Human Practices
Our project relies heavily on the feedback of the people who we want to affect with our product, like farmers and phytopathologists. Because of that, we sought feedback from them, to find out how we could improve the applicability of the test in-field and to get an overview of how much demand there is for our test.
23.04.2021 Farmers Market
To get in contact with a lot of potential customers we went to a local farmers market. We wanted to know whether our test could be useful to the farmers and if there is meaningful demand for a product like this.
We gave them a quick overview about our project and most of them instantly liked our idea. Many of them were really interested in talking to us and giving us some advice. We contacted them later to have deeper conversations through emails and via calls. They were able to give us really important information about their situation. For them, the main point would be the price. One test was estimated to cost around 7€ and depending on the scenario a lot of tests might be needed. Learn more about our statement of cost here. At this point we still had no clear vision for our test's final application, but this made us aware that relying on the test alone to test a whole field, was not an option. We therefore thought about different ways to integrate our test into a wider application.
08.06.2021 Dr. Manuel Sommer
Dr. Manuel Sommer is working for Bayer as Project Manager for Strategy and Business Consulting. That is why we contacted him to determine in which direction our project's marketing could go. He helped us with some more brainstorming and we ended up with a few possible directions. Since farmers know their fields best, the only option here would be to go with the pathogen forecast idea, to warn surrounding farmers about possible pathogenic spread. The problems we would face with this idea are that we would have to test every other day, to get data that would actually be able to forecast the spread. And with such a high frequency of testing, there is also the cost factor that needs to be taken into account. Another idea was to become more ecological by using our test, to enable more precise pesticide application. This would probably also lead to less resistances. Florists and gardeners are also a possible target group for our test due to the specific market and high prizes that come with it. The pressure of keeping the plants healthy and knowing exactly what they need is really high. But here we would either have the problem that the florist might not have a high disease pressure on the flowers and it is a small market or the gardener has too many different plants and our aptamers probably won’t work for every species with the same affinity. Last but not least, private individuals could be another target group, but here we also would have the problem, that there are too many different plants that would all need their separate aptamers.
As one can see, it was important for us to decide, because as listed above, with every different direction there arose different and new questions that had to be addressed separately. In the end we decided to go for 3 possible scenarios for farmers that either have expensive seedlings, need regular field inspections or want to differentiate between two or more pathogens. More detailed information can be found here.
15.07.2021 Interview Bavarian Farmer
We interviewed a farmer who lives in Bavaria and primarily works with strawberries. He wanted to stay anonymous. For almost an hour, we talked through all the aspects of our project and its applicability in-field. He gave us a lot of insights into how he normally tries to defend his crops from pathogens. Every two weeks, an appraiser visits his fields and inspects the plants visually. Once symptoms occur, curative or preventive phytosanitary measures are applied. So our project would provide an advantage by detecting the stress before the occurrence of visual symptoms. The sooner a farmer knows what he has to deal with on his field, the better he can protect it. Another very interesting point is that if you sell products to the retail trade, you must follow very strict regulations. Farmers are allowed to only use a certain amount of substance groups to defend their crops. Our test can aid farmers to stay in these regulations due to limiting unnecessary spraying of pesticides.
16.07.2021 Interview Florian Kamlah
Another farmer we talked to is Florian Kamlah, who owns 2 hectares of land in Munich and sells his organic produce in his shop. He loved our idea and thinks it can help a lot of farmers, but it is not as interesting for him. There are two reasons for that: His crop-plants and his cultivation method. He doesn’t grow his crop plants in monocultures, but instead has divided his field into many compartments.
In addition to his land, he has a few greenhouses, in which he again has no monoculture. If a plant pathogen appears in his field, its spread is limited due to the lack of host plants. In addition, he plants lots of salads, which are very cheap and grow very fast. One young salad plant costs 15 cents and grows in 2-4 weeks. So, if he sees a spreading pathogen, he can simply grow new salads, which is why our test is not interesting for him. We need to focus on monocultures and crops that take a long time to grow or are expensive to cultivate.
02.07.2021 Robert Bossmann
In July some members of our team participated in a tractor tour, organized by local farmer Robert Bossmann. The trip under the motto "Agriculture you can touch!" aimed to showcase and explain sustainable agriculture at the hand of Mr. Bossmann’s fields. He gave his unfiltered opinion on the discussion of ecological vs. conventional agriculture in regards to crops, his fields and the impact on surrounding fauna and flora. The group was also accompanied by Elke Löpke, head of the regional, biological station "Haus Bürgel". She shared her knowledge on the environmental properties of the floodplain the fields are located in.
