Let’s 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 Preventive tree cutting implies high costs Ceratocystis platani • Supposed introduction: American landing in 1944 • Blocks sap flow • No effective treatment 3
Ethical questioning Mankind Nature Beauty Science in the Observation and service of art and modification of beauty ? nature Synthetic Biology Our approach 4
SubtiTree to save our heritage Our chassis: Bacillus subtilis, an endophyte bacterium of the plane tree 5
SubtiTree to save our heritage Our chassis: Bacillus subtilis, TARGET an endophyte bacterium of the plane tree Mediated by a chimeric protein targeting chitin components 5
SubtiTree to save our heritage TARGET Mediated by a chimeric protein targeting chitin components Chitin-derived compounds Chitin 5
SubtiTree to save our heritage BIND Mediated by a chimeric protein anchoring in both fungal cell wall and peptidoglycan Chitin 5
SubtiTree to save our heritage BIND Mediated by a chimeric protein anchoring in both fungal cell wall and peptidoglycan Chitin 5
SubtiTree to save our heritage Chitin ERADICATE Production of different anti- fungal peptides 5
SubtiTree to save our heritage 5
1- TARGET Concept McpA McpA McpA VCD T BBa_1364004 N- Acetylglucosamine SubtiTree ² Glucose NAG NAG Extracellular Intracellular VCD McpA Chimeric V. cholerae B. subtilis protein 6
1- TARGET Concept McpA McpA McpA VCD T BBa_1364004 N- Acetylglucosamine SubtiTree ² Glucose NAG NAG Extracellular Intracellular VCD McpA Chimeric V. cholerae B. subtilis protein 6
1- TARGET Tests Bacillus 250mM GLC subtilis Plate tests WT Plug-in-pond protocol ² Tube tests Motility test in capillary Capillary assay Bacteria swimming to the top of a tip (Imperial College iGEM team 2011) 7
1- TARGET Tests WT Bacillus subtilis Glucose ² Galactose NAG Final results WT Bacillus subtilis naturally attracted to NAG! 8
2- BINDING Concept BBa_1364005 CWB Linker CBD T LytC, B. subt. GbpA, V. chol. ² Peptidoglycan Chitin SubtiTree 9
2- BINDING Tests Chitin-coated magnetic beads ² Engineered B. subtilis 10
2- BINDING Tests ² Engineered B. subtilis 10
2- BINDING Tests ² 10
2- BINDING Tests Beads covered with B. subtilis (confocal microscopy) ² 100 µm 10
2- BINDING Tests ² 10
2- BINDING Tests ² 10
2- BINDING Tests ² 10
2- BINDING Tests ² 10
2- BINDING Tests ² 10
2- BINDING Tests 1,E+05 = 170x 1,E+04 Number of bacteria per mL 1,E+03 ² 1,E+02 1,E+01 1,E+00 WT SubtiTree 10
3- ERADICATE Concept in situ production of antifungal peptides SubtiTree ² BBa_1364008 BBa_1364019 BBa_1364009 GAFP-1 D4E1 EcAMP T Inhibit hyphae elongation Mannose-binding lectin Sterol binding 11
3- ERADICATE Tests Setting up the testing protocol Commercial peptides Improve experimental conditions Copper sulfate Balance the growth WT bacteria ² 12
3- ERADICATE Tests Setting up the testing protocol Commercial peptides Improve experimental conditions Copper sulfate Balance the growth WT bacteria Tests with transformed bacteria Expressing 1, 2 or 3 antifungal peptides ² 12 SN: Culture supernatant P: cells
Let’s look back on the first steps Proof of concept Modeling Target - Bind - Eradicate Assembling the 3 modules 13 Tests with model plants
in planta Tests 80 % of sites showing necrosis 70 60 50 40 30 20 10 Nicotiana benthamiana 0 ² Mock WT D4E1 D4E1 + GAFP-1 WT SubtiTree 14
in planta Tests 80 % of sites showing necrosis 70 60 50 40 30 20 10 Nicotiana benthamiana 0 ² Mock WT D4E1 D4E1 + GAFP-1 WT SubtiTree 14
Modeling Concept When to treat the trees ? What will happen to SubtiTree during winter ? Cardinal temperature model of Rosso (1992) Bacterial growth ² Tree model Volume: 30m 3 Density: 650kg/m 3 Bacteria inoculum: 10mL at 10⁹ cfu/mL Two conditions: Toulouse 15
Modeling Results Bacterial cycle as a function of temperature to Average temperatures in Toulouse predict the active period of SubtiTree ² Growth stops Bacteria die Subtitree death Subtitree action time Best inoculation time is March 16 Subtitree will not survive to winter
What about now? The future of SubtiTree BBa_K314200 BBa_K314201 ² Further studies Binding Induction of Regulation specificity expression 17
Communication Communication tools Newsletters iGEM Toulouse presentation poster iGEM Toulouse presentation flyer SubtiTree presentation flyer Social networks ² 18
Communication Communication tools Newsletters iGEM Toulouse presentation poster iGEM Toulouse presentation flyer SubtiTree presentation flyer Social networks ² Oral presentations Agora Journées de l’ Ecole Doctorale BSB Exposcience Novela BioSynSys conference 18
Communication Communication tools Newsletters iGEM Toulouse presentation poster iGEM Toulouse presentation flyer SubtiTree presentation flyer Social networks ² Oral presentations Agora Journées de l’ Ecole Doctorale BSB Exposcience Novela BioSynSys conference Press 14 articles 18
Communication Communication tools Newsletters iGEM Toulouse presentation poster iGEM Toulouse presentation flyer SubtiTree presentation flyer Social networks ² Oral presentations Agora Journées de l’ Ecole Doctorale BSB Exposcience Novela BioSynSys conference Press 14 articles 1 radio interview 18
Communication Communication tools Newsletters iGEM Toulouse presentation poster iGEM Toulouse presentation flyer SubtiTree presentation flyer Social networks ² Oral presentations Agora Journées de l’ Ecole Doctorale BSB Exposcience Novela BioSynSys conference Press 14 articles 1 radio interview 1 tv interview 18
Public perception PUBLIC SUPPORT Crowdfunding ² Local & GOVERNMENTAL SCIENTIFIC ORGANIZATIONS INSTITUTION National Agricultural Ministry INRA Town Halls 19
Achievements Ethical point of view 20
Achievements Proof of concept of the modules 16 new BioBricks Ethical point of view A new integrative plasmid for B. subtilis (BBa_K1364021) Characterized BBa_K1364009, BBa_K1364013 and BBa_K1364005 Improved BBa_K1162001 by adding STOP codon Created and improved new protocols 20
Achievements Tests in planta Proof of concept of the modules Ethical point of view 20
Achievements Toward plane trees (Modeling) Tests in planta Proof of concept of the modules Ethical point of view 20
Achievements Toward plane trees (Modeling) Tests in planta Proof of concept of the modules And had a Ethical point GREAT summer ! of view 20
Acknowlegments Our institutions Sponsors Lab equipment support 21
Thank you for your attention 22
Let’s save our trees with SubtiTree!
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