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Diagnosing bacterial Diagnosing bacterial Diagnosing bacterial Diagnosing bacterial infections with viruses infections with viruses infections with viruses infections with viruses (Modular Construction of Bacteriophage for Diagnostic

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Diagnosing bacterial Diagnosing bacterial Diagnosing bacterial Diagnosing bacterial infections with viruses infections with viruses infections with viruses infections with viruses

(Modular Construction of Bacteriophage for Diagnostic Systems) (Modular Construction of Bacteriophage for Diagnostic Systems) (Modular Construction of Bacteriophage for Diagnostic Systems) (Modular Construction of Bacteriophage for Diagnostic Systems)

BIRKBECK BIRKBECK BIRKBECK BIRKBECK iGEM iGEM iGEM iGEM 2015 2015 2015 2015

  • A. Mirzarafie-Ahi, E. Parris, S. Craig, B. Steijl, L. Prout
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We are We are We are We are Birkbeck Birkbeck Birkbeck Birkbeck, University of London , University of London , University of London , University of London

“London’s evening university”

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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Meet the Meet the Meet the Meet the Owligos Owligos Owligos Owligos

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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Infectious diseases in the developing world Infectious diseases in the developing world Infectious diseases in the developing world Infectious diseases in the developing world

Infectious disease Detection

Speed Cost

Treatment

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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Case study: TB and antibiotic resistance Case study: TB and antibiotic resistance Case study: TB and antibiotic resistance Case study: TB and antibiotic resistance

Source: World Health Organisation (2012) Images: World Health Organisation

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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What do you consider to be the main challenges in the eradication of TB and drug resistant strains?

  • Dr. Sanjib Bhakta

Director of Mycobacteria Research Laboratory at Birkbeck/UCL

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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Are there any problems you frequently encounter with diagnosis?

  • Dr. Muhammad Jawad Khan

The Brooke Hospital Pakistan

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses

“The enemy of my enemy is my friend”

Tail Baseplate Short tail fibres

Lambda phage

  • E. coli
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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses

4.5 h

Jacobs et al (2012) Journal of Clinical Microbiology, 50(4), 1362; doi: 10.1128/JCM.06192-11

Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses

TB

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses

  • 1. Patient provides

sample

  • 2. Sample mixed with

phage solution

  • 3. Signal in presence
  • f pathogen

GFP

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etc.

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses Fighting infectious disease with viruses

  • 1. Patient provides

sample

  • 2. Sample mixed with

phage solution

  • 3. Signal in presence
  • f pathogen

GFP

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Plan of Attack Plan of Attack Plan of Attack Plan of Attack – – – – Developing Modularity Developing Modularity Developing Modularity Developing Modularity

using Bacteriophage lambda (arch enemy of E.coli) as chassis

phage genome tail fibre protein (stf)

facilitates initial attachment

  • f virion to E.coli outer

membrane protein C (OmpC)

tip attachment protein J

binds to E.coli maltoporin (LamB)

tail fibre assembly protein (tfa)

chaperone – facilitates tail fibre protein folding

phage protein shell

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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lambda phage short tail fibres

  • E. coli receptor
  • E. coli mutant

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Directed Evolution of Short Tail Directed Evolution of Short Tail Directed Evolution of Short Tail Directed Evolution of Short Tail Fibres Fibres Fibres Fibres

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lambda phage short tail fibres

  • E. coli receptor

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Directed Evolution of Directed Evolution of Directed Evolution of Directed Evolution of Short Tail Short Tail Short Tail Short Tail Fibres Fibres Fibres Fibres

  • E. coli mutant
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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

  • E. coli receptor

engineered lambda phage short tail fibres

  • E. coli mutant

Directed Evolution of Directed Evolution of Directed Evolution of Directed Evolution of Short Tail Short Tail Short Tail Short Tail Fibres Fibres Fibres Fibres

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity

Circuit Development

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity

Circuit Development

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity

Circuit Development

1, 2, 3: T7 Express E. coli 4, 5, 6: T7 Express E. coli + cl-Crocircuit 7, 8, 9: DH10β E. coli + cl-Cro circuit 10, 11: DH10β E. coli Figure 1. Analysis of cI/Cro circuit in DH10β E. coli alongside control samples showing successful expression of the cI repressor under a constitutive promoter and no expression in T7 Express E. coli cells.

