SubtiTreeLet’s save our trees with SubtiTree

Trees endangered

1. TARGETN-AcetylGlucosamine is cell wall component of canker fungus. To induce chemotaxis targeting the fungus, we constructed a synthetic NAG receptor, based on 2 existing receptors from Vibrio cholerae and B. subtilis. We improved experimental protocols and developed new ones and shown that a natural B. subtilis strain is attracted to NAG.

2. BINDTo promote fixation of SubtiTree to kill the fungus, we designed binding module (BBa_K1364005), composed of a chimeric protein with 2 domains use to associate bacterial peptidoglycan and fungal chitin .In presence of the binding module, there is an attachment on the chitin beads, used to mimic the fungal cell wall.

3. ERADICATETo kill the pathogen, fungicide triple therapy is achieved with 3 peptides producing genes ( EcAMP-1, D4E1 and GAFP-1) organized in operon. So far, we validated the eradicate module containing D4E1 and GAFP-1 (DG) (BBa_K1364013).

The “Canal du Midi” is a UNESCO World Heritage masterpiece, located in the heart of our city and wandering through Southern France. Unfortunately, this legacy is threatened by the canker disease: a fungal plant pathogen, Ceratocystis platani, is devastating the plane trees bordering the “Canal du Midi”. The Toulouse team engaged in the iGEM 2014 competition, to defend the plane trees forming a gorgeous natural arch and protect the natural beauty of this extraordinary example of civil engineering. We took advantage of Bacillus subtilis, a bacterial strain naturally present in the trees to create a genetically modified bacterium able to kill Ceratocystis platani in plane trees.

MEET THE TEAM

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Advisors

INSTITUTIONS

CROWDFUNDING

Modeling

SpreadingAchievements

In this work, we modified Bacillus subtilis to create SubtiTree, a strain equipped with three genetic modules: chemotaxis (swim toward the fungus), binding (attach to fungi cell wall) and fungicides (kill the pathogen).

A critical validation of the project is SubtiTree’s ability to kill the fungus in planta. We tested on Nicotiana benthamiana the capacity of SubtiTree to reduce leaves necrosis and infection spreading.

We modeled the impact of Toulouse’s temperature on B. subtilis growth inside trees during one year. The optimum condit ion for treatment injection is in March, growth will continue during the summer and the cold winter temperatures wi l l stop SubtiTree’s development.

The use of SubtiTree directly in plane trees imposes environmental control of its putative dissemination: our smart bacterium must stay in plane trees, live no more than one year and avoid horizontal DNA transfer.

Modules design and validation

CONTACT

igemtoulouse2014@gmail.com

iGEM Toulouse 2014

@iGEM_Toulouse

FRANCE

Toulouse

• Tips capillary system• Improving swimming conditions • Microscopic observations of SubtiTree (in green) on chitin beads

• Attachment of SubtiTreeto chitin beads • SubtiTree kills Trichoderma reesei

Black: B. subtilis growth during one year. Red: average temperature in Toulouse area. Blue: threshold at 10°C under which growth decreases.Dotted blue line: threshold at 4°C below which bacteria die.

• In planta test with SubtiTree

TARGETAbility to move

towards agradientof NAG

ERADICATEAntifungal activity

on Petri dishesand in planta

BINDBinding to thefungus chitin

• B. subtilis WT • SubtiTree

Added 16 new parts on Registry

Added new integrative plasmid PsBbs4E for B. subtilis (BBa_K1364021)

Improved BBa_K1162001 (Pveg - EcAMP-1) by adding STOP codon (BBa_K136401)

Characterized BBa_K1364009 (Pveg-D4E1), BBa_K1364013 (Pveg-D4E1-GAFP1), BBa_K1364005 (Binding)

New ethical point of view for synthetic biology: protect the beauty of nature

Phase 1: in Petri dishes

Phase 2: in planta

Phase 3: in plane treesNext step !

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Validation in planta

Labastide d’Anjou Montréal Ventenac-en-Minervois

Gilles Truan Brice Enjalbert Florence Bordes Kaymeuang Cam

Diane Emeline Mathieu Florie

Camille Aurélie Laureen Manon

Fanny Pierre Abdel