Lets save our trees with SubtiTree! Opening synthetic biology to - - PowerPoint PPT Presentation

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Lets save our trees with SubtiTree! Opening synthetic biology to - - PowerPoint PPT Presentation

May 18, 2023 45 likes •559 views

Lets save our trees with SubtiTree! Opening synthetic biology to new challenges 2 A more local scale 2 The Canal du Midi Southwest of France 42,000 plane trees UNESCO 2 Canal du Midi and Canker Plane trees lining the Canal

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Let’s save our trees with SubtiTree!

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Opening synthetic biology to new challenges

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A more local scale

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The “Canal du Midi” Southwest of France

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UNESCO 42,000 plane trees

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Ceratocystis platani

  • Supposed introduction:

American landing in 1944

  • Blocks sap flow
  • No effective treatment

Canal du Midi and Canker

Plane trees lining the Canal Preventive tree cutting implies high costs

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Mankind

Observation and modification of nature

Nature

Synthetic Biology

Beauty

Science in the service of art and beauty ?

Our approach

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Ethical questioning

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Our chassis: Bacillus subtilis, an endophyte bacterium of the plane tree

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SubtiTree to save our heritage

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Mediated by a chimeric protein targeting chitin components

TARGET

Our chassis: Bacillus subtilis, an endophyte bacterium of the plane tree

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SubtiTree to save our heritage

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Mediated by a chimeric protein targeting chitin components

Chitin-derived compounds Chitin

TARGET

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SubtiTree to save our heritage

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Mediated by a chimeric protein anchoring in both fungal cell wall and peptidoglycan

Chitin

BIND

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SubtiTree to save our heritage

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Mediated by a chimeric protein anchoring in both fungal cell wall and peptidoglycan

Chitin

BIND

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SubtiTree to save our heritage

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Production of different anti- fungal peptides

Chitin

ERADICATE

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SubtiTree to save our heritage

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SubtiTree to save our heritage

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McpA VCD

Concept

McpA McpA

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N- Acetylglucosamine SubtiTree

Chimeric protein VCD

  • V. cholerae

McpA

  • B. subtilis

Glucose

NAG NAG

Extracellular Intracellular

1- TARGET

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BBa_1364004

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McpA VCD

Concept

McpA McpA

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N- Acetylglucosamine SubtiTree

Chimeric protein VCD

  • V. cholerae

McpA

  • B. subtilis

Glucose

NAG NAG

Extracellular Intracellular

1- TARGET

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BBa_1364004

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Tube tests

Motility test in capillary

Plate tests

Plug-in-pond protocol

Tests

1- TARGET

Capillary assay

Bacteria swimming to the top of a tip (Imperial College iGEM team 2011)

7 250mM GLC

Bacillus subtilis WT

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Glucose NAG Galactose

WT Bacillus subtilis

Final results

WT Bacillus subtilis naturally attracted to NAG!

Tests

1- TARGET

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Concept

CWB Linker CBD T LytC, B. subt. GbpA, V. chol. Peptidoglycan Chitin

SubtiTree

2- BINDING

BBa_1364005

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2- BINDING

Tests

Chitin-coated magnetic beads Engineered B. subtilis

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2- BINDING

Tests

Engineered B. subtilis

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2- BINDING

Tests

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2- BINDING

Tests

Beads covered with B. subtilis (confocal microscopy)

100 µm

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2- BINDING

Tests

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2- BINDING

Tests

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2- BINDING

Tests

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2- BINDING

Tests

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2- BINDING

Tests

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2- BINDING

Tests

1,E+00 1,E+01 1,E+02 1,E+03 1,E+04 1,E+05 WT SubtiTree

Number of bacteria per mL

= 170x

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² EcAMP GAFP-1 D4E1

Inhibit hyphae elongation Mannose-binding lectin Sterol binding

T in situ production of antifungal peptides

SubtiTree

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3- ERADICATE

Concept

BBa_1364019 BBa_1364008 BBa_1364009

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Setting up the testing protocol

