Geneticall Genet ically engineer engineered ed bact bacter - PowerPoint PPT Presentation

Geneticall Genet ically engineer engineered ed bact bacter eriopha iophage ge for or dia diagnos gnosis is of of whooping hooping cough cough Josh Fass 1 , Jacqueline Grimm 2 , Syed Hassan 3 , John Hubczak 4 , Joseph Muldoon 2,5 , Shaun Moshasha 5,6 , Omar Raza 2 and Alexander Zorychta 1 Departments of Biomedical Engineering 1 , Biology 2 , Computer Science 3 , Chemical Engineering 4 , Chemistry 5 , and Physics 6 University of Virginia

Background: Whooping Cough o Highly infectious respiratory disease o Caused by the bacterium Bordetella pertussis o Significant cause of infant mortality o Early antibiotic treatment can relieve symptoms and reduce transmittance

Background: A Resurgent Threat o Incidence has been on the rise in the US since the 1980s Major epidemics in 2005, 2010, 2012 (NEJM, 2012) • Incidence of Pertussis per 100,000 in the US, 1980-2011

Background: A Resurgent Threat 16 million cases worldwide in 2008 o • 95% in the developing world • 195,000 child deaths (WHO, 2011) Whooping Cough data from Mortality and Burden of Disease in Member States of the WHO , 2002 Disability-adjusted Life Year data per 100,000 inhabitants

Background: Current Diagnostics Inadequate o Clinical • Differential diagnosis § Early symptoms nondescript o Lab • PCR § False positives § Expensive equipment & expertise • Culturing § Requires several days § Sensitivity decreases with symptom duration • Serology § Requires 2-8 weeks after cough onset

Epidemiological Model µ N kR S bIS/N E aE I vI R µ E µ I µ R µ S α I S uscep&ble ¡ E xposed ¡ kT T I nfected ¡ T reated ¡ µ T R ecovered ¡

Model: Early Detection = Reduced Infection Infected Pop (Pop = 1000) Infection Peak Current Situation Impact ¡of ¡ Faster ¡Diagnosis ¡

Design Goals: The Ideal Diagnostic Specific. Rapid Affordable Low Tech Easy to use

Design Goals: Specific o Bacteriophages are viruses which infect bacteria o They are naturally specific to a certain host o We worked with: § T7 phage and E. coli § BPP-1 phage and Bordetella

Design Goals: Rapid o Pertussis symptoms initially resemble a cold or flu o Increases the amount of time individuals unknowingly spread the disease o Goal : same-day results

Design Goals: Implementable Anywhere Affordable. Easy to Use. Low Tech

Pregnanc gnancy Tes est! !

What is hCG? Human Chorionic Gonadotropin (hCG): Hormone produced during pregnancy, detected by pregnancy test Pregnancy Tests are: Sensitive. Cheap. Available in any clinic. Low-Tech.

The Idea 1. Engineer a host-specific bacteriophage to produce hCG upon infection of host bacterium 2. Apply engineered phage to clinical sample 3. Determine presence of host bacteria by pregnancy test

The Idea: Engineering the Phage hCG on plasmid hCG in viral genome Transformation Transformation Expression of modified phage hCG Expression

The Idea: Clinical Application

Results: BioBricks o Human chorionic gonadotropin, beta subunit ( hCG- β ) o Synthetic chorionic gonadotropin, beta subunit ( synCG- β ) o Promoter + RBS + synCG- β o Lysin bbp9

Results: Pregnancy Test Detects hCG- β Posi<ve ¡result ¡ Deposit ¡ sample ¡ Flow ¡ Test ¡ Control ¡ band ¡ band ¡

Results: Pregnancy Test Detects synCG- β Lysis by sonication Positive results Lysis by T7 infection

Results: Pregnancy Test Controls Positive results Positive control Stock hCG dimer Apply directly Solubilize and fold; both bands are fainter to test Negative control No hCG present No ¡band ¡

