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