Commercial peptides Copper sulfate WT bacteria Improve experimental conditions Balance the growth

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3- ERADICATE

Tests

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Tests with transformed bacteria

Expressing 1, 2 or 3 antifungal peptides

Setting up the testing protocol

Commercial peptides Copper sulfate WT bacteria Improve experimental conditions Balance the growth

SN: Culture supernatant P: cells

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3- ERADICATE

Tests

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Proof of concept Target - Bind - Eradicate

Tests with model plants Modeling Assembling the 3 modules

Let’s look back on the first steps

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in planta Tests

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Nicotiana benthamiana

10 20 30 40 50 60 70 80

Mock WT D4E1 D4E1 + GAFP-1

% of sites showing necrosis

WT SubtiTree

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in planta Tests

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Nicotiana benthamiana

10 20 30 40 50 60 70 80

Mock WT D4E1 D4E1 + GAFP-1

% of sites showing necrosis

WT SubtiTree

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Modeling

Concept

When to treat the trees ? What will happen to SubtiTree during winter ?

Cardinal temperature model of Rosso (1992)

Tree model  Volume: 30m3  Density: 650kg/m3  Bacteria inoculum: 10mL at 10⁹cfu/mL Two conditions: Bacterial growth Toulouse

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Results

Growth stops Bacteria die

Bacterial cycle as a function of temperature to predict the active period of SubtiTree

Subtitree action time

Best inoculation time is March Subtitree will not survive to winter

Subtitree death

Average temperatures in Toulouse

Modeling

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What about now?

The future of SubtiTree

Binding specificity Induction of expression Regulation Further studies

BBa_K314200 BBa_K314201

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Communication Communication tools

Newsletters iGEM Toulouse presentation flyer iGEM Toulouse presentation poster SubtiTree presentation flyer Social networks

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Communication Oral presentations

Agora Journées de l’Ecole Doctorale BSB Exposcience BioSynSys conference Novela

Communication tools

Newsletters iGEM Toulouse presentation flyer iGEM Toulouse presentation poster SubtiTree presentation flyer Social networks

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Communication

Press

14 articles

Oral presentations

Agora Journées de l’Ecole Doctorale BSB Exposcience BioSynSys conference Novela

Communication tools

Newsletters iGEM Toulouse presentation flyer iGEM Toulouse presentation poster SubtiTree presentation flyer Social networks

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Communication

Press

14 articles 1 radio interview

Oral presentations

Agora Journées de l’Ecole Doctorale BSB Exposcience BioSynSys conference Novela

Communication tools

Newsletters iGEM Toulouse presentation flyer iGEM Toulouse presentation poster SubtiTree presentation flyer Social networks

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Communication

Press

14 articles 1 tv interview 1 radio interview

Oral presentations

Agora Journées de l’Ecole Doctorale BSB Exposcience BioSynSys conference Novela

Communication tools

Newsletters iGEM Toulouse presentation flyer iGEM Toulouse presentation poster SubtiTree presentation flyer Social networks

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Public perception

Local & National

INRA Agricultural Ministry Town Halls PUBLIC SUPPORT GOVERNMENTAL ORGANIZATIONS SCIENTIFIC INSTITUTION

Crowdfunding

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Achievements

Ethical point

  • f view
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Achievements

Ethical point

  • f view

Proof of concept of the modules 16 new BioBricks A new integrative plasmid for

  • B. subtilis (BBa_K1364021)

Improved BBa_K1162001 by adding STOP codon Characterized BBa_K1364009, BBa_K1364013 and BBa_K1364005 Created and improved new protocols

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Achievements

Ethical point

  • f view

Tests in planta Proof of concept of the modules

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Achievements

Ethical point

  • f view

Tests in planta Toward plane trees

(Modeling)

Proof of concept of the modules

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And had a GREAT summer !

Achievements

Ethical point

  • f view

Tests in planta Toward plane trees

(Modeling)

Proof of concept of the modules

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Acknowlegments

Sponsors Our institutions Lab equipment support

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Thank you for your attention

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Let’s save our trees with SubtiTree!