We participated to learn more about agriculture first hand and to get in contact with our target audience. While we were at it we also presented our project idea to Mr. Bossmann. He was interested in our project and praised our engagement. He mentioned that a quick test for early pathogen differentiation could be really helpful in practise. Farmers could decide more easily on how and when to treat their crops. Ultimately his feedback helped to further visualize our project. We want to thank Mr. Bossmann and Ms. Löpke again for the fun and educational tractor trip.
Figure 1: From left to right: Elke Löpke, our members Moritz, Christian, and Vincent and farmer Robert Bossmann after the tractor tour.
18.08.2021 Prof. Dr. Benjamin Stich
In August Prof. Dr. Stich agreed to meet with us and discuss matters concerning local farmers. He is the head of the institute of Quantitative Genetics and Genomics of Plants at the Heinrich-Heine University in Düsseldorf. We asked him if he could give us advice on a PCR that was unsuccessful but he referred us to his colleague Prof. Dr. Zeier, who has more expertise in this area.
We then moved on to possible target plants and pathogens. We were advised that specialized crops are a good starting point for detection but that we should keep the option to add more cultures. On the other hand, we were told to focus on only one pathogen. Dr. Stich proposed some options about more specific plants and plant diseases to look at. A possible target could be ornamental plants, but since we are aiming to make agriculture more sustainable, we knew that that was not an option for us.
Furthermore, pathogens that affect potatoes were proposed. The advantages about potato as a plant to be tested are that it is affected by many pathogens (e.g. Phytophthora infestans) and are harvested only once a year. Because of that, prevention of disease could be very attractive to farmers, to avoid losing their crops.
After the talk with Prof. Dr. Stich, we thought about options for detection and how to include that in our storytelling. We concluded that it would be the smartest to focus on plants that local farmers in our area are planting and that are mostly affected by pathogens.
19.08.2021 Annika Baumann
Another farmer we talked to was Annika Baumann, who is the managing director of the distribution department from a farm in Bavaria that specializes in asparagus and berries cultivation. She confirmed the meaningfulness of our applications but pointed in a specific direction regarding the pathogen type: For her, the biggest use case would be the detection of notifiable plant diseases, because of their high infectiousness and the impact that they can have on a field in the long term. Some can stay in the ground even after the whole field is destroyed. Phytophthora is a notifiable plant disease but causes symptoms looking the same as ones caused Xanthomonas and some other pathogens. Additionally, once the pathogen is visible, it is already too late, and the pathogen has spread through the field. Knowing if it is a notifiable disease or not makes a big difference for the farmer and the applied countermeasures. Another aspect she pointed out is the decrease of "unnecessary" pesticide use. Our system enables more targeted phytosanitary measures and is therefore beneficial for the environment.
10.08.2021 Marius Wetzler
Marius Wetzler is an expert for pest management at the agricultural office of the "Bodenseekreis". He is responsible for advising farmers in pathogen related manners. He sees huge potential in our test for identifying and differentiating specific pathogens. There are different potential applications. One would be the constant surveillance of fields with multispectral imaging followed by one or more tests in case the vegetational indices drop. In this case the test would need to cover a broad variety of species but would give a very early response to an infection.
In another approach the tests would function as a supportive tool for experts in pathogen identification. They can generally identify the most common plant disease by eye. But for rare diseases their confidence drops making it necessary to send samples to the lab. But this only works in later stages of the disease and takes up to one month. Here our test can provide a fast and easy to use tool to get a quick result. But even in case of the more common pathogens our test can provide additional confidence for a relatively low cost making it worthwhile for farmers if they can save on phytosanitary means. This obviously requires a very high confidence of our test.
As in his region mostly pome fruit are cultivated we went into detail on apple pathogens. Here a common example would be the apple scab caused by the fungus Venturia inaequalis. Another significant pathogen is the bacterium Erwinia amylovora which causes the apple blight. This disease is extremely contagious and can destroy whole orchards in a single season. Due to this an early and highly confident detection method would bring a huge saving potential.
Integrated Human Practices
At the beginning of the year, our goal was to build a test to identify and differentiate stress in plants. For that purpose, we aimed to build a Lateral flow test that utilizes aptamers instead of antibodies. Our targets were supposed to be stress-related proteins in plants. With the help of drones, we wanted to measure the photosynthetic activity of the field to apply our stress-test in. As we had little to no experience in dealing with aptamers and LFAs, we contacted experts on these topics.
Throughout the year, our initial idea changed and our project evolved thanks to the feedback we received. How and why can be read here.
23.04.2021 Dr. Marcus Menger
We met with Dr. Menger in mid-April. We wanted to meet him because he is one of the leading experts in aptamer technologies and aptamer biosensors and works at the Fraunhofer Institute for cell therapy and immunology in Potsdam.
