Discover:

• Interested in the issue of food security during January 2022, we were acutely aware of issues in the global wheat supply chain as a result of conflict in Ukraine. This led us to conduct research into the different vulnerabilities in wheat production, as a valuable staple crop, evaluating avenues for increasing crop yield, reducing yield loss to disease/climate change.


• We quickly learnt about wheat stem rust, caused by the fungus Puccinia striiformis tritici (Pgt), one of the most devastating plant diseases affecting the crop. In 1998, a highly virulent race of wheat stem rust emerged and rendered up to 80% of the world’s wheat varieties vulnerable to stem rust. A stem rust outbreak took place in Sicily in 2016, affecting thousands of hectares of both durum and bread wheat, in the same year one to two million hectares of spring wheat in Western Siberia were hit causing average yield losses of 30-40% on regional scale.


• The reemergence of stem rust is also supported by climate change, as it primarily favors warmer conditions for infection. The rise in temperatures, coupled with a rapid increase in global wheat consumption (a 60% increase in demand by 2050) means that a novel technique for fighting against wheat rust is sorely needed.


• The most effective ways to control stem rust are the use of resistant cultivars and the timely use of an appropriate dose of fungicides. The changing nature of rust pathogens outwits the use of resistant cultivars, and the use of fungicides is associated with environmental problems.


• Speaking to farmers in Europe, such as Paolo Petrilli, we learnt that farmers perform up to 8 sprays of fungicides on their crop as both a preventative and curative measure, with epidemic level outbreaks happening regardless.

‍Define:

• The key problem identified is the vulnerability of wheat crops to stem rust.


• The unmet need is for an approach that enables crop protection as soon as stem rust spores land on the crop plant, eliminating the pathogen before it can invade host tissue at which point treatment is ineffective. The challenge here is detection, as synthetic fungicides if applied at the right time can kill the pathogen.

‍Develop:

• Our initial idea was to address wheat rust through a paired detection and pathogen elimination system. We thought of engineering the chassis bacteria Bacillus subtilis to produce lipopeptides toxic to Pgt upon recognition of ribitol (a unique sugar released during germination of Pgt).


• This bacteria is known to be a rhizosphere promoting bacteria and creates a biofilm coating the roots. It is already used to produce biofungicides, and is ideal for real world applications as it can survive in extremely harsh soil conditions. It is an endophytic bacteria that has already shown to have some success fighting against stem rust. It also has GRAS status, so safe to coat plants that are later consumed.


• In subsistence, the idea would consist of a sense and response system where expression of antifungal lipopeptides takes place upon detection of ribitol via an inducible promoter.

Discover:

• We went back to Paolo Petrilli for feedback on our idea. Although he found it interesting, through our discussion we realized that at the moment stem rust was not the primary disease farmers were concerned about.
• Through further literature review, we identified that although the threat of stem rust was large and it is one the most dangerous diseases to crops, other milder fungal diseases were more prevalent and thus of greater immediate concern.
• Speaking to experts in our own university such as Prof Pietro Spanu and Prof Colin Turnbull, we learnt of some impracticalities of the idea of using B. subtilis with the idea of harnessing its biofilm as a delivery mechanism. This is due to the thickness of the biofilm and how our biomarker could be easily accessible to the B. subtilis cells.
• Furthermore, they highlighted that although the bacteria is known to form a biofilm around the roots, it may not do so on the aerial parts of the plants and its presence there could affect plant functions. They also pointed out that this idea may not be robust and would likely not behave as designed in the field due to the vulnerability of the cells to changing climatic conditions.
• Speaking to members of the public at the Great Exhibition Road festival, we also identified a key concern regarding ingesting food products containing foreign DNA.

‍Define:

• Stem rust poses a grave risk but is not yet highly prevalent.
• Early detection of the next epidemic could prevent a global catastrophe in wheat production.
• Spraying cells as a prophylactic measure is likely to fail in the real world, due to highly variable environmental conditions that may disrupt the population present on the surface of the plant.
• Spraying of engineered bacteria poses a biocontainment, regulatory and public perception issue.
• The key challenge is thus developing an early warning system for stem rust.

