BACTAIL: Interactive Poster (Grant)

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

iGEM IONIS

BacTail

BacTail

EXPERIMENTAL APPROACH

BUDGET & TIMING

CONTEXT

• Overconsumption antibiotics
• Antimicrobial Resistance
• 10 million deaths per year by 2050
• Bacteriophage inspired concept
• Synthetic biology

Workflow of the first part of the BacTail project

First part of the project = 2823,24€

March-July = Bibliography & Protocol write-up, Dry Lab June-September: Wet Lab

EXPECTED OUTCOME

AIMS

• Double Kill Switch
• Software Design
• Galenic form
• Pharmacological factors

• Design a plasmid to insert into chassis

• Specific recognition and binding to pathogenic resistant bacteria

Schematic representation of the distal LTF gene part 37 at the surface of our chassis E. coli K12 ΔOmpC

• Trigger the lysis of the target bacteria

ABSTRACT

COLLAB.

REFS.

Sup'Biotech Interactive Scientific Poster ©

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

BacTail

TEAM

Thomas

Albane

Renaud

Giulia

Max

Louis

Lucie

David

Charlotte

Elliot

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

CONTEXT

BacTail

• 20th century: Antibiotics = Major healthcare breakthrough
• Penicillin discovery in 1928 (Alexander Fleming)
• Block reproduction of bacteria or destroy them
• Antibiotic Resistance : bacteria develop an immunity to drugs designed to kill them
• One of the biggest threats to global health
• Deadly consequences
• Pneumonia, tuberculosis, blood poisoning or gonorrhea can now potentially kill

2000-2015ATB consumption rate increased by 39%

30% of antibiotics prescribed by doctors are unnecessary (USA)

DDDs per 1,000 inhabitants per day

[1] Global antibiotic consumption by country income classification: 2000–2015DDD=Defined daily doses

[1] IQVIA MIDAS, 2000–2015, IQVIA Inc.

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

CONTEXT

BacTail

10 million deaths per year by 2050 if no solution found

No new famillies of antibiotics launched in 25 years

In France: costs 350$ million / year

Resistance to Antibiotics

High level of ocean pollution linked to antibiotics

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

CONTEXT

BacTail

• Bacteriophages
• Natural predators of bacteria
• 10 different families
• Specifically recognize then infect a bacterium --> lysis
• Myoviridae use their Long Tail Fibers to link target’s membrane receptors
• Specificity determined by tip of the LTF (gp37)
• Phage Therapy:
• Lack of knowledge
• Phage multiplication uncontrollable – difficult to modify
• Delivery barriers
• Benefit-Risk balance unknown

[1] Schematic representation of a T4 bacteriophage specific to E. coli and Long Tail Fiber gene and protein organization

[2] Crystallography schematic representation of a distal Long Tail Fiber protein organization

[1] Pherecydes pharma (translation FREN) and Islam, Mohammad Z., et al. 2019 [2] Sergio G. Bartual et al. PNAS 2010;107:47:20287-20292

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

AIMS

BacTail

Genetically modify Escherichia coli (E.coli) in order to fight against bacterial strains (target) considered as resistant to antibiotics. The genetic modification involves the integration of bacterial recognition genes from bacteriophages, giving the capacity to recognize and lyse the targeted bacteria.

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

AIMS

BacTail

1- Plasmid design and construction for the LTF expression 2- Confirmation of expression and localization of the LTF 3- Confirmation of the binding to the target cell and specificity evaluation

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

EXPERIMENTAL APPROACH

BacTail

Expressing the LTF region gp37 on the bacterial surface

Some examples of gene functions:

A BioBrick with the ability to execute this task, is inserted between a prefix and suffix region of a high copy number plasmid, pSB1A3

• LPP-OmpA: Address the fusion protein on the surface
• LTF-gp37: Recognize the pathogenic target
• Chaperonne proteins gp38 and 57a: Ensure the right folding

Fig : Scheme of the BioBrick allowing the LTF expression on the outer membrane

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

EXPERIMENTAL APPROACH

BacTail

Plasmid construction

• Different fragments are assembled by Hifi Assembly Method
• E.coli K12 ΔOmpC, our model organism, is rendered competent before proceeding with the CaCl2 transformation step
• Then culture on a selective media

Fig: Final plasmid construct

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

EXPERIMENTAL APPROACH

BacTail

Characterization

Verification that LTFs are well expressed on the outer membrane of the bacteria

• TEV cleavage → Free the end of the complex (HisTag-RFP-Linker-gp37)
• Affinity Chromatography→ Purification of the complex
• SDS PAGE → Confirm presence of complex (molecular weight)

Measurements

• Quantification of the level of fluorescence → ImageJ

• Testing different strengths of promoter and RBS combinations
• Evaluate the expression of our construct

Fig: Schematic representation of the Characterization protocol

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

BUDGET & TIMING

BacTail

GANTT CHART

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

BUDGET & TIMING

BacTail

BUDGET

• First part of the project → Total consumables = 2823,24€

• iGEM + sponsors = free products
• E.coli K12 ΔOmpC
• Integrated DNA Technologies (IDT)
• T4 phage
• Bacillus subtilis

