Ag-goat-ination: Combating Antibiotic Resistance through Diagnostic Agglutination Assays iGEM 2014 Giant Jamboree 10/31/14
Our Team 1 2 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 Worcester Polytechnic Institute 3
Road Map • Our Motivation • Choosing a Pathogen Background • Surface Display of Proteins • Agglutination • Project Summary Our Main • Future Uses Project • Beyond the Bench • Practices • Improving characterization of existing BioBrick Subprojects Worcester Polytechnic Institute 4
Our Motivation Antibiotic Resistance in the U.S According to the 2013 CDC Report: Antibiotic Resistance Threats in the United States (CDC,2013) 5 Worcester Polytechnic Institute
Our Motivation The Longitude Prize 2014 (Longitude Prize, 2014) 6 Worcester Polytechnic Institute
Our Motivation Roots of Antibiotic Resistance Pew Charitable Trusts, 2013 7 Worcester Polytechnic Institute
Our Motivation Misdiagnosis of Livestock Infections � 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. Worcester Polytechnic Institute 8
Choosing a Pathogen Caprine Arthritis Encephalitis Virus (CAEV) Global Impact: Disease Characteristics: � Goat population is � Slow to cause visible symptoms approximately 1,005,600,000 worldwide (FAOSTAT, 2013) � Symptoms are nebulous � Reported cases of CAEV � If undetected, spreads quickly around the world through a herd Have your goat or sheep herds ever been affected by CAEV? 2 9 Worcester Polytechnic Institute
Road Map • Our Motivation • Choosing a Pathogen Background • Surface Display of Proteins • Agglutination • Project Summary Our Main • Future Uses Project • Beyond the Bench • Practices • Improving characterization of existing BioBrick Subprojects Worcester Polytechnic Institute 10
Ag-goat-ination Our Direct Agglutination Assay Add E. coli expressing Centrifuge blood sample antigen on the cell to isolate plasma surface to plasma + Draw blood from a goat or Check for agglutination in the blood sample Worcester Polytechnic Institute 11
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 GFP BclA Structure Cell Surface Display of Protein using BclA (Park et al.,Microbial Cell Factory, 2013) (Park et al.,Microbial Cell Factory, 2013) Worcester Polytechnic Institute 12
Surface Display of Proteins CAEV p28 Expression YFP Expression BBa_K1423004 BBa_K1423005 Codon optimized Transformed E. Coli Codon optimized BclA-CAEV p28 in BclA-YFP in Transformed E. Coli oligos expressing BclA and oligos psB1C3 plasmid psB1C3 plasmid expressing BclA and CAEV p28 on the cell YFP on the cell surface surface Worcester Polytechnic Institute 13
Surface Display of Proteins Microscopy Proof of Principle GFP INP-YFP BclA-YFP Cell Surface Display of Protein using BclA (Park et al.,Microbial Cell Factory, 2013 ) Internal Expression of GFP INP External Expression of YFP BclA External Expression of YFP (BBa_I20260) (BBa_K523013) (BBa_K1423005) Worcester Polytechnic Institute Contrast Normalized 14
Ag-goat-ination Our Direct Agglutination Assay Add E. coli expressing Centrifuge blood sample antigen on the cell to isolate plasma surface to plasma + Draw blood from a goat or Check for agglutination in the blood sample Worcester Polytechnic Institute 15
Agglutination Basic Concept � Blood Typing � Latex Bead Agglutination (Blood Typing Game, 2014) 5 (Agglutination, 2009) Worcester Polytechnic Institute 16
Agglutination Direct Agglutination Assay Agglutinating Not Agglutinating With Matching Antibody- With Non-matching Antigen Pair Antibody-Antigen Pair Worcester Polytechnic Institute 17
Agglutination Our Results GFP-G1 GFP-8H11 Antibody Antibody Key BclA-CAEV (BBa_K1423004) (-) Titer: Positive to Internal GFP >1:5,000 (BBa_I20260) (+) Overnight Results INP-YFP (BBa_K523013) Adams et. Al, PLOS Neglected Tropical Diseases, 2012 BclA-YFP (BBa_K1423005) Worcester Polytechnic Institute 18
Project Summary Our Genetically Engineered Machine: From the Farm to the Lab: Reagent Cost ELISA Diagnosis: $1.50-$1.90 per sample 3 Our Diagnostic Tool: $0.003 per sample Worcester Polytechnic Institute 19
Further Uses of Our Design � � YFP YFP � � CAEV CAEV � Other Viruses � Other Viruses � Other Bacteria � Other Bacteria � � Future Pathogens Future Pathogens 6 Worcester Polytechnic Institute 20
Road Map • Our Motivation • Choosing a Pathogen Background • Cell Surface Expression • Agglutination • Project Summary Our Main • Future Uses Project • Beyond the Bench • Practices • Improving Characterization of Existing BioBrick Subprojects Worcester Polytechnic Institute 21
Beyond the Bench Survey of Farmers � Distributed a survey to farmers across New England n=29 � Results: Yes 33% � 27% of farmers who No had goats or sheep said that their herds 60% I might have been affected consider it. 7% CAEV � 93% of respondents Would you use a blood test that utilizes would consider using Bacteria ( E. coli ) constructed using our diagnostic tool synthetic biology for early detection of livestock diseases such as CAEV? Worcester Polytechnic Institute 22
Practices � 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 Link to our curriculum: http://2014.igem.org/wiki/images/9/99/ WPI_Introduction_to_Synthetic_Biology_Curriculum.zip Worcester Polytechnic Institute 23
Better Characterization of a BioBrick BBa_J45199 Banana Odor Generator Created by 2006 MIT iGEM Worcester Polytechnic Institute 24
Better Characterization of a BioBrick Isoamyl Alcohol Isoamyl Acetate pArs pCon DT DT RBS ATF1 ATF1 RBS BBa_K1423006 BBa_K1423007 Banana Odor Generator with Banana Odor Generator with Heavy Metal Inducible Promoter Constitutive Promoter Worcester Polytechnic Institute 25
Better Characterization of a BioBrick Isoamyl Isoamyl Alcohol Acetate Detritus from liquid LB Worcester Polytechnic Institute 26
Better Characterization of a BioBrick Concentrations of Isoamyl Acetate from pArs Normalized to ConP Worcester Polytechnic Institute 27
Summary � 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) Worcester Polytechnic Institute 28
Worcester Polytechnic Institute
Questions? Email us at: igem2014@wpi.edu Email us at: igem2014@wpi.edu • Our Motivation 5,6,7,8 • Choosing a Pathogen 9 Background • Surface Display of Proteins 12,13,14 • Agglutination 16,17,18 • Project Summary 19 Our Main • Future Uses 20 Project • Beyond the Bench 22 • Practices 23 • Improving characterization of existing BioBrick 24,25,26,27 Subprojects Worcester Polytechnic Institute
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