Solution
A plant able to protect crops by producing mating disruption pheromones when
intercropped with the affected plant species.
We present you
The newest pest management method. Safe, sustainable and environmentally
friendly.
OBJECTIVEReproduce insect sexual pheromone biosynthesis pathway in plants
WHICH INSECT?
Moths (Lepidopterans)
OBJECTIVEReproduce insect sexual pheromone biosynthesis pathway in plants
WHICH INSECT?Moths (Lepidopterans)
WHY?• High impact as a worldwide plague
Worldwide presence of Helicoverpa armigera & Helicoverpa zea
OBJECTIVEReproduce insect sexual pheromone biosynthesis pathway in plants
WHICH INSECT?Moths (Lepidopterans)
WHY?• High impact as a worldwide plague• Chemical simplicity of the pheromones
(Z)-11-Hexadecen-1-ol
(Z)-11-Hexadecenal (Z)-11-Hexadecenyl acetate
H. a
rmig
era
A. tra
nsitella
E. alatus
16:CoA
Z11-16:CoA
DesaturaseAtrΔ11
ReductaseHarFAR
Acetyl transferase
EaDAcT
Alcohol Oxidase
FAO
(Z)-11-Hexadecen-1-ol
Alcohol
(Z)-11-Hexadecenyl acetate
Acetate
(Z)-11-Hexadecenal
Aldehyde
N. benthamiana
Original Organisms
Escherichia coli
Multigenic assembly
Agrobacterium tumefaciens / Rhizobium radiobacter
Nicotiana benthamiana
NOT PREVIOUSLY ACHIEVED
Goldenbraid standard ≠ Biobricks standard
Parts adaptation with help from NRP_UEA_Norwich
3 parts sent to the registry:
DesaturaseAtrΔ11
ReductaseHarFAR
Acetyl transferase
EaDAcT
Bba_K1554001 Bba_K1554002 Bba_K1554003
Original Organisms
Escherichia coli
Multigenic assembly
Agrobacterium tumefaciens / Rhizobium radiobacter
Nicotiana benthamianaAgroinfiltration
How can we know if the plant is producing our desired pheromones?
VOLATILE ORGANIC COMPOUNDS ANALYSIS
Headspace Solid-Phase Micro Extraction- Gas Chromatography/ Mass Spectrometry
Results:
20.00 25.00 30.00 35.00 40.00 45.00 50.00
100000020000003000000400000050000006000000700000080000009000000
1e+071.1e+071.2e+071.3e+071.4e+071.5e+071.6e+071.7e+071.8e+071.9e+07
2e+072.1e+072.2e+072.3e+072.4e+072.5e+07
Time-->
Abundance
TIC: Control P19 GFP 300914.D\data.ms
20.00 25.00 30.00 35.00 40.00 45.00 50.00
100000020000003000000400000050000006000000700000080000009000000
1e+071.1e+071.2e+071.3e+071.4e+071.5e+071.6e+071.7e+071.8e+071.9e+07
2e+072.1e+072.2e+072.3e+072.4e+072.5e+07
Time-->
Abundance
TIC: Ruta CIS hoja 300914.D\data.ms
(Z)-11-Hexadecen-1-ol
Alcohol
(Z)-11-Hexadecenyl acetate
Acetate
More results:
(Z)-11-Hexadecenal
Aldehyde
INSECT-PHEROMONE INTERACTION - ELECTROANTENNOGRAPHY
Moth antenna connection to two electrodes
INSECT-PHEROMONE INTERACTION - ELECTROANTENNOGRAPHY
Detection of antenna electrical signals in response to pheromones
What about the metabolic burden?
16:CoA
Existing substrate
How much product?
Pheromone
PREDICTIVE TOOL
MODEL
Flux Balance Analysis (FBA)a mathematical approach for analyzing the flux of reactions through a metabolic network.
RESULTS:
OBJECTIVE : Maximize Palmitic acid flux (substrate) and Biomass
Coupled production yield
Substrate vs Biomass
OBJECTIVES• Reduce metabolic cost for the plant• Increase release ratio• Avoid uncontrolled pheromone release
WHERE?
OBJECTIVE: selective expression and pheromone production
GLANDULAR TRICHOMES SPECIFIC PROMOTER
WHEN?
OBJECTIVE: Genetic switch implementation to controlpheromone production
INDUCTOR: CuSO4
• Inexpensive• Easily obtainable• Ease of use by the farmer
0
0,2
0,4
0,6
0,8
1
PNos CuSO4 - CuSO4 1mM CuSO4 10mM
Nor
mal
ized
Flu
c/R
luc
x7.8
x3.6
LUCIFERASE ASSAY
• Firefly luciferase as a gene expression reporter
• Assay after induction with different CuSO4 concentrations
How much pheromone is our plant able to produce in reality?
How much do we need to get the results we want? Is it enough?
DIFUSSION PROCESS
Pheromones are molecules that can undergo a diffusion process moving away from it source?
HEAT DIFUSSION EQUATION
2D
Temporal and spatialdistribution of
pheromone concentration
How do we solve it?
Numerically with theEuler method
Solution
MOTHS RESPONSE
• Males Follow the gradientErratic random flight
• Females Erratic random flightPheromone emission
Netlogo
OBJECTIVE: Ensure the Sexy plant to be completely safe in the field for living beings and the environment
NEEDS:• This plant is not the consumed plant • Unable to spread itself without control• Unequivocally identified in the field
✓✓✓
IDENTITY PRESERVATION
MALE STERILITY
Anthocyanin accumulation
Anther-specificpromoter (Ta29)and Barnase (RNAse)
A Sexy Plant able to produce moth sex pheromones
2 genetic modules to control pheromone release Trichome specific expression Functional genetic switch
2 genetic modules to make a completely safe plant Identity preservation + Male-sterility
Detectable pheromone-insect interaction
Analysis of pheromone production capabilities of our plant metabolic network.
A Simulation Environment for the pheromone diffusion and moth response behaviour
Policy & Practices:Interaction with involved stakeholders and social diffusion of Synthetic Biology
• Bayer CropScience• National research Council (CSIC)• Agricultural Centre for Chemical Ecology (CEQA)• Cooperatives for Agrofood industry (FECOAV)
Collaborations: NRP_UEA_Norwich: Conversor and chromoproteins (biosafety) NRP_UEA_Norwich and Cambridge-JIC: Plants for iGEM Workshop Sheffield – Lab notation Paris Bettencourt – Newsletter and MOOC High School
Interlab Study
Submitted Parts: AtrΔ11 HarFAR EaDAcT Ta29 Biosafety module (Barnase + Blue Chromoprotein) Biosafety module (Barnase + Yellow Chromoprotein) Omega undercover
• Department of Crop and Forest Sciences - UDL• Sustainable Agriculture Group - UPV• Institute of Plant Molecular and Cell Biology (IBMCP).• School of Agricultural Engineering (ETSIAMN)
VICERRECTORADO DE ALUMNOS Y EXTENSIÓN
UNIVERSITARIA
ETS DE INGENIERÍA AGRONÓMICA Y DEL
MEDIO NATURAL