Human Practices

Introduction

During the course of our project, there were several moments that let us realize that iGEM is not just having an idea. It involves designing an experiment and making it work. Especially as students without much experience, we had to rely on inputs from experts in the field. The feedback we got from different people more than once changed our plans for the project. When we first gathered as a newly assembled iGEM-team, we did a lot of brainstorming to come up with a good project idea. Our ideas covered directed evolution, working with fungi, genetically engineering plants, CRISPR/Cas9, OMVs, plant immunity, and others. Relatively soon, we agreed on working with plant immunity, since we wanted to make use of the fantastic resources at the UZH's Department of Plant and Microbial Biology. Additionally, Prof. Zipfel, leading plant immunity expert and PI of last year’s UZH iGEM team, offered to us a lab space after hearing about the course of our project. After some more brainstorming, we knew that we also wanted to incorporate bacterial outer membrane vesicles (OMVs), which are buddings of the outer membrane of gram-negative bacteria, into our project. Prof. Eberl’s lab, also located in the Department of Plant and Microbial Biology, happens to focus on said OMVs, which came in quite handy for us.
As our idea of combining plant immunity with OMV research started to take shape, we came up with two main questions for our project:

Can we use OMVs to trigger the outer immune system (PTI) of the plant?
Can OMVs deliver molecules into a plant cell and therefore trigger the inner immune system (ETI)?

We hypothesized that if the answer to either questions above is yes, we then could build a bacterium that could somehow sense pathogens and produce these OMVs on demand, in order to trigger the plant’s immune response before the pathogen could cause too much damage.

The Importance of BOOM V

“Globally, the situation [in agriculture] is very unsatisfying. It is perhaps not as catastrophic as it is sometimes portrayed, but it definitely doesn’t look good.”
Prof. Beat Keller from the University of Zurich, August 2021 [1]

In Switzerland and all around the globe, food safety is essential. Pathogens are a frightening danger to our harvests and we need to protect our crops from them. For this, we mainly focus on synthetic pesticides which bring big problems with them such as emerging resistant pathogens, decreases in biodiversity and pollution of groundwater. Our project aims at strengthening the plant’s own immune system with bacterial vesicles. This is a brand new philosophy, and just like organic wine grower Christian Zündel put it in an interview with us [2]:

As soon as we see a pathogen, we shoot. That’s our way of handling problems. I don’t think that this attitude is sustainable. We need to change our mindset about handling pests in agriculture.

The Swiss Academy of Sciences mentioned "the negative effects of pesticides on non-target organisms, on biodiversity, and especially on water bodies. (...) Numerous evidence is available in Switzerland that legal requirements and ecotoxicologically based environmental quality criteria have been exceeded for a long time. This leads to significant (...) costs for society." [3]
Knowing about these problems with pesticides and supported by the opinions of the farmers we talked to, we realized that new approaches to combat pathogens need to be developed. At the same time, the number of independent pesticide production companies in Switzerland has decreased over the last few years, and the production of seeds and pesticides are often combined in the same company which leads to bigger dependency on a few suppliers [4].
Prof. Beat Keller confirms this: „Globally, genetic engineering today is mainly used by big companies. They focus a lot on the aspect of herbicide resistance and of course, the use of herbicides is in principle somewhat problematic.“ [1]
This led us to investigate the entrepreneurial aspects of our project. Council of States member Ruedi Noser suggested going abroad if we wanted to turn our project into a start-up [5]:

In Switzerland, the admission procedure for a GEO-product takes very long and is extremely expensive. This could quickly become fatal for a small start-up. But the market isn’t bound to national borders, and I think your project could have huge potential.

So although we are in a country that banned the commercial use of GEOs, we are trying to use the technologies of genetic engineering and synthetic biology to develop a creative alternative to strengthen our plants. The farmers which produce our everyday food seem very enthusiastic about our project and are keen on using our OMVs.

Our Experts

Prof. Cyril Zipfel - PI and expert in plant immunity

Recommended using only the outer immune system (PTI) of plants as the ETI immune response might be too strong and damage the plants
Recommended using flg22 and elf18 as an elicitor
Recommended using reactive oxygen species (ROS) burst assays as an assay for immune response instead of seedling growth inhibition assays, as flg22 works better with ROS assays
Told us that although spraying our vesicles onto plants would be the most suitable option in Switzerland, we should not limit ourselves in terms of engineering a bacterial system that would be released into fields where they could sense pathogens and start vesicle production

Prof. Leo Eberl - Expert in bacteria-plant interaction and membrane vesicles

Recommended considering cytoplasmic membrane vesicles (CMVs) in addition to outer membrane vesicles (OMVs) as they contain cytoplasmic content, which can be used to deliver cargoes into plant cells.
Recommended and provided us some plant growth-promoting bacteria.