The insights he gave us are immeasurable. He advised us to rethink our buffer- and target approach. The conditions in SELEX should resemble the application setting as much as possible. Sodium chloride concentrations can for example impact the three-dimensional structure of the oligonucleotides and therefore impact the affinity of an aptamer to its target. This also includes post-translational modifications of our target proteins, which lead us to think about changing our expression host for synthesizing protein targets for SELEX to one more similar to Arabidopsis thaliana. He helped a lot with fine-tuning our protocol on many levels with his expert knowledge of aptamers and SELEX.
23.04.2021 Prof. Dr. Alga Zuccaro
Prof. Dr. Zuccaro is an expert in plant-fungi interactions and works at the university of cologne. She helped us to bulletproof our whole idea with lots of critical questions that we hadn’t thought about before. We thought about changing our expression system from E. coli to an eukaryotic cell, which ties in to the point that Dr. Menger already mentioned. As a bacterium, E. coli is missing the ability to modify proteins after translation, which impacts their three-dimensional structure. As we extract the proteins from plants in our final application, it made sense to also express our proteins for SELEX in plants to match the modification pattern. This could lead to an aptamer that works for the E. coli proteins, but not for the proteins that are present in the plant.
She also confirmed our idea, that we could grind up the plant samples in a suitable buffer to extract the proteins, before applying them to our test strip. This extraction step is important so the proteins can freely bind to our aptamers.
Another very important point was target selection. We had to determine the timespan during which the proteins are expressed and if that correlates with the time we plan our test to be used in. If our target is only expressed for a short time, or if it's expressed for too long, we would get a higher error rate. This led us to modify and extend our proof of concept.
Figure 2: Some of our members in our meeting with Prof. Zuccaro.
29.04.2021 Prof. Dr. Markus Kollmann & Dr. Christopher Blum
We talked to Prof. Dr. Kollmann and Dr. Blum, they are experts in the field of Mathematical Modelling of Biological Systems. We talked with them about aptamer binding predictions. Due to our lack of knowledge in this specific topic, we were pleased to have their contact to discuss our future steps.
We gave them a quick overview of our ideas in modelling, based on iGEM projects from the past and our research and started to rethink our ideas.
Even though we use DNA aptamers in our SELEX, we discussed that we should start our modelling journey by using RNA aptamers for our in-silico SELEX. The reason was quite trivial: we were not able to find enough data, as only little research in this direction had previously been made. Our hope was that we could convert RNA into DNA data and that these protein-complexes might respond in similar ways. In our following research we learned that this is not possible.
Luckily, Prof. Dr. Kollmann and Dr. Blum were really interested in our modelling idea, so they offered us help in finding matching proteins for our project and checking the proteins we already found for our LFA.
Regarding the programming part of our project, we also told them about our plan to predict waves of infection in fields with an interactive forecast. Our IT expert Ferhat created this map for us and the only thing left was to find data to display the pathogen spread more accurately. The map can be found here. Unfortunately, we had to drop this idea after a few attempts, because in the end we had too little data.
10.05.2021 Prof. Dr. Jürgen Zeier
On the 10th of May, we had the chance to get more detailed information on the protein candidates we could use, that would only be expressed, when the plant is infected by Pseudomonas syringae.
Prof. Dr. Jürgen Zeier is a professor at the Heinrich-Heine university, who specializes in plant-pathogen interactions. We presented our project with special emphasis on the plant's response during pathogen induced stresses such as the stresses caused by P. syringae. When we told him about our four initial proteins, he said that two of them, the Pathogenesis-related protein 1 (PR1) and the UDP-glycosyltransferase 74F2 (UDP 74F2), could be good candidates for our project, due to their high expression during pathogen infection with P. syringae. The other two, FKBP-type peptidyl-prolyl cis-trans isomerase family protein and rotamase CYP 3, were not suitable for us, as they lack the required expression pattern. Furthermore he showed us an article from Gruner et al. 20131, in which microarray data about expression patterns of different proteins during pathogen infections was collected.
He also addressed our question if we could use salicylic acid and its already existing aptamers to test out and improve our test before we generate our own aptamers. He suggested that we also had to consider the jasmonate pathway and its proteins. The jasmonate pathway is also very important in the pathogen response of Arabidopsis thaliana and therefore the proteins would be good for our project too. By including an expression marker for the jasmonate pathway into our test as an additional line we could differentiate between a necrotrophic and biotrophic infection. Generally an infection by a necrotrophic pathogen triggers a defense response via the jasmonate pathway, while an infection by a biotrophic pathogen induces a salicylic acid dependent immune response.