+with IPTG (0.4 µg/ml [final]) ‡ with IPTG (2.0 µg/ml [final])

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity

Circuit Development

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity

Circuit Development

1, 2, 3: tfa composite part (control) 4, 5, 6: TetRcomposite part (control) 7, 8, 9: TetR-regulated tfa circuit in pSΒ1A3 10: DH10β (control) 11, 12, 13: TetR-regulated tfa circuit in pSΒ1K3 Figure 2. Analysis of TetR-regulated tfa (tail fibre assembly) circuit in DH10β E. coli alongside control samples showing successful expression of the tfa protein under TetR regulation.

*with anhydrous tetracycline (aTc) (0.4 µg/ml [final])

†with aTc (2.0 µg/ml [final])

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Future Work: Future Work: Future Work: Future Work: Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity Changing bacteriophage specificity

Directed Evolution Strategy

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New Parts submitted to the Registry New Parts submitted to the Registry New Parts submitted to the Registry New Parts submitted to the Registry

basic “modular” parts basic “modular” parts basic “modular” parts basic “modular” parts

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements Name Construct Description Status Part Goal ORF-314

Basic part Complete BBa_K1846000

stf gene

Basic part Complete BBa_K1846004

tfa gene

Basic part in production

J gene

Basic part in production

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New Parts submitted to the Registry New Parts submitted to the Registry New Parts submitted to the Registry New Parts submitted to the Registry

composite parts and circuits composite parts and circuits composite parts and circuits composite parts and circuits

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements Name Construct Description Status Part Goal tfa construct

tail fibre assembly construct Composite part Complete BBa_K1846001

P(Cat)- TetR construct

construct for the production of TetR Composite part Complete BBa_K1846003

TetR-tfa circuit

circuit to control tail fibre assembly protein Composite part Complete BBa_K1846007

cI-cro circuit

circuit to control lytic and lysogenic cycles of lambda phage Composite part Complete BBa_K1846005

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Infectious diseases in the developing world Infectious diseases in the developing world Infectious diseases in the developing world Infectious diseases in the developing world

Infectious disease Detection Treatment

Speed Cost

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

What do you consider to be the main challenges in the eradication of TB and drug resistant strains?

  • Dr. Sanjib Bhakta

Director of Mycobacteria Research Laboratory at Birkbeck / UCL

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Science Busking Science Busking Science Busking Science Busking

We used fun props and games to illustrate key busking at the local William Tyndale primary school in North London. concepts in sanitation and genetics to an audience of 9- & 11-year olds. We did science

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About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Conclusion Conclusion Conclusion Conclusion

  • We isolated phage parts that are key for specificity
  • We set up circuits to:
  • regulate assembly of the new parts
  • control timing for the assembly of recombinant phages
  • We submitted new parts to the Registry
  • We discussed the importance and challenges of disease

detection in remote and deprived communities with specialists

  • We inspired the new generation of iGEMers by engaging primary

school children in synthetic biology games

  • We contributed to the standardisation of measurement by taking

part in the Interlab Study

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Advisors

  • Yan Kay Ho
  • Pedro Galvao Tizei

Instructors

  • Dr Vitor Bernardes Pinheiro
  • Dr Jane Nicklin

About us - Background - Plan of attack - BioBricks - Conclusions - Acknowledgements

Our team

  • Sean Ross Craig
  • Elliott Parris
  • Rachel Wellman

Attributions Attributions Attributions Attributions

Part-time Team Members

  • Wayne Pires (Birkbeck iGEM team founder)
  • Mervyn Richardson (outreach participant)
  • Shapoor Mohamadi (outreach participant)
  • Tim Walker (policy and practice advisor)
  • Alberto Aparicio (policy and practice advisor)
  • Ariana Mirzarafie-Ahi
  • Barbara Steijl
  • Luba Prout
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Acknowledgements Acknowledgements Acknowledgements Acknowledgements

