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Team: UNSW_Australia
iGEM Year: 2022
Track: Food and Nutrition
Project Name: Catching a Cereal Killer: Developing Antifungal Peptide Candidates for the Treatment of Cereal Rust Infections
Cereal rust infections caused by fungi of the Puccinia genus presents a significant global burden resulting in the loss of wheat yields, posing implications for international economies and risks the issue of food insecurity. With growing concerns of the increased incidence of more virulent strains resistant to commonly used antifungal drugs, the search for novel alternatives to treating this problem is a priority. Recently, antifungal peptides have been shown to be promising and powerful candidates due to their specificity and efficacy. In alignment with this current research, we employed bioinformatic approaches to scour the highly understudied fungi genome databases and published literature to identify two potential fungal targets, PstSCR1 and Pst_12806, known to be involved in the fungal pathogenesis. After locating known binding partners found naturally in plants, SERK3B and TaISP, respectively, we aimed to design and measure the binding affinity of our predicted peptide sequences as potential antifungal treatments.
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Describe what work your team members did and what other people did for your project.
Our team was facilitated by Dr. Dominic Glover, organising everything from funding our lab and supplying resources to offering extensive guidance along the journey.
Our team was split into 5 groups, distributed as following:
Team leads were responsible for the facilitation, organisation, and delegation of tasks to each team’s members. Dry Lab tasks included protein modeling and BLAST searching.
Wet Lab involved culturing and assaying proteins. Human Practices consulted experts and academics concerning Cereal Rust. Wiki & Design created graphics and onboarded the wiki itself.
Education & Communication engaged in student outreach and inter-team collaboration. Beyond our team we were aided by PhD student Gustave Severin, as well as a number of academics
including but not limited to Associate Professor Till Boecking, Professor Matthew Kearnes, Dr. Megan Lenardon, Professor Robert Park, and Dr. Lucy Carter. Each of them was instrumental
in guiding our initial research as well as provided consultation throughout the process of our experiments.
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Describe how and why you chose your iGEM project.
In light of climate change, resistance to fungicides and the recent floods in Australia, many farmers have been subjected to a more prominent spread of fungal rust diseases through crops.
The 2022 UNSW iGEM project aims to implement the voices of a number of user groups (e.g. farmers, academics, rust experts, etc.) to develop a method of controlling stem rust disease.
This has been achieved by developing a range of inhibitory peptides which are predicted to bind to the active site of effector proteins secreted by the fungal pathogen.
Read more over here page.
Make a useful contribution for future iGEM teams.
The 2022 iGEM team has conducted various experiments with fungal and plant proteins using a lac operon system, creating new parts which we used in our project.
Conducting in depth literature research as well as discussing our methods with academics via meetings allowed us to create a trustworthy and efficient final product.
In doing this future teams can learn and benefit from our experiment.
Read more over here.
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Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.
We aimed to complete a full iteration of the engineering cycle through the testing of binding strength between the fungal proteins and our inhibitory peptides in the
laboratory. Following the design of the inhibitory peptides from the dry lab team, the wet lab would express and purify fluorescently labelled versions of the peptides
and fungal proteins in the lab. A FRET assay would have been performed to indicate the binding strength and provide information that could be fed back into the dry lab
for improvement of the peptide design. Due to time constraints this was not possible so the wet lab pursued a slightly deviated engineering cycle. We designed the
plasmids for the fungal proteins’ natural binding partners in the plants and synthesised these in the lab. Our FRET assay will provide information about the binding
strength between these proteins, and hence, can provide valuable information for the improvement of peptide design henceforth.
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Collaborate with one (or more) 2022 iGEM team(s) in a meaningful way.
Collaboration remained at the forefront of our iGEM journey this year, where we nurtured strong and rewarding relationships with teams both local and international.
Such teams included The King’s School, University of Newcastle and University of Sydney. Earlier in the year, we hosted a virtual meet-up to introduce the teams to
each other and their respective projects and set the scene for a collaborative and supportive environment to come. Here, we established links to inform each other
of the experiments we were undertaking and aided in validating a few of the experimental procedures. Interestingly, with The King’s School’s ties to the agricultural
community, they enabled us to get in contact with relevant user groups that assisted us in developing and changing how we approach our solution. Towards the end, we
hosted another virtual meet-up to exchange feedback to aid in bettering our projects in preparation for the Jamboree. This meet-up was also useful in gauging how our
projects have evolved since the beginning meet-up and explore how much we've learnt along the way.
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Explain how you have determined your work is responsible and good for the world.
A diverse range of stakeholders were contacted in order to ensure our project was considered responsible and beneficial to society. A people-centered approach was
initiated which guaranteed we understood the cultural and social constructs of our project. The four stakeholder groups we chose to concentrate on were academics,
government officials, traditional owners and businesses which correlated with our cause. Consulting with social scientists and environmental scientists allowed our
team to explore ethical, social and environmental values, ensuring our project was considered ethical and equitable. Additionally, performing multiple meetings with
individuals associated within the environmental, social and scientific field allowed us to appreciate and learn a wide range of perspectives on using synthetic
biology to assist in wheat crop growth. Advice on solutions in applying synthetic biology on wheat and how to deal with the long term effects of this also ensured
that our project would not only be beneficial to society now but would not be harmful in the future.
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Explain how you would implement your project in the real world.
