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

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

Dec 20, 2022 349 likes •688 views

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 ,

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SLIDE 1

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

Genet Geneticall ically engineer engineered ed bact bacter eriopha iophage ge for

  • r dia

diagnos gnosis is of

  • f whooping

hooping cough cough

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SLIDE 2

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

Background:

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SLIDE 3
  • 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

A Resurgent Threat

Background:

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SLIDE 4

A Resurgent Threat

Background:

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

  • 16 million cases worldwide in 2008
  • 95% in the developing world
  • 195,000 child deaths (WHO, 2011)
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SLIDE 5
  • Clinical
  • Differential diagnosis

§ Early symptoms nondescript

  • Lab
  • PCR

§ False positives § Expensive equipment & expertise

  • Culturing

§ Requires several days § Sensitivity decreases with symptom duration

  • Serology

§ Requires 2-8 weeks after cough onset

Current Diagnostics Inadequate

Background:

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SLIDE 6

S E I R T bIS/N aE vI kR µN µS µE µI µR αI kT µT

Suscep&ble ¡ Exposed ¡ Infected ¡ Treated ¡ Recovered ¡

Epidemiological Model

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SLIDE 7

Infection Peak

Infected Pop (Pop = 1000)

Current Situation Impact ¡of ¡ Faster ¡Diagnosis ¡

Model:

Early Detection = Reduced Infection

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SLIDE 8

Rapid

Affordable

Easy to use

Low Tech Specific. The Ideal Diagnostic

Design Goals:

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SLIDE 9
  • Bacteriophages are viruses

which infect bacteria

  • They are naturally specific to a

certain host

  • We worked with:

§ T7 phage and E. coli § BPP-1 phage and Bordetella

Specific

Design Goals:

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SLIDE 10
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SLIDE 11

Rapid

  • Pertussis symptoms initially

resemble a cold or flu

  • Increases the amount of time

individuals unknowingly spread the disease

  • Goal: same-day results

Design Goals:

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SLIDE 12

Low Tech Easy to Use. Affordable.

Implementable Anywhere

Design Goals:

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SLIDE 13

Pregnanc gnancy Tes est! !

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SLIDE 14

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.

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SLIDE 15
  • 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

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SLIDE 16

hCG on plasmid Transformation hCG Expression hCG in viral genome Transformation Expression of modified phage

Engineering the Phage

The Idea:

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SLIDE 17

Clinical Application

The Idea:

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SLIDE 18
  • Human chorionic gonadotropin, beta subunit (hCG-β)
  • Synthetic chorionic gonadotropin, beta subunit

(synCG-β)

  • Promoter + RBS + synCG-β
  • Lysin bbp9

BioBricks

Results:

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SLIDE 19

Deposit ¡ sample ¡ Flow ¡ Control ¡ band ¡ Test ¡ band ¡

Posi<ve ¡result ¡

Pregnancy Test Detects hCG-β

Results:

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SLIDE 20

Lysis by T7 infection Lysis by sonication

Positive results

Pregnancy Test Detects synCG-β

Results:

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SLIDE 21

Positive control

Stock hCG dimer

Negative control

No hCG present

Positive results Apply directly to test

Solubilize and fold; both bands are fainter

No ¡band ¡

Pregnancy Test Controls

Results:

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SLIDE 22

hCGβ synCGβ hCGβ hCGβ synCGβ synCGβ P ¡RBS P ¡RBS

PCR-amplify BclI digest

T7 genome

BclI digest

hCGβ hCGβ P ¡RBS synCGβ P ¡RBS synCGβ

Ligate fragments to assemble modified genomes

1 2 3 4 Transform cells with genome

Host assembles phage particles Amplify and isolate phage Isolate genome

term term term term

T7 Bacteriophage Modification

Results:

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SLIDE 23

0.5 1 1.5 2 1 2 3 4 5 Culture OD600 Time after Transformation (hours)

Transformation of BL21 pLysS E. coli

Negative control (no DNA) T7 Genome

Host Manufactures Phage

Results:

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SLIDE 24

Human Practices

  • Local outreach
  • Collaboration and cooperation
  • Metaphors analysis
  • Intellectual property analysis
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SLIDE 25

Stimulating interest in synthetic biology

  • 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

Local Outreach

Human Practices:

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SLIDE 26

NTNU Trondheim’s iGEM Matchmaker

  • Offered and solicited skills

Grenoble BioBrick Safety Sheet

  • Met through Matchmaker
  • Provided feedback
  • Won Safety Commendation

International Collaboration

Human Practices:

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SLIDE 27
  • Metaphors actively shape our approaches to

problems

  • SynBio relies heavily on metaphors from

electrical engineering

  • Other possibilities?
  • Overcoming functional fixedness
  • How can insights from cognitive science help us

design better metaphors?

Metaphors and Design Intuition

Human Practices:

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SLIDE 28
  • IP needs to provide incentives for development

without hampering innovation

  • Status quo inhibits follow-on research
  • A sui generis framework which addresses the unique

aspects of synbio

  • Needs large combinations of parts
  • Living things vs. machines

SynBio and Intellectual Property

Human Practices:

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SLIDE 29

Summary

  • Novel diagnostic for pathogenic bacteria
  • hCG-β
  • 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
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SLIDE 30

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

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SLIDE 31

Ques Questions ions?

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SLIDE 32

Wor

  • rks

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) ¡ ¡ ¡