Results: T7 Bacteriophage Modification 1 2 hCG β synCG β Isolate genome PCR-amplify T7 genome BclI digest BclI digest P ¡RBS term P ¡RBS term hCG β hCG β synCG β synCG β 4 Transform cells with genome 3 Ligate fragments to assemble Host assembles phage particles modified genomes Amplify and isolate phage hCG β P ¡RBS term hCG β synCG β P ¡RBS synCG β term

Results: Host Manufactures Phage Transformation of BL21 pLysS E. coli 2 Culture OD 600 1.5 1 Negative control (no DNA) 0.5 T7 Genome 0 1 2 3 4 5 Time after Transformation (hours)

Human Practices o Local outreach o Collaboration and cooperation o Metaphors analysis o Intellectual property analysis

Human Practices: Local Outreach Stimulating interest in synthetic biology o Personal outreach, coordinated presence at SynBio-related events Flash Seminar o A “learning flash mob” o Organized with John Arras, a member of the Presidential Bioethics Commission

Human Practices: International Collaboration NTNU Trondheim’s iGEM Matchmaker o Offered and solicited skills Grenoble BioBrick Safety Sheet o Met through Matchmaker o Provided feedback o Won Safety Commendation

Human Practices: Metaphors and Design Intuition o Metaphors actively shape our approaches to problems o SynBio relies heavily on metaphors from electrical engineering o Other possibilities ? o Overcoming functional fixedness o How can insights from cognitive science help us design better metaphors?

Human Practices: SynBio and Intellectual Property o IP needs to provide incentives for development without hampering innovation o Status quo inhibits follow-on research o A sui generis framework which addresses the unique aspects of synbio • Needs large combinations of parts • Living things vs. machines

Summary o Novel diagnostic for pathogenic bacteria o hCG- β • Designed in silico and synthesized • Assembled as a BioBrick device • Detectable by $1 pregnancy tests o Next steps • Model confirmation • Complete T7 phage modification • Expand our approach to detect pertussis and other organisms

Acknowledgements Advisors: Drs. Keith Kozminski, Inchan Kwon, and Jason Papin Kelly Lab: Dr. Kimberly Kelly, Dr. Siva Dasa, Dr. Jeff Smith, Stephanie Thomas, Dustin Bauknight, Jaymes Beech, Lindsey Brinton, Marc Seaman, Soo Shin, & Rachel Smith Dr. Eric Hewlett Dr. Gina Donato Past iGEMers: Arjun Athreya, Yanzhi Yang, Jacqueline Niu

Ques Questions ions?

Wor orks ks Cit ited ed World ¡Health ¡Organiza<on ¡(2011) . ¡ Pertussis . ¡Retrieved ¡from ¡ h?p://www.who.int/immuniza&on/topics/pertussis/en/index.html. ¡ Cherry, ¡J. ¡D. ¡(2012). ¡ Epidemic ¡Pertussis ¡in ¡2012 ¡-­‑ ¡The ¡Resurgence ¡of ¡a ¡Vaccine-­‑ Preventable ¡Disease. ¡ The ¡New ¡England ¡Journal ¡of ¡Medicine , ¡ 267 (9), ¡785–787. ¡ Centers ¡for ¡Disease ¡Control ¡and ¡Preven<on ¡(2012) . ¡Photos ¡of ¡Pertussis. ¡Retrieved ¡ from: ¡h?p://www.cdc.gov/pertussis/about/photos.html. ¡( Photo ¡of ¡infant ¡with ¡ pertussis ) ¡ h?p://en.wikipedia.org/w/index.php?&tle=File:PhageExterior.svg&page=1. ¡Author: ¡ Adenosine. ¡( Phage ¡illustra<on ) ¡ Thiel, ¡K. ¡(2004) . ¡Old ¡dogma, ¡new ¡tricks-­‑-­‑21st ¡Century ¡phage ¡therapy. ¡Nature ¡ Biotechnology, ¡22(1), ¡31–6. ¡( Ly<c ¡cycle ¡diagram ) ¡ h?p://www.babymed.com/sites/default/files/pregnancy_test_posi&ve.jpg ¡Author: ¡ BabyMed.com. ¡( Pregnancy ¡test ¡picture ) ¡ ¡ ¡