NRP-UEA iGEM Where? Meet Our Team Cara Deal Alistair Middlemiss - - PowerPoint PPT Presentation

NRP-UEA iGEM

Where?

Cara Deal Jessica Gray Alistair Middlemiss Jack Day Mischa Spiegel Steven Monsey

Meet Our Team

• 7.2 Billion
• 9 Billion by 2050
• Approximately 215,000 born every day
• 842 million undernourished worldwide.
• We need more food for now and for the

future!

References

• S. Preston; Heuveline, P; Guillot, M. (2001) Demography: Measuring and Modelling Population

Processes, Blackwell, p131 Food and Agriculture Organisation

• Major factor in the reduction of

food production globally.

• Estimated 35-40% of all rice, maize,

wheat, and potato crops are lost to pests and disease.

• Crop disease causes loss in

approximately 15% of pre-harvested crops.

• We need to make the most out of
• ur farmland.

References: Oerke E C (2005). Crop Losses to Pests, Journ Agricultural Sci, vol 144, p31-43

• Constant agrochemical use can potentially:
• Be harmful to biodiversity
• Pollute the environment
• Be expensive for farmers
• Pose a health risk for us
• Cause resistance in pathogens

References:

• McManus P S; Stockwell V O; Sundin G W; and Jones A L (2002) Antibiotic use in pla

nt agriculture, Ann Rev Phytopathology, col 40, p443-465

• Graslund S and Bengtsson B (2001). Science of the total environment, vol 280, p93-1

31

Photo credit: canaries.co.uk

A diagnostic sentinel plant that will act as a biosensor for plant-pathogen interactions

• Visible chromoprotein output signal

specific for pathogen present

• Farmers can spray selectively, and

develop/choose a more resistant crop for the following year

• Not consumed as part of the food

chain

What Is The Green Canary?

• Allows for cloning of multi-gene constructs in a single,

1 step digestion- ligation reaction.

• Uses type IIS Restriction endonucleases.
• Cleaves downstream of the recognition sequence, leaving

a scar-less construct using enzymes BsaI and BpiI

Golden Gate Cloning

Golden Gate Modular Flipper

• We have submitted a collection of plant-specific promoter,

CDS and terminator parts

• The improved Bba_J04450 (RFP) contains two divergently
• rientated BsaI sites between the 4 BioBrick restriction

enzyme sites which allows the Golden Gate modules to be dropped into the BioBrick vector in a one-step digestion-ligation Golden Gate reaction.

What Pathogens Does Green Canary Respond To?

• Xanthomonas oryzae
• Disease found worldwide and particularly destructive in the

rice-producing regions in Asia.

• Xanthomonas campestris
• Xanthomonas campestris pv. vesicatora is a bacterium that

causes bacterial leaf spot (BLS) on peppers and tomatoes.

• We were able to create and express a variety of circuits in

plants that were switched on or up-regulated in respo nse to hormonal signals of pathogen invasion.

Promoter Selection:

Methyl Jasmonate

• Activates the pdf1.2 promoter, driving gene expression.

Salicylic Acid

• Activates the PR1 promoter, driving gene expression.

Plant Hormones

Chassis

Escherichia coli Agrobacterium tumefacians Nicotiana benthamiana

Nicotiana benthamiana

• Used in plant pathology due to large number of

plant pathogens (viruses, bacteria, fungi, oomycetes) that can successfully infect it.

• Easily genetically transformed
• Amenable to facile methods of transient protein

expression

Green Canary

Promoter – PR1

A: Untreated B: + Salicylic Acid

Pro:PR1 CDS:GFP TER:OCS Pro:PR1 CDS:GFP TER:OCS

Promoter – PDF1.2

A: Untreated B: +Methyl Jasmonate

Pro:PDF1 CDS:GFP TER:OCS PDF1 CDS:GFP TER:OCS

TALES

(Transcription Activator Like Effectors)

A: B:

Pro:BS3 CDS:GFP TER:OCS Pro:BS3 CDS:GFP TER:OCS Pro35S

CDS:AvrBS3

TER:OCS

Reporters - BAX

A: Negative control (water) B:

Pro:35s CDS:BAX TER:OCS

Green Canary?

Pro:BS3 CDS:BLUE TER:OCS PRO

CDS:chloro- phyllase

TER:OCS

Promoter that responders to any pathogen or large group of pathogens PR1 or synthetic BS3/XA promoter with multiple recognition sequences for different TALES

Pro:XA

CDS:YELLOW

TER:OCS

Promoters that respond to effectors secreted by specific pathogens

CDS:RCCR RNAi

degreening Diagnosis:

+

Avr Xa TALE e.g. Avr BS3 TALE Any pathogen:

All circuits made but we ran out of time to make multi- gene assembly and test in plants!

Experimental Achievements

 Created a proof of concept transient expression biosensor system in response to Xanthamonas orzyae and Xanthamonas campestris TALE’s.  Submitted 13 number of new and improved parts to the iGEM registry of standard parts  Created a Golden Gate Modular Flipper to allow future iGEM teams to clone in Golden Gate but submit in Bio Brick standard.  Created a Request for Comments (RFC) with Cambridge and Valencia iGEM teams to raise the profile of Golden Gate and plant syntheticbiology  Created a collection of 5 plant-specific iGEM parts to be used by future teams

Future Applications

We hope that the technology can be extended to enable the sentinel to:

• De-green
• Re-green
• Express different chromoproteins
• Detect different pathogens

Future Plans

Considerations for a business plan:

• Suitability
• Logistics
• Cost

Future Applications

Other points for considerations:

• Different plants
• Plant growth

requirements

• Number

Future Work

Policy & Practices

Key Questions:

• Can the Green Canary viably help to solve food security issues?
• Is the Green Canary safe to use in interaction with other crops?
• What is the public opinion on our project?
• Do young people know about the issues that affect their futures?
• Set out to answer these questions by engaging the public
• Process of gaining ethical approval for data gathering
• Set the foundations for further NRP-UEA iGEM teams

Can We Grow The Green Canary?

• Farming community
• Focused on food

security

• Presented our project
• Interactive

demonstrations

• Received feedback on
• ur project
• Success! We were asked

Food For Thought

• More focused and in

depth on discussing issues

• Food Security, Genetic

Modification

• Great feedback on Future

Applications, pros and cons of our topic and GM

• Science Cafés will

continue at The CUT!

Science Café

Anglia Farmer

Workshops designed to teach young students (14/15 years

• f age) about:
• Bacteria
• Diseases
• Food Security
• Synthetic Biology/GM

Activities included:

• Swabbing
• Thumb prints
• Spread of infection
• Discussion & Debate

The Hewett School

• Ethical guidelines and procedures differ

between countries and institutions

• At UEA, ethics approval needed for use

and protection of human opinions and data

• We propose that iGEM set universal

Ethical guidelines to ensure all teams conduct themselves in the correct manner

A Lesson Learnt…

Laura Bowater (MED)

• Advice on Science Communication

Tom Shakespeare (MED)

• Advice on research ethics

Mark Wilkinson (MED)

• Advice on research ethics

Kay Yeoman (BIO)

• Advice on Policy and Practices

Mark Youles (TSL)

• Golden Gate Cloning Expertise

Matt Hutchings (BIO)

• Providing the team shirts

Acknowledgments