Our proposed end users are organisations and farmers who want to prevent crop rusts and thus protect food safety. We plan to assemble peptides that are able to control
the growth of Puccinia spp. in E. coli and make them into a spray, which is easy for farmers to use. In order to implement our project into the real world, we needed to
evaluate the effect of the artificial peptide on the growth of wheat in different environments after verifying the effect of the peptide on the target protein,
including optimal conditions and hard conditions. Furthermore, we have spoken to academics, government officials, traditional owners and businesses which correlated
with our cause to get the social license to use synthetic biology to address the benefits of rust.
The main safety considerations are to protect the microbial diversity of the soil and to prevent unintentional invasions by our modified species that are not expected.
To reduce this risk, we plan to incorporate a kill switch into our design to ensure that our antifungal drug remains controlled and that it will stop working under the
necessary conditions. Moreover, ensuring the stable expression of our designed peptides in the spray is a challenge we need to address.
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Demonstrate how your team responded to your human practices reflections, research, and/or engagement. You should show how your activities impacted your project purpose, design and/or execution.
Our human practices and proposed implementation sections concentrated on resolving the conflict between scientific and public mindsets. Through our silver Human Practices work, consulting with
our main stakeholder groups has allowed us to achieve this conflict by shaping our project based on the values of these stakeholders. The human practices team's work presents a way in which we
can create a sense of security to wheat farmers, including government and non-government organisations. Not only this, but the increase of food security can lead to a safer sense of security to
the public economically. Additional academics who looked through the workflow of our wet lab and dry lab methodology allowed us to improve our present goals and form stronger solutions. Consulting
with a large and diverse range of academics from differing fields of science through our silver human practices work, allowed us to steer towards the most efficient solutions of our project and modify
accordingly. Although all stakeholders discussed the topic of using GMOs to solve the issue of cereal rust, each conversation reaffirmed a positive outlook on our solution.
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Use modeling to gain insight into how your project works or should be implemented. Explain your model's assumptions, data, parameters, and results in a way that anyone could understand.
Our team developed exploratory modelling to identify essential target proteins in Puccinia graminis that when inhibited would result in cell death. We highlighted the assumptions on the modelling
page. Alongside an in depth writeup of the data, parameters and results that then followed from this model and how it helped our team to identify suitable target proteins. On our modelling
page we also comment on how these models can be implemented by future iGEM teams emphasising the most important challenges we faced so that future teams may implement the model better than
ourselves. From the target proteins found in the aforementioned model, we then used other pre-existing exploratory modelling approaches to identify active sites. Using these active sites our team was then
able to develop inhibitor protein sequences for our team to test in the lab.
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Develop and implement education, science communication, and/or outreach materials related to synthetic biology.
Our aim this year was to broaden the understanding of synthetic biology within the general audience which spanned from children to university students and beyond. The main initiative we undertook was a
program aimed at educating students about synthetic biology and biotechnology by drawing connections to their school syllabus. We created a presentation and complementary Kahoot! which we presented at
two schools, East Hills Boys High School and Strathfield Girls High School. To further aid our purpose we had an ongoing post series on our Instagram accounts titled, “Pathogen of the Week '' which covered
important facts about a new pathogen every week. Throughout the year we collaborated with numerous other iGEM teams in their communication initiatives such as the iGEM McGill with their Bacteria Book,
iGEM Tec Chihuahua with their Comic Book and iGEM KU Leuven in their podcast initiative. Finally we also created and published a Synthetic Biology magazine in collaboration with iGEM at William and Mary,
iGEM Groningen, iGEM Crete and iGEM IONIS. This magazine includes articles on biology projects around the world as well as activities such as crosswords and colouring pages. Through this magazine we aimed
to reach a wide variety of audiences and provide education as well as entertainment.
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The UNSW iGEM 2022 team believes that integrated human practices involves resolving the conflict between scientific and public mindsets. Our nature-based design began by seeking advice from ethicists
and environmental scientists in order to understand the complex relationship between synthetic biology and the world. Our team believed that communicating with a diverse range of stakeholders was
essential in order to understand this relationship. The research and progress of our project was heavily influenced by the beliefs and advice of stakeholders includings, Traditional Owners, Agricultural
companies, Governments and Non-government organisations, and academics. Conversations with these stakeholders was vital for the human practices team. Using their perspectives, we were given guidance,
allowing us to develop the safest and effective technology for our solution. Conversing with different stakeholders resulted in a proposal that abided with multiple values, ensuring a responsible and
successful solution to cereal rust on wheat.
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Modelling was an integral component of our project this year as it allowed us to determine the behaviour of our proteins and peptides before synthesis within the lab. Our team attempted to find unique
protein targets through trying to annotate poorly annotated proteins using Blastp and Genemania. This analysis allowed us to determine protein targets that weren’t highly conserved among species but did
remain vital to the functioning of the organism. These fungal protein targets were modelled on Alphafold to determine the best site for attachment of a His tag without interference to the protein’s
functioning. Furthermore, these PDB models were utilised in the computational design of inhibitory peptides. The Alphafold output of highest confidence was input into a docking software to simulate the
binding of random sequences, with the active site annotated manually for specific binding. These simulations yielded a series of peptides that were rerun on docking simulations to evaluate their performance.
We hope that, in the future, the peptides we have created through our modelling approaches will be tested in the wet lab.
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