Prof. Beat Keller - Expert in molecular plant biology and resistance

Informed us on the current state of genetically modified organisms (GMOs) in Switzerland and globally, and the stance of the public towards it.
Told us about the administrative processes our project would need if we would want to turn it into a real-world application
Recommended using RNAi for the fusion experiments, where we would test if vesicles can fuse with plant cells and deliver their content
Referred us to Prof. Olivier Voinnet (ETHZ) as an expert in RNAi

Prof. Olivier Voinnet - Expert in RNA silencing at the ETHZ

Advised us in our fusion experiment and helped to set up the experimental plan
Advised us to use a different construct than we originally planned to use to heighten our chances of seeing a fusion, if one would occur
Provided supervision, protocols, plasmids, and plants for the fusion experiment
Is currently helping us evaluate the results from our fusion experiment and helping us with planning and conducting our follow up experiments

Rudy Studer - Winegrower

Told us that pesticides applied to plants are often washed away by rain, making us aware of this possible issue with our project
Informed us on the current state of genetically modified organisms (GMOs) in Switzerland and globally, and the stance of the public towards it.
Showed us the need for a solution for the problems we’re addressing with our project
Confirmed to us the problem with emerging pesticide-resistant pathogens
Told us that our product would probably be used by him and other farmers

Roberto Mozzini - Farmer

Showed us the need for a solution for the problems we’re addressing with our project
Told us that our product would probably be used by him and other farmers
Explained the current situation in Swiss agriculture
Tells us that the prizes for synthetic pesticides have increased
Thinks that at some point in the future, genetically engineered plants will be cultivated in Switzerland, too

Christian Zündel - Winegrower, studied plant biology at ETHZ

Mentioned the difficulty of making field trials with our OMVs
Showed us the need for a solution for the problems we’re addressing with our project
Explained the current situation in Swiss agriculture

Ruedi Noser - Council of States, Entrepreneur

explained the political backgrounds behind the Swiss ban on commercially cultivating GM-derived products
Recommends going abroad because the admission processes for GM-products in Switzerland are too complex for a small start-up
Is positive our project has big potential from an entrepreneurial point of view

Alessandro Franchini - Promega

Gave us the input to use smaller bioreactors which produce OMVs locally in farms, as opposed to producing them in a large scale, as the OMV production is not infinitely scalable
Suggested thinking about lyophilisation of our technology for easier distribution and transportation
Advised us on options to detect whether vesicles enter the plant cells:
- suggested using a luciferase assay such as NanoBiT®
- suggested putting a plasmid in membrane vesicles and then attempting to detect the genes from the plasmid in the plant cells

How our experts influenced the course of our project

When we first pitched our idea to Prof. Cyril Zipfel, he found it ambitious to do in the given timeframe, yet doable in the sense of the logic behind it. However, he said that maybe it wouldn’t be desirable to trigger the plant’s inner immune system (ETI) as this often leads to a very strong immune response and often to cell death, which would mean that we might actually kill the plants with our system, which was obviously not what we wanted. Due to this feedback, we decided to focus on the activation of the outer immune system (PTI) of the plants.
Our goal was no longer to trigger the ETI in plants, however, the curiosity remained if the OMVs could fuse with plant cells for other purposes. We came up with a new approach that could benefit from OMVs fusing with plant cells: RNA silencing. Instead of triggering the ETI with the fusing vesicles, we could deliver substances that silence RNA and therefore could interfere for example with viral reproduction. But how could we achieve that? We needed more inputs on the properties of vesicles and decided to talk to Prof. Leo Eberl.

During our talk with him, he suggested not only looking at outer membrane vesicles (OMVs) but also considering cytoplasmic membrane vesicles (CMVs). CMVs, as he explained to us, are vesicles that form through an apoptotic process, in which the bacterial cell lyses and fragments itself into multiple small CMVs.
The advantage of these CMVs is that they contain the cytoplasm of the bacterium, whereas the outer membrane vesicles only contain the periplasm of the bacteria. He said that it could prove easier to engineer these vesicles since our inserted plasmid would be translated into the cytoplasm and automatically end up in the vesicles. Thanks to Prof. Eberl’s feedback, we decided to include CMVs besides the OMVs into our project. Our course had changed as follows:

Both OMVs and CMVs would be used to induce the PTI.
Due to the fact that CMVs contain cytoplasm, which facilitates the loading of cargo into the vesicles, they would be our main actor in testing the fusion between plant membrane and bacterial membrane.

Now that we had the information that we needed about vesicles, we needed to find out more about RNA interference (RNAi). Upon researching more about the topic and its possibilities, we came across Prof. Olivier Voinnet (ETHZ), an RNAi expert at the ETH. We talked to him about our idea to test the fusion between plant cells and bacterial vesicles. He helped us design and set up the experiments in a fashion that would allow us to detect a fusion even in a single cell in case it would happen. We implemented a signal amplification technique that Prof. Voinnet had developed during his research that was based on the principle of RNAi. Unfortunately, despite the great help we received from Prof. Voinnet, we couldn’t find evidence that CMVs could fuse with plant cells yet. We decided to shift the focus of our project away from the fusion to inducing the PTI with our engineered vesicles. From now on, our main project goal was to investigate whether OMVs could be a candidate for an alternative to synthetic pesticides by inducing the plants’ PTI and whether we thereby could design a plant-associated bacterium that would secrete those OMVs once a pathogen is sensed.