‍Develop:

• Our idea evolved to having an E. coli sensor with a hardware device that could be installed in farms. In summary, Pgt spores present in the air would land on the device, once these germinate, E. coli cells immobilised on the surface of the device would begin producing a colour reporter. The farmer would then notice a change in colour and applies a biofungicide, such as existing B. subtilis sprays, in response.

Discover:

• At this stage we began to speak to farmers in the UK, members of the National Farmers Union, as well as farmers in India. We soon realized the intense financial stressors facing farmers, and it was clear that they would not be suitable end users of a device like the one ideated in the previous stage. Such an expensive and complex preventative measure for a problem they are not sure they will face is not attractive. This is especially true for resource poor farmers.
• We also learnt that farmers today often use integrated pest management systems, involving crop rotations. This means that a single farm will vary its production between different grains for example every other or every five years. Each crop is also vulnerable to a multitude of fungal pathogens not only one.
• Will Dickinson, a UK farmer, also highlighted how throughout his career, he has seen the rise of resistance to synthetic fungicides build. We also learnt that mid-sized farming enterprises in the UK have access to advanced technological systems such as aerial imaging of their farms to inform application of fungicide in order to optimize the same and limit consumption. The same was not the case for farmers in India. This pointed us towards another problem of a lack of an equitable solution to fungal pathogens. A point also highlighted by a Dr. Sridhar Bhavani from the CIMMYT (International Maize and Wheat Improvement Center) who spoke to us about the plight of farmers in Kenya.
• Dr. Bhavani also shared his knowledge and experience with failed biofungicide trials in Mexico, due to their inefficacy in high disease pressure conditions and lack of persistence in the crop for example due to precipitation and high temperatures.
• Speaking to Prof. Tom Ellis, we learnt about B. subtilis’s ability to form endospores, which are a dormant form of the bacteria that is highly robust and durable. He suggested this might be an interesting delivery mechanism that is less vulnerable to changing climatic conditions.
• Exploring literature, we also learnt of the grave issue of fungal pathogen emergence.

‍Define:

• Farmers are in an arms race against a myriad of phytopathogens.
• There is a need for a solution with high efficacy against multiple pathogenic strains as well as potential emerging ones, that is cost-effective and accessible. Need for a solution that is not specific to a single crop. Issue of timeliness of biocontrol agent deployment remains.

‍Develop:

• At this stage, we began forming an idea for a broadspectrum biofungicide consisting of B. subtilis spore system that could be sprayed on crops. This would also address issues in terms of costs, ease of storage and transportation.
• The modes of action of B. subtilis cells present bioactivity, such as production of antifungal lipopeptides and up regulation of plant innate immunity, are not race specific.
• As we were unfamiliar with this system we still faced two challenges at this stage which caused us to go back to the Discover phase: how to ensure we produced cells or bioactive compounds in a timely fashion, thereby targeting both pathogens when they are most vulnerable, and how to achieve robust biocontainment.

Discover:

• At this stage we performed an extensive literature review to understand how to achieve sense and respond in a spore system, where metabolic activity is heavily reduced. We began to learn about the germination process and the functionality of germinant receptors.
• We also began to search potential biomarkers that could be universal to all fungal phytopathogens and that are unlikely to be subject to evolutionary change, so as to appropriately target the risk of pathogen emergence.
• Speaking to experts such as Dr. Guy Polturak, we identified chitin as the best candidate, also thanks to its function as an inducer of plant immune response.

‍Develop:

• We finally arrived to our project idea: a non-toxic, broad-spectrum bio-fungicide, consisting of a B. subtilis bacterial spore system that eliminates the time lag between diagnosis and treatment. Our Project Description page includes a full explanation of the solution.

‍Deliver:

• With an idea informed by feedback from different stakeholders, we now began to work on our experimental design. This also involved reaching out to academics with expertise in this chassis and the types of experiments we wanted to conduct such as directed evolution.
• Our achievements are summarized in our Results and Proof of Concept pages.
• We also continued to reach out to stakeholders as part of the AREA framework, trying to engage all groups who would be impacted by our project, anticipate risks, and address their concerns. An outcome of this work was our Product Risk Assessment.

Bio: Prof Tait works at the University of Edinburgh where she specialises in bioregulation, policymaking, stakeholder attitudes and communication. She is a Founding Director of the Innogen Centre, which focuses on safe innovation, and is a member of the Regulatory Horizons Council.

