Ag-goat-ination: Combating Antibiotic Resistance through Diagnostic Agglutination Assays. igem 2014 Giant Jamboree 10/31/14

Transcription

1 Ag-goat-ination: Combating Antibiotic Resistance through Diagnostic Agglutination Assays igem 2014 Giant Jamboree 10/31/14

2 Our Team 1 2

3 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 3

4 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 4

5 Our Motivation Antibiotic Resistance in the U.S According to the 2013 CDC Report: Antibiotic Resistance Threats in the United States (CDC,2013) 5

6 Our Motivation The Longitude Prize 2014 (Longitude Prize, 2014) 6

7 Our Motivation Roots of Antibiotic Resistance Pew Charitable Trusts,

8 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. 8

9 Choosing a Pathogen Caprine Arthritis Encephalitis Virus (CAEV) 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 Have your goat or sheep herds ever been affected by CAEV? 2 9

10 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 10

11 Ag-goat-ination Our Direct Agglutination Assay Centrifuge blood sample to isolate plasma + Add E. coli expressing antigen on the cell surface to plasma Draw blood from a goat or Check for agglutination in the blood sample 11

12 Surface Display of Proteins Using BclA " INP is currently used for cell surface targetting (BBa_K by Edinburgh 2011) " BclA originates from Bacillus anthracis " The BclA N-Terminal Domain leads to cell surface localization GFP BclA Structure (Park et al.,microbial Cell Factory, 2013) Cell Surface Display of Protein using BclA (Park et al.,microbial Cell Factory, 2013) 12

13 Surface Display of Proteins CAEV YFP p28 Expression Expression BBa_K BBa_K Codon optimized Codon optimized oligos oligos BclA-CAEV BclA-YFP in p28 in psb1c3 psb1c3 plasmid plasmid Transformed E. Coli expressing Transformed BclA E. and Coli CAEV expressing p28 on BclA the and cell YFP surface on the cell surface 13

14 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 (BBa_I20260) Contrast Normalized INP External Expression of YFP (BBa_K523013) BclA External Expression of YFP (BBa_K ) 14

15 Ag-goat-ination Our Direct Agglutination Assay Centrifuge blood sample to isolate plasma Add E. coli expressing antigen on the cell surface to plasma + Draw blood from a goat or Check for agglutination in the blood sample 15

16 Agglutination Basic Concept " Blood Typing " Latex Bead Agglutination (Blood Typing Game, 2014) (Agglutination, 2009) 5 16

17 Agglutination Direct Agglutination Assay Agglutinating With Matching Antibody- Antigen Pair Not Agglutinating With Non-matching Antibody-Antigen Pair 17

18 Agglutination Our Results Key (-) (+) Adams et. Al, PLOS Neglected Tropical Diseases, 2012 BclA-CAEV (BBa_K ) Internal GFP (BBa_I20260) INP-YFP (BBa_K523013) GFP-G1 Antibody GFP-8H11 Antibody Titer: Positive to >1:5,000 Overnight Results BclA-YFP (BBa_K ) 18

19 Project Summary Our Genetically Engineered Machine: From the Farm to the Lab: ELISA Diagnosis: Our Diagnostic Tool: Reagent Cost $1.50-$1.90 per sample $0.003 per sample 3 19

20 Further Uses of Our Design " YFP " CAEV " Other Viruses " Other Bacteria " Future Pathogens 6 20

21 Road Map 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 21

22 Beyond the Bench Survey of Farmers " Distributed a survey to farmers across New England n=29 " Results: " 27% of farmers who had goats or sheep said that their herds have been affected CAEV " 93% of respondents would consider using our diagnostic tool 60% 33% 7% 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? 22

23 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: WPI_Introduction_to_Synthetic_Biology_Curriculum.zip 23

24 Better Characterization of a BioBrick BBa_J45199 Banana Odor Generator Created by 2006 MIT igem 24

25 Better Characterization of a BioBrick Isoamyl Alcohol Isoamyl Acetate pars pcon RBS ATF1 DT RBS ATF1 DT BBa_K BBa_K Banana Odor Generator with Banana Odor Generator with Heavy Metal Inducible Promoter Constitutive Promoter 25

26 Better Characterization of a BioBrick Isoamyl Alcohol Isoamyl Acetate Detritus from liquid LB 26

27 Better Characterization of a BioBrick Concentrations of Isoamyl Acetate from pars Normalized to ConP 27

28 Summary " Designed an improved diagnostic tool for infections " Created a new surface expression tag BioBrick (BBa_K ) " 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) 28

29

30 Questions? us at: Background Our Motivation 5,6,7,8 Choosing a Pathogen 9 Our Main Project Surface Display of Proteins 12,13,14 Agglutination 16,17,18 Project Summary 19 Future Uses 20 Subprojects Beyond the Bench 22 Practices 23 Improving characterization of existing BioBrick 24,25,26,27