (CDC,2013)

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Our Motivation

Antibiotic Resistance in the U.S

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Worcester Polytechnic Institute

(Longitude Prize, 2014)

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Our Motivation

The Longitude Prize 2014

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Worcester Polytechnic Institute

Pew Charitable Trusts, 2013

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Our Motivation

Roots of Antibiotic Resistance

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Worcester Polytechnic Institute

Better diagnostics are needed for all livestock

infections

Blanket antibiotic treatment often occurs when

a virus is misdiagnosed as a bacterial infection

Our goal: Create a tool that can accurately

diagnose livestock infections in order to prevent the misuse of antibiotics.

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Our Motivation

Misdiagnosis of Livestock Infections

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Worcester Polytechnic Institute Global Impact:

Goat population is

approximately 1,005,600,000 worldwide (FAOSTAT, 2013)

Reported cases of CAEV

around the world Disease Characteristics:

Slow to cause visible symptoms Symptoms are nebulous If undetected, spreads quickly

through a herd

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Caprine Arthritis Encephalitis Virus (CAEV)

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Have your goat or sheep herds ever been affected by CAEV?

Choosing a Pathogen

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Worcester Polytechnic Institute

Background

Our Motivation
Choosing a Pathogen

Our Main Project

Surface Display of Proteins
Agglutination
Project Summary
Future Uses

Subprojects

Beyond the Bench
Practices
Improving characterization of existing BioBrick

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Road Map

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Worcester Polytechnic Institute

Draw blood from a goat Centrifuge blood sample to isolate plasma

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Check for agglutination in the blood sample Add E. coli expressing antigen on the cell surface to plasma

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Ag-goat-ination

Our Direct Agglutination Assay

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Worcester Polytechnic Institute

Surface Display of Proteins

Using BclA

INP is currently used for cell surface targetting

(BBa_K523013 by Edinburgh 2011)

BclA originates from Bacillus anthracis The BclA N-Terminal Domain leads to cell surface localization

BclA Structure

(Park et al.,Microbial Cell Factory, 2013)

Cell Surface Display of Protein using BclA

(Park et al.,Microbial Cell Factory, 2013)

GFP

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Worcester Polytechnic Institute

Codon optimized

ligos

Transformed E. Coli expressing BclA and CAEV p28 on the cell surface

Surface Display of Proteins

CAEV p28 Expression

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BclA-CAEV p28 in psB1C3 plasmid

BBa_K1423004 BBa_K1423005

BclA-YFP in psB1C3 plasmid Transformed E. Coli expressing BclA and YFP on the cell surface Codon optimized

ligos

YFP Expression

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Worcester Polytechnic Institute

BclA-YFP

BclA External Expression of YFP (BBa_K1423005)

GFP

Internal Expression of GFP (BBa_I20260)

INP-YFP

INP External Expression of YFP (BBa_K523013)

Cell Surface Display of Protein using BclA

(Park et al.,Microbial Cell Factory, 2013)

Contrast Normalized

Surface Display of Proteins

Microscopy Proof of Principle

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Worcester Polytechnic Institute

Draw blood from a goat Centrifuge blood sample to isolate plasma

r

Check for agglutination in the blood sample Add E. coli expressing antigen on the cell surface to plasma

+

Ag-goat-ination

Our Direct Agglutination Assay

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Worcester Polytechnic Institute

(Agglutination, 2009)

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Blood Typing Latex Bead Agglutination

(Blood Typing Game, 2014)

Agglutination

Basic Concept

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Worcester Polytechnic Institute

Agglutinating With Matching Antibody- Antigen Pair Not Agglutinating With Non-matching Antibody-Antigen Pair

Agglutination

Direct Agglutination Assay

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Worcester Polytechnic Institute

(-) Key (+)

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Agglutination

Our Results

Adams et. Al, PLOS Neglected Tropical Diseases, 2012

BclA-CAEV Internal GFP INP-YFP BclA-YFP GFP-G1 Antibody GFP-8H11 Antibody Titer: Positive to >1:5,000 Overnight Results

(BBa_K1423004) (BBa_I20260) (BBa_K523013) (BBa_K1423005)

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Worcester Polytechnic Institute

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Our Genetically Engineered Machine: From the Farm to the Lab:

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Reagent Cost

ELISA Diagnosis: $1.50-$1.90 per sample Our Diagnostic Tool: $0.003 per sample

Project Summary

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Worcester Polytechnic Institute

Other Bacteria
Other Bacteria
Other Viruses
Other Viruses
Future Pathogens
Future Pathogens

Further Uses of Our Design

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YFP
CAEV
YFP
CAEV

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Worcester Polytechnic Institute

Background

Our Motivation
Choosing a Pathogen

Our Main Project

Cell Surface Expression
Agglutination
Project Summary
Future Uses

Subprojects

Beyond the Bench
Practices
Improving Characterization of Existing BioBrick

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Road Map

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Worcester Polytechnic Institute

Beyond the Bench

Survey of Farmers

Distributed a survey to farmers across New

England

Results:
27% of farmers who

had goats or sheep said that their herds have been affected CAEV

93% of respondents

would consider using

ur diagnostic tool

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33% 7% 60% Yes No I might consider it.

Would you use a blood test that utilizes Bacteria (E. coli) constructed using synthetic biology for early detection of livestock diseases such as CAEV? n=29

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Worcester Polytechnic Institute

Interlab Study
Science Project and

Equipment Camp (SPeC) at Wachusett Regional High School

Intro to Biotech Lecture
Biotech Club Lecture
Women in Science Day

Camp

23 Link to our curriculum: http://2014.igem.org/wiki/images/9/99/ WPI_Introduction_to_Synthetic_Biology_Curriculum.zip

Practices

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Worcester Polytechnic Institute

Better Characterization of a BioBrick

BBa_J45199 Banana Odor Generator Created by 2006 MIT iGEM

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Worcester Polytechnic Institute

Better Characterization of a BioBrick

BBa_K1423006 BBa_K1423007 Banana Odor Generator with Banana Odor Generator with Heavy Metal Inducible Promoter Constitutive Promoter

RBS RBS ATF1 ATF1

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DT DT pArs pCon Isoamyl Alcohol Isoamyl Acetate

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Worcester Polytechnic Institute

Better Characterization of a BioBrick

Isoamyl Alcohol Isoamyl Acetate

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Detritus from liquid LB

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Worcester Polytechnic Institute

Better Characterization of a BioBrick

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Concentrations of Isoamyl Acetate from pArs Normalized to ConP

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Worcester Polytechnic Institute 28

Designed an improved diagnostic tool for infections
Created a new surface expression tag BioBrick

(BBa_K1423005)

Proof of principle experiments for cell surface

expression and direct agglutination assay

Outreach and educational activities
Improved characterization of the banana odor

generator (BBa_J45199) and heavy metal inducible promoter (BBa_J33201)

Summary

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Worcester Polytechnic Institute

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Worcester Polytechnic Institute

Questions?

Email us at: igem2014@wpi.edu

Background

Our Motivation 5,6,7,8
Choosing a Pathogen

9 Our Main Project

Surface Display of Proteins

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Agglutination

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Project Summary

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Future Uses

20 Subprojects

Beyond the Bench

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Practices

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Improving characterization of existing BioBrick 24,25,26,27