During an interview for our YouTube series with winegrower Rudy Studer, he told us that one problem he faces regularly with pesticides is that they are often washed away by the rain. If we wanted to make our project a success and create a good alternative to current pesticides, why not tackle that problem as well? Because we are using engineerable vesicles we have the advantage over regular pesticides in that we can implement structures to our vesicles that help reversibly “attach” them to the plant tissue. To achieve this we decided to build in a cellulose-binding domain on the surface of our vesicles. Our OMVs now included not only the immune response triggering epitopes but also a structure, that would enrich the OMV at the cell wall, where the plant's immune receptors are located. With this we hope to achieve two things: 1) Less washing off of the OMVs through rain due to attachment to the plant cell wall and 2) a higher immune response the OMVs can elicit because we bring the epitopes closer to the plant immune receptors. To find out what results we gathered from testing this structure, go to Results.

Since synthetic biology uses genetic engineering as a tool and our project required the release of a genetically engineered organism, we interviewed Prof. Beat Keller regarding the current state of GMOs in plants in Switzerland and around the world. He gave us valuable arguments which supported the applicability and usefulness of our project, which confirmed that we were on track in trying to solve a real-world problem. Prof. Keller asked: “One of the first things to consider would be how the admission procedure for field trials for a product like your genetically engineered vesicles would look like. How long would the admissions procedure take? And which kind of admissions would you need?” [1]
He knows too well how tedious such admissions can be, as he himself had to deal with them for field trials with genetically engineered plants for decades. In Switzerland, there are a lot of requirements that need to be met before starting a field trial for a new product, especially in the agricultural sector and especially then when they involve GMOs. Only after many successful trials, a product will be permitted to be sold.
Winegrower Christian Zündel confirmed this: "You would first need to test your product in the field. Us farmers probably can’t afford to do these trials, but that’s why we have [...] these research institutes. But also, often these ideas work wonderfully in the lab, but on the field, it’s a different story. But I would definitely try to use them if they work." [2]

How did we integrate this feedback into our project? We recognized that, unless the laws around GMOs in Switzerland changed, there was no way to implement a pathogen-sensing, OMV-secreting, genetically modified bacterium in Swiss agriculture anytime soon - or at least not commercially. However, that doesn’t mean it couldn’t be implemented somewhere else. Ruedi Noser, council of states and entrepreneur, confirmed our sentiment: He recommended developing our endeavor abroad, as the admissions for GM-products are stricter and more complex in Switzerland compared to some other countries. We decided to adapt our project into two parallel narratives: One that would include the release of the genetically modified bacteria that releases OMVs upon danger, and one that would consist of solely spraying harvested OMVs from the genetically modified bacteria onto the fields. The latter probably wouldn’t fall under the GMO laws, because OMVs aren’t genetically engineered organisms but rather the products of a GMO. With these two parallel narratives, we could adapt to the countries' current GMO regulations and allow the application of our project even in countries with strict GMO regulations.

Now that we knew that at least in Switzerland the option of spraying OMV’s is the only feasible one, we had to find out how we could implement that in the real world. We spoke to Alessandro Franchini from Promega, who recommended using smaller bioreactors that produce OMVs locally in farms as opposed to on a large scale. We liked this idea since a decentralized production would also mean less dependence from monopoles on the side of the farmers.

Summary

Based on all the talks with our experts the best and most feasible course regarding the implementation of our project in the real world is as follows:

We follow a parallel approach: On one hand, we could release an engineered bacterium into the field where it releases OMVs when a pathogen is sensed, and on the other hand we could harvest OMVs and spray them onto the fields when the season for a certain pathogen is nearing.
The technology behind OMVs acting as an alternative to synthetic pesticides is based on the activation of an innate plant immune response, the PTI, rather than the activation of the ETI or the implementation of RNAi.
We took into account the problem farmers face with rain washing away their pesticides and decided to include a cellulose-binding domain in our project to hopefully equip our OMVs with better resistance to rain.
In the case of spraying OMVs, farmers could produce the OMVs themselves in a smaller bioreactor which would allow a decentralized OMV production.

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[1] Research, Regulations and Resistance: An Interview with Beat Keller by iGEM UZH 2021, 10. October 2021
[2] An Interview with Christian Zündel by iGEM UZH 2021 in August 2021
[3] Swiss Academics Factsheet: Pestizide: Auswirkungen auf Umwelt, Biodiversität und Ökosystemleistungen, Vol. 16, No. 2, 2021
[4] "Ernährungssicherheit der Schweiz", 2021, Agroscope Science, p. 11
[5] Ständerat, Self-Sufficieny and Start-ups: An Interview with Ruedi Noser by iGEM UZH 2021, 14. October 2021