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

EXPECTED OUTCOME

BacTail

Double Kill Switch 1- Lysis response when recognition of the target 2- Prevent dissemination of the chassis

Software development - Given pathogenic bacteria - Identify associated LTF - Design key plasmid

Pharmacological Factors Assessment Pharmacodynamic + pharmacokinetics Optimal concentration Route of administration

Galenic Form Oral administration → Resistance to gastric acid

LET'S FIGHT ANTIMICROBIAL RESISTANCE TOGETHER

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

BIBLIOGRAPHY

BacTail

(1) Dunne, M., & Loessner, M. J. (2019). Modified Bacteriophage Tail Fiber Proteins for Labeling, Immobilization, Capture, and Detection of Bacteria. In Foodborne Bacterial Pathogens (pp. 67-86). Humana Press, New York, NY.(2) University of Jyväskylä - Jyväskylän yliopisto. (2020, March 27). Forgotten tale of phage therapy history revealed: Phage therapy was successfully used against dysentery and staphylococcal infections. ScienceDaily. Retrieved April 27, 2020 from www.sciencedaily.com/releases/2020/03/200327113647.html (3) de Kraker, M. E. A., Stewardson, A. J., & Harbarth, S. (2016). Will 10 Million People Die a Year due to Antimicrobial Resistance by 2050? PLOS Medicine, 13(11), e1002184. https://doi.org/10.1371/journal.pmed.1002184 (4) More, J. A. F. D. (2018). OECD Health Policy Studies This series of publications analyses the organisation and performance of health systems, and factors explaining performance variations. Studies are conducted on such topics as coordination of care, pharmaceutical pricing, long-term care and disability, health workforce and international migration of health workers, information and communications technologies in health care, and the economics of prevention. English Also available in: French. (5) Yoichi, M., Abe, M., Miyanaga, K., Unno, H., & Tanji, Y. (2005). Alteration of tail fiber protein gp38 enables T2 phage to infect Escherichia coli O157: H7. Journal of biotechnology, 115(1), 101-107. (6) ETH Zurich. (2019, November 4). Synthetic phages with programmable specificity. ScienceDaily. Retrieved April 27, 2020 from www.sciencedaily.com/releases/2019/11/191104112838.html (7) Trojet, S. N., Caumont-Sarcos, A., Perrody, E., Comeau, A. M., & Krisch, H. M. (2011). The gp38 adhesins of the T4 superfamily: a complex modular determinant of the phage’s host specificity. Genome biology and evolution, 3, 674-686. (8) United Nations, Department of Economic and Social Affairs, Population Division (2019). World Population Prospects 2019: Highlights (ST/ESA/SER.A/423). https://population.un.org/wpp/Publications/Files/WPP2019_Highlights.pdf (9) Chaired by Jim O’Neill,(2016). Tackling Drug-Resistant Infections Globally: final report and recommendations. https://amrreview.org/sites/default/files/160525_Final% 20paper_with%20cover.pdf (10) Boldt, J.Life as a Technological Product: Philosophical and Ethical Aspects of Synthetic Biology. Biol Theory 8, 391–401 (2013). https://doi.org/10.1007/s13752-013-0138-7 (11) Christine Noiville, http://www.hautconseildesbiotechnologies.fr/fr (12) Bensaude Vincent, B. Ethical Perspectives on Synthetic Biology. Biol Theory 8, 368–375 (2013). https://doi.org/10.1007/s13752-013-0137-8 (13) https://www.benchling.com (14) Gouv.fr, (2019). Les organismes génétiquement modifiés (OGM), https://www.ecologique solidaire.gouv.fr/organismes-genetiquement-modifies-ogm-0 (15) https://2019.igem.org/Safety/Final_Safety_Form

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

ABSTRACT

BacTail

The massive and repeated use of antibiotics has recently led to the appearance of resistant bacteria for which no treatment exists. This is a major health issue to which the iGEM IONIS 2020 team is trying to respond. This project consists in designing several BioBricks to procure three main abilities to the engineered bacteria. First, expressing Long Tail Fibers of bacteriophages on the bacterial surface to recognize and bind the specific target. Then, setting-up the precise target lysis. And last, the self- regulation via a kill switch to prevent its dissemination to the environment.

Elliot COQUEREL, Giulia CRISEO, Charlotte DUTEIL, Renaud EYNARD, Albane MABRO, Lucie PESENTI

COLLABORATIONS & ACKNOWLEDGEMENTS

BacTail

• Integrated DNA Technologies (IDT) → 20,000 bases of free DNA synthesis

• Professor Frank Delvigne at the University of Liège → Contributions in microbiology

• Dr Ansaldi working at bacterial chemistry laboratory → Phage T4 DNA

• Geneious & SnapGene→ Free licenses

• Benchling → Free accounts

• MathWorks→ Free MATLAB software