NRP-UEA iGEM
Where?
Meet Our Team Cara Deal Alistair Middlemiss Steven Monsey Jessica Gray Mischa Spiegel Jack Day
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
M ajor 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 our 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?
Golden Gate Cloning 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 Modular Flipper We have submitted a collection of plant-specific promoter, CDS and terminator parts The improved Bba_J04450 (RFP) contains two divergently orientated 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.
Plant Hormones 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.
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: Pro35S TER:OCS CDS:AvrBS3 Pro:BS3 CDS:GFP TER:OCS B: Pro:BS3 CDS:GFP TER:OCS
Reporters - BAX A: Negative control (water) Pro:35s CDS:BAX TER:OCS B:
Green Canary? All circuits made but we ran out of time Promoter that responders to any pathogen or large to make multi- group of pathogens gene assembly PR1 or synthetic BS3/XA promoter with multiple and test in plants! recognition sequences for different TALES Any pathogen: CDS:chloro- CDS:RCCR PRO TER:OCS degreenin g phyllase RNAi + Pro:BS3 CDS:BLUE TER:OCS Diagnosis: Pro:XA TER:OCS e.g. Avr BS3 CDS:YELLOW TALE Promoters that respond to effectors Avr Xa secreted by specific pathogens TALE
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
Future Plans We hope that the technology can be extended to enable the sentinel to: De-green Re-green Express different chromoproteins Detect different pathogens
Future Applications Considerations for a business plan: Suitability Logistics Cost
Future Work Other points for considerations: Different plants Plant growth requirements Number
Policy & Practices
Can We Grow The Green Canary? 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
Food For Thought Farming community Focused on food security Presented our project Interactive demonstrations Received feedback on our project Success! We were asked
Science Café 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!
Anglia Farmer
The Hewett School Workshops designed to teach young students (14/15 years Activities included: of age) about: • Swabbing • Bacteria • Thumb prints • Diseases • Spread of infection • Food Security • Discussion & Debate • Synthetic Biology/GM
A Lesson Learnt… 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
Acknowledgments 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
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