Most importantly Prof. Dr. Zeier was able to kindly provide us with a P. syringae strain. We were provided with information on how to handle this pathogen by the PhD student Ipek Yildiz. We required this knowledge to infect Arabidopsis thaliana as this was our model-interaction. Read more about our Protein expression here. Additionally, we later used this strain as targets in our cell-SELEX approach. Read more about our SELEX-process here.
Figure 3: Some of our members in our meeting with Prof. Zeier.
12.05.2021 Prof. Dr. Günter Mayer
In the beginning of May we had a meeting with Prof. Dr. Mayer. He works at the Rheinische Friedrich-Wilhelms university in Bonn and is a very eminent scientist in the field of chemical biology and aptamers. In retrospect, the meeting with Prof. Mayer was one of the most important ones of our iGEM year. The main reason for this was his insight that our test would not work the way we intended it to. The original plan was for two bands to be seen in the event of a negative event, since the gold-conjugated aptamer would bind to its complementary tag and to the poly-A tag on the membrane. If the test result was positive only one band should be seen, since the aptamer, once it has bound to the target, can no longer bind to its complementary sequence. On this point, Prof. Mayer gave us his concern that we would very likely always see two bands, regardless of whether a target is present or not. This is due to the fact that not all aptamers could bind to a target and thus a second band would always arise in the test. That can be prevented in the lab with carefully adjusted substance quantities, but under natural conditions, as they would occur on the field, it would certainly appear. This would result in false negatives.
Additionally, his references to his publications were very helpful234. He gave us more than satisfactory answers to questions about protocols. Among other things, we have a reference to one of his aptamer libraries from him. His libraries have been tested in such a way that their primer binding sequences are as insensitive to parasite DNA as possible, which greatly reduces the chance of misamplications in the PCR.
In summary, it can be said that Prof. Mayer had a significant influence on the choice of our test and the implementation of our SELEX process. Without his helpful hints, we would have encountered massive problems during the implementation of our project.
Figure 4: Some of our members in a meeting with Prof. Mayer.
26.07.2021 Prof. Dr. Meltem Avci-Adali
At the beginning of April, we had a meeting with Prof. Dr. Avci-Adali. She works in the university hospital Tübingen and researches oligonucleotide applications. We had an extremely productive call and were able to make a few very important decisions due to her advice. One key point that she made us aware of were the advantages and disadvantages of DNA over RNA aptamers for our applications. Even though RNA aptamers have more spatial formations, they are not as stable as DNA aptamers, and they are more expensive. Our focus was not only set on stability for storage purposes but also on functionality. In the plant cell, RNases are more abundant than DNases, meaning that the RNA aptamers would degrade more quickly, and therefore potentially compromise test results. Another key idea that she sparked was to use already established aptamers in addition to SELEX. That way, our detection team was able to work on their proof of concept immediately, giving us a huge time advantage.
Figure 5: Some of our members presenting our project to Prof. Avci-Adali.
04.09.2021 Dr. Katja Muders
Next, we talked to plant breeders, another potential target group besides Phytopathologists, farmers, and plant-appraisers. We talked with Dr. Muders, who works for NORIKA, a company that breeds potatoes. We talked a lot about how plant breeders screen their plants before selling them in very tight intervals with antibody-based tests, and how our system is better or worse than the potential competitors. The conclusion from the meeting is summarized as followed: Aptamers can be produced cheaper than antibodies. We can theoretically provide a product for a fraction of the costs. We are more versatile than Antibody tests. If we scale our project, we could not only do pathogen detection but do tests that indicate a lack of water, for example. We can do tests for viroids that do not have a protein membrane, and therefore no antibodies can be generated.
25.06.2021 Prof. Dr. Ralf Voegele, Dr. Stefan Thomas, Christian Trautmann
A pivoting point in our project was marked by the meeting with phytopathologists from the University of Hohenheim. They explained different kinds of drone cameras and algorithms to us that can be used to measure photosynthetic activity, including that some drones and algorithms can differentiate stress already. This means that we had to change the direction of our project, away from detecting stress in general but more towards pathogen detection and using drones to detect them earlier than possible with the naked eye.
They did advise us to focus on special crops because they are often the most expensive ones when it comes to purchasing, nurturing and growing them. One of the PhD students, Christian Trautmann, works on the farm of his parents, so he is not only a science expert but has practical expertise as well. He was very fond of our idea, especially regarding disease differentiation, when visual differentiation becomes difficult, and lab analysis needs to be done to verify the involved pathogen. These kinds of tests are expensive and can take up to 6 weeks until the farmer gets a result. During this time the pathogen can spread and lead to very high economical damage. From the time of planting a hectare of strawberries until the harvest, a farmer invests 20,000 Euros before they can sell a single strawberry. Our test could provide a result in 15 minutes for an affordable price and therefore enable the fast application of phytosanitary measures.