‍Key insights: We initially approached Prof Tait for advice on a bioregulatory framework. Having reviewed existing bioregulation, such as the Nagoya and Cartagena protocols, we wanted to create a new framework that would address the gaps in what exists already.

‍Reflections: Prof Tait shared that the issue with bioregulation is that there is too much regulation, rather than the lack of a common framework. Instead, she recommended product-based regulation, which is more specific and straightforward than the current process-based strategy.
We also asked Prof Tait about how we could label our product. She suggested positive labelling; being transparent and clear about the synthetic biology processes used, and also explicitly showing how these products benefit the environment.

‍Next steps: We decided to work on a thorough product risk assessment rather than an overall process framework. This assesses the ethical and biological implications of our particular product. We also incorporated positive labelling into our MVP label design and marketing strategy.

Liz Truss and Rishi Sunak
Bio: Liz Truss and Rishi Sunak were the candidates for Prime Minister here in the United Kingdom. The National Farmers Union held hustings events for both PM candidates, giving members the opportunity to pose questions. Hearing from them helped us gain insight into how politicians work with the agricultural sector, and the future direction of bioregulation.

‍Key Insights: Liz Truss, who is now the UK Prime Minister, highlighted the issue of food insecurity given the recent Ukraine grain crisis. Upon being asked about regulation, Liz asserted that there was “too much red tape” around farming, and that she was interested in simplifying environmental schemes to instead focus on food production. Rishi Sunak was even more open and pro-innovation. He also suggested deregulation and proposing gene editing as a means of making crops more resilient.

‍Reflections: It seems the regulatory landscape in the UK is moving in a less strict direction, following Brexit and the appointment of new leadership. This is positive for us, as it means our product may be more easily commercialised in the UK.

‍Next steps: Speak with manufacturers such as Bingchun Wang to see what regulation is involved in making a real product.

Bio:
Paolo Petrilli is a medium-scale, organic farmer based in Italy.

‍Key Insights:
We spoke to Paolo throughout our project. He shared that sulphur fungicides are currently being used to treat stem rust, but they are not an ideal solution as they are expensive and inhibit plant growth. Sulfur is toxic to wheat plants and inhibits their growth, decreasing leaf size which is important for photosynthesis. Chemical fungicides are also labour-intensive, requiring up to 8 applications per season.

‍Reflections: A new solution to fungal diseases is sorely needed, given that current solutions are not only bad for the environment, but also decrease yield and are expensive, which impacts farmers directly.

‍Next steps: Investigate biofungicides, which are more environmentally friendly and not toxic to the plant. Research early detection, and how it can play a role in more effective disease treatment.

Will Dickinson and Josh Redford

Bio: The National Farmers Union is the central hub of the agriculture space in the UK, representing over 46,000 farms and businesses such as Cross Farm, owned by Will Dickinson. We were very grateful for the opportunity to meet Will Dickinson, owner of Cross Farm and NFU council delegate, and Josh Redford, a NFU county advisor.

‍Key Insights: Josh provided great insight into the agricultural landscape in the UK. We learned that farmers work closely with agronomists, scientists who advise farmers on what products to use on their crops and how to combat disease. We also visited the Rothamsted Research Institute, where GMO crop trials were taking place - these were fenced off to ward off anti-GMO protesters.
Will explained that the wheat in his farm was most severely affected by yellow rust. He typically applies 2-3 rounds of chemical fungicides per season, and intensive re-sprays of the entire field are required if any outbreaks occur. Crops have become increasingly susceptible to fungal diseases in recent years, as hotter and drier conditions make crops more vulnerable.
We were also very happy to learn that Will was open to the idea of biofungicides and genetic engineering - in fact, he has already adopted digestate, a biodegradable feedstock, for his crops.

‍Reflections: This visit reconfirmed to us that stem rust has a significant impact on farmers worldwide, and is becoming more severe with global warming. We also found that farmers may be more open and receptive to bioengineered products than expected - instead, regulators and policymakers are the ones with a stricter stance.
The fact that full-scale, costly re-application is required once an outbreak occurs also further supports the need for a sense-and-respond system that does not require frequent re-application.