  • Technion, Haifa, Israel
  • University College London, UK
  • Westminster University, London, UK
  • University of Kent, Canterbury, UK
  • Biohackspace, London, UK
  • Amoy University, Xiamen, China
  • Aalto University, Helsinki, Finland
  • Tianjin University, China
  • Aix Marseille Université, France
  • Macquarie University, Sydney, Australia
  • Trinity College Dublin, Ireland
  • Prof David Latchman (Birkbeck sponsorship)
  • Prof Darren Nesbeth (lab training & UCL sponsorship)
  • Prof Gabriel Waksman (team foundation support)
  • Dr Nicholas Keep (team foundation support)
  • Bilkis Banu Kazi (lab management and assistance)
  • Prof John Ward (phage assay training & provision of

phages and host strains)

  • Henry De Malmanche (phage assay training)
  • Prof Elena Orlova (lambda & general phage

information)

  • Dr Renos Savva (advice on bacteriophages in biotech)
  • Dr Salvador Tomas (lab space provision)
  • Dr Sanjib Bakhta (TB advice)
  • Lewis Moffat (wiki advice)
  • Dr Richard Rayne (lab space provision)
  • Tim Hoe (administration)
  • Sandra Codlin (lab space provision)
  • Alexey Tomsov (gene provision)

Thanks to Collaborations

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References References References References

  • Echols, H., & Murialdo, H. (1978). Genetic map of bacteriophage lambda. Microbiological

Reviews, 42(3), 577–591.

  • Friedman, D. I., & Court, D. L. (2001). Bacteriophage lambda: Alive and well and still doing

its thing. Current Opinion in Microbiology, 4(2), 201–207. doi:10.1016/S1369- 5274(00)00189-2

  • Haggard-Ljungquist, E., Halling, C., & Calendar, R. (1992). DNA sequences of the tail fiber

genes of bacteriophage P2: Evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages. Journal of Bacteriology, 174(5), 1462–1477.

  • Hashemolhosseini, S., Stierhof, Y. D., Hindennach, I., & Henning, U. (1996).

Characterization of the helper proteins for the assembly of tail fibers of coliphages T4 and λ. Journal of Bacteriology, 178(21), 6258–6265.

  • Hendrix, R. W., & Duda, R. L. (1992). Bacteriophage lambda PaPa: not the mother of all

lambda phages. Science (New York, N.Y.), 258(5085), 1145–1148. doi:10.1126/science.1439823

  • Jacobs et al (2012) Journal of Clinical Microbiology, 50(4), 1362. doi: 10.1128/JCM.06192-

11

  • Montag, D., Schwarz, H., & Henning, U. (1989). A component of the side tail fiber of E.

coli bacteriophage λ can functionally replace the receptor-recognizing part of a long tail fiber protein of the unrelated bacteriophage T4. Journal of Bacteriology, 171(8), 4378–4384.

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Questions Questions Questions Questions

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Changing bacteriophage Changing bacteriophage Changing bacteriophage Changing bacteriophage specificity specificity specificity specificity Supplementary Information Supplementary Information Supplementary Information Supplementary Information

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Changing bacteriophage specificity

  • Introduction of short tail fibre and selection

marker ampR genes into the Enterobacteria phage λ gt11 vector (lambda DNA) that lacks tail fibre genes

  • Packaging of the recombinant DNA into λ

proteins from a packaging extract.

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Changing bacteriophage specificity

  • Transformation of Y1088 E.coli strain

(hsdR-, hsdM+) with genetic circuits controlling the production of (i) assembly protein assisting with the folding of the short tail fibre, and (ii) lysogenic/lytic bacteriophage cycles

  • Infection of bacteria with the assembled

phage

  • Replication of the recombinant phage via

the E.coli machinery

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Changing bacteriophage specificity

  • Production of new

recombinant virions with variant short tail fibres by the infected E.coli.

  • Lytic cycle is activated
  • Recombinant phages

erupt from E.coli cells

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Changing bacteriophage specificity

  • Assessment of the

recombinant phage infectivity on E.coli BW25113 lamB/ompC double knock-out strain.

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Changing bacteriophage specificity

  • Successfully infected cells

are expected to proliferate

  • n Amp-supplemented

plates, suggesting altered tail fibre range.

  • Colonies are picked and

phage DNA is purified. Subsequent rounds of selection are performed.