Genetically engineered bacteriophage for

diagnosis of whooping cough

Josh Fass1, Jacqueline Grimm2, Syed Hassan3, John Hubczak4,

Joseph Muldoon2,5, Shaun Moshasha5,6, Omar Raza2 and Alexander Zorychta1
Departments of Biomedical Engineering1, Biology2, Computer Science3, Chemical Engineering4, Chemistry5, and Physics6

University of Virginia
 Whooping Cough
Highly infectious respiratory
disease

Caused by the bacterium

Bordetella pertussis

Significant cause of infant

mortality

Early antibiotic treatment can

relieve symptoms and reduce
transmittance
 A Resurging Threat
16 million cases worldwide in 2008
95% in the developing world
195,000 child deaths (WHO, 2011)
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
 Existing Diagnostics Are
Inadequate
Clinical
Differential diagnosis
Early symptoms nondescript

Lab
PCR
Rapid and sensitive, but prone to false positives
Requires expensive lab equipment
Culturing
High specificity, but takes several days
Sensitivity decreases with symptom duration
Serology
Can confirm diagnosis 2-8 weeks after cough onset
 Model shows impact of faster diagnosis

N
kR

S bIS/N E aE I vI R

S E I R
I

Susceptible kT
Exposed T
Infected
Treated
Recovered
T
Early detection can dramatically
reduce incidence of infection
Peak of
infections
Infected Pop (Pop = 1000)

Current (w/o intervention)

Infection periods (days)

Desired (w/ intervention)

 A New Whooping Cough
Diagnostic Must Be
Specific
Rapid
Affordable
Low-tech
Easy to use
Specific
Bacteriophages are viruses
which infect bacteria

They are naturally specific to

a certain host

We worked with
T7 phage and E. coli
Bacteriophage BPP-1 and
Bordetella
Lytic cycle
Quick
Pertussis symptoms can
initially resemble a cold or
flu

This increases the amount

of time individuals
unknowingly spread the
disease

Goal: same-day results

 Implementable anywhere

Affordable

Low-tech

Easy to use
Pregnancy Test
 What is hCG?
hCG is a hormone produced during
pregnancy detected by a pregnancy test
Pregnancy tests are...
Sensitive
Cheap
Available in any clinic
 The Idea
Engineer a host-specific bacteriophage to
produce hCG upon infection of host
bacterium
Apply engineered phage to clinical sample
Determine presence of host bacteria by
pregnancy test
 Developing the Modified
Phage

hCG on plasmid
hCG in viral genome

Transformation
Transformation

Expression of modified phage

Expression of hCG
Clinical Application
 BioBricks

Human chorionic gonadotropin, beta subunit (hCG-)

Synthetic chorionic gonadotropin, beta subunit
(synCG-)

Promoter + RBS + synCG-

Lysin bbp9
 Pregnancy test detects hCG

Positive result
Deposit
sample

Flow

Test Control
band band
 Pregnancy tests detect synCG

Lysis by sonication

Positive results

Lysis by T7 infection
 Pregnancy test controls

Positive results

Positive control
Stock hCG dimer

Apply directly to test Solubilize and fold;

both bands are fainter

Negative control
No hCG present

No band
 T7 phage modification
1 2 hCG synCG

Isolate genome

T7 genome
PCR-amplify

BclI digest BclI digest

hCG P RBS hCG term synCG P RBS synCG term

3 4
Ligate fragments to assemble modified genomes Transform cells with genome
Host assembles phage particles
hCG Amplify and isolate phage

P RBS hCG term

synCG

P RBS synCG term

 Transformation of BL21 pLysS E. coli
2
Optical Density of Cells

1.5

Negative control
0.5 (no DNA)

T7 Genome

0
1 2 3 4 5

Time after Transformation (hours)

Human Practices
 Overview

Local outreach

Collaboration and cooperation

Metaphors analysis

Intellectual property analysis

 Local Outreach
Stimulating interest in synthetic biology by
personal outreach, coordinated presence at
synbio-related events

Flash Seminar
A learning flash mob
Organized with John Arras,
a member of the Presidential Bioethics
Commission
 Collaboration
NTNU Trondheims iGEM Matchmaker
Offered and solicited skills

Grenoble BioBrick Safety Sheet

Met through Matchmaker
Provided feedback
Won Safety Commendation

Grenoble
Metaphors and design intuition
Inadequate conceptual framings can
bias designers toward functional
fixedness
Example: junk DNA
Counter example: our project
Pregnancy test to test for whooping cough
Reductionist framing can help overcome
functional fixedness
Synbio & Intellectual Property
Status quo
Patents mismatch with technical reality
Perfect storm of issues biotech and software patents
Strongly incentivizes certain kinds of development
Inhibits follow-on research: tragedy of anticommons

Abolishing gene patents

Twice as popular as status quo
Would strongly hinder translation
Sui generis (custom-built) framework
Tailor-made solutions to synthetic biologys unique
challenge to patent policy
 Summary
Novel diagnostic for pathogenic bacteria
hCG-
Designed in silico and synthesized
Assembled as a BioBrick device
Detectable by $1 pregnancy tests

Next steps
Model confirmation
Complete T7 phage modification
Expand our approach to detect pertussis and other
organisms
 Acknowledgments
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, and
Soo Shin

Dr. Eric Hewlett

Dr. Gina Donato
Past iGEMers: Arjun Athreya, Yanzhi Yang,
Jacqueline Niu
Questions?
 Bibliography
1. World Health Organization (2011). Pertussis. Retrieved from http://www.who.int/
immunization/topics/pertussis/en/index.html.

2. Cherry, J. D. (2012). Epidemic Pertussis in 2012 - The Resurgence of a Vaccine-

Preventable Disease. The New England Journal of Medicine, 267(9), 785787.