‍Next steps: Our dry lab team contacted agronomists that Josh Redford referred to us, to find out more about how higher concentrations of B. subtilis from our product might influence the microbiome.

Bio:
Karnal, once the birthplace of the Green Revolution in India, is famous for its world class agricultural research institutions and is a popular hub for agriculture in Northern India. We are extremely grateful for the opportunity to have interviewed Farmers, in person, from various land holding backgrounds, experts from the top Agricultural Institutes of India: IARI (Indian Agricultural Research Institute) & IIWBR (Indian Institute of Wheat and Barley Research) as well as Pesticide/fungicide retailers.

‍Key Insights:
As identified by the experts from IARI, the main fungal infections affecting wheat from the Northern plains of India (eg. Karnal) are the Yellow rust and Leaf stem rust infections. Most farmers prefer using preventive sprays which, at present, are mostly chemical fungicides. Trichoderma and Pseudomonas are the only 2 well known broad spectrum biofungicides that have been used in Karnal. Biofungicides are most commonly used in specific regions of India such as Gujarat, Madhya pradesh and Rajasthan- that grow crops for export to European countries where usage of chemical fungicides on crops are banned.
As a few experts from IIWBR had discussed, there are 4 main concerns with current biofungicides:

1. Storage: Shelf life of current biofungicides is low.
2. Awareness: Farmers are not aware of biofungicides and their properties.
3. Efficacy, time bound results and delivery system: Unclear with biofungicides.
4. Performance of biofungicides: Influence of temperature and and ideal climatic conditions makes biofungicides hard to control/maintain.

Experts have also noticed that there is a discrepancy between the efficacy of biofungicides in the lab vs on the fields- Several factors including maintenance and handling of the biofungicides factors into its performance.

‍Reflections: Based on some of the points discussed with experts and from the opinions of farmers, we’ve come to reflect on our biofungicide to ensure that it is in line with the main concerns and addresses issues such as Shelf life, Awareness, Efficacy and Performance.

‍Next steps: We are looking at spreading awareness about our biofungicide through various social media platforms, presentations, middle school talks and scientific exhibitions as well as reach out to the poor farmers through the National Farmer’s Union in the UK. With the use of bacterial spores, we have tackled the issue with shelf life of biofungicides. We aim to take steps to maximise efficiency of our biofungicide as best as possible and work with a microbiome modelling tool as well as field trials to improve performance.

WashU St Louis 2018 iGEM Team
Elizabeth Johnson
‍16/03

Bio: Elizabeth was a team lead of the WUSTL 2019 iGEM team, who inspired our project. They worked on a ribitol-sensing system in B. subtilis to respond to wheat stem rust.

‍Key Insights: The team wasn’t able to test the sensitivity of their ribitol sensor, due to not having the ability to clone. Their ribitol transport construct worked, and we had a look at its sequence on the registry. Other parts couldn’t be tested due to being lost. They shared a report on the genetic circuit for ribitol detection.
Human Practises wise, the team found going to Uganda and Ethiopia to speak to stakeholders most helpful. They prepared questions for farmers and learnt that most farmers didn't know about resistance genes in their crop so wouldn’t know which strains would be virulent. This informed their decision to try a more general detection mechanism.

‍Reflections: We must investigate ribitol sensing more - we initially assumed that it worked as a sensor from WUSTL’s Wiki, but it seems that it was not fully tested. On-site stakeholder research will be key to our Human Practices.

‍Next Steps: We dug further into ribitol as a biosensor and found that there was actually not much literature supporting it as a biosensor specific to stem rust. This eventually led to us shifting to using chitin as a biomarker rather than ribitol. We prepared questions for farmers and travelled to Haryana and Harpenden to speak to them. We also spoke to Paolo Petrilli, a farmer in Italy, online.

Bayer
Dr Julia Stevens, Dr Keith Merrill and Dr Partha Ramaseshadri
Bio:
Bayer is a major synthetic biology pharmaceutical company, with agriculture as one of its specialisms. We spoke to Dr Keith Merrill and Dr Julie Stevens, disease platform leads, facilitated by Dr Partha Ramaseshardri.

Key Insights:
The academics suggested that stickiness of B. subtilis to crops could be an issue, especially in the face of changing conditions like rain and the spraying of other pesticides. They also raised questions about whether our system would be overwhelmed by very high volumes of stem rust. Regarding regulation, they mentioned that our product could still be considered as GMO, as despite foreign DNA being removed a foreign protein is still expressed. They recommended running allergenicity and toxicity tests on our sequences with Compare Database, to see whether our product would be approved by regulatory bodies.

Reflections:
Given that the stability of B. subtilis staying on crops is key to our project and its philosophy of fewer applications, it is imperative we make it a priority to research this further. With bioregulation, we should see how other experts think our product is defined (GMO or not), and gauge public perception of it. We should also test our sequences as we go.

Next Steps:
Regarding durability, we reached out to postdocs at Imperial studying B. subtilis to confirm its stickiness on plants.
We ran tests on our sequences on Compare Database. Our CotZ Chitinase, CotG and CotG Linker Chitinase constructs received % similarity results >35%, meaning they are not considered as a cross-reactive risk.

The Great Exhibition Road Festival is an annual science and arts festival held in London. It is targeted for members of the public from all backgrounds and ages, making science approachable for those who do not have a background in it.

We applied for a stall at the festival, under the title ‘Genetic Engineering: a new power tool in biology’. From February to the festival date in June, we worked closely with Mimmi Mårtensson, a Public Engagement Programmes Coordinator for the event, on our stall activities. Together we ensured that our stall would be interactive, interesting and approachable to anyone who came by.

The main activity we ran at a stall was a product ranking game. Visitors were shown a set of everyday household items, including bike tyres, plastic cups and tomatoes, and were asked how comfortable they would be using these items if they were genetically modified.

Visitors largely were less comfortable with the food items being genetically modified, citing concerns about ingesting something toxic or carcinogenic, although some were not able to explain their reasoning very much. Items that they would need to come into less direct contact with, like the bike tyres, were more well received.

Younger people and STEM students were more likely to be ok with items being genetically modified, with some saying that all of them were ok. There were a few individuals who were quite strongly against all items being genetically modified, all from older generations. When we explained why genetic engineering was being used, for instance as a more environmentally friendly option, they agreed with the need for solutions but rejected genetic engineering as something they would accept. Instead, they said they would opt for other solutions, for example “re-use, reduce, recycle” or “if we sort our politics out food insecurity won’t be a problem anymore”. To them genetic engineering was a very last resort.

We also created a poster introducing the topic of bioengineering so visitors could learn more about it, and provided brochures on our project for those interested in speaking to us about our research. Many individuals seemed to initially not receive our project well, assuming that we were genetically modifying wheat plants themselves. However, once we explained that we were genetically engineering a biofungicide and not the wheat genome, people seemed relieved and were more positive about our project. This supports our decision to work on a biofungicide with self-digesting plasmid technology, as it appears to be more acceptable to the general public.

Going forwards, our experience at the Festival has informed us to:

• Be clear in communicating about risk and bioengineering, dispelling myths e.g. “GMOs cause cancer”
• Make it clear in the promotion of our product that it is a biofungicide without foreign DNA, as this makes it more acceptable to the public
• Communicate to the public why we are using bioengineering to solve world issues, and why existing solutions like political change and environmental consciousness might not realistically be enough
• Focus on improving public perception of GM foods specifically.

We are very grateful to have had the opportunity to present at the Festival and speak to so many people - over 2500 individuals walked through the section our stall was in, and it was a great success for us.

Consumers are particularly important, as they are the ones who may eventually ingest crops grown with Sporadicate; they are the ones who will decide whether to buy these food products, and they are the constituency that informs the direction in which regulation of bioengineered products evolves.

Thus we decided to conduct a consumer opinion survey to better understand the different perspectives present in the general public, and identify what the primary public opinion in regards to the use of genetically engineered organisms and synthetic biology is.

Here is our report!

Bio: Mayor’s Fund for London is a local charity with a major focus on food insecurity. They work closely with young Londoners from low-income backgrounds, investigating and addressing the causes of undernourishment.

‍Key Insights: Their representative, Jade Harris, shared that there are now over 70,000 suffering from food insecurity in London. She helped us define food insecurity as a cycle, rather than the feeling of hunger - families may feel it more during the school holidays or while heating bills also need to be paid. She also explained that there is no governmental intervention, so both temporary and root cause approaches like ours are needed.