Latest revision as of 23:40, 17 October 2018

Integrated Human Practices

Applications

Since our system could be used to switch any gene or combination of genes ON and OFF and we engineered it to work both in plants and in yeast, the potential uses are manifold. They reach from controlling individual genes to creating whole logical pathways. Therefore, we did a lot of brainstorming and also talked to farmers and scientists to identify some of the most useful applications.

Original Idea

The idea we started out with was that we wanted to be able to turn resistances against pests and diseases in crops on when they are needed to ensure the survival of the plants, but turn them off when they are not, in order to prevent or at least slow down the development of resistant strains. One example for this is the expression of BT by the plant. However, once we’d decided on using a toggle switch to solve this problem, we quickly realised that this kind of tool could also be used in a host of other applications.

Fundamental research

When we explained the goal of our project to plant molecular biologists Jürgen Kleine-Vehn and Sascha Waidmann on 19th of June, because we wanted their help in working with plants they were immediately interested and pointed out that our system didn’t just have agricultural applications but could also be very useful in the lab, as it would allow the temporary repression of individual genes with only a single dose of a signal molecule. Currently this is achieved either by total knock-outs of the genes of interest, which aren’t always viable, or by permanently applying a signal molecule like ethanol or estradiol, which also influence the plants in other ways and can therefore make it difficult to distinguish between the effects of the signal molecule and the effects of the repression. While our system, at least when using signal molecules and not liposomes to deliver the signal, also uses those same signal molecules their use would be short term and the repression of the gene would persist long after the effect of the signal molecule would have worn off, which would be an improvement.

Late frost

We also started looking for common reasons for harvest failures in news reports and one of the most devastating ones we found was late frost. This refers to a prolonged cold period in late spring, after the fruit trees have started blooming and is a growing problem in Austria and other countries in central Europe due to climate change. [1] When it happens the cold temperature kills off the blooms of the tree and thereby hinder fruit from forming. Our system could help to prevent this problem, by suppressing the genes that trigger blooming until a time where late frost becomes very unlikely. We also talked to a farmer who has been affected by this problem:

Interview with Hannes Weingartmann

29th of August afternoon at Puch near to Weiz. Meeting with Hannes Weingartmann talking about GMO, modern organic agriculture, the problems with variable climate and drinking delicious grape juice.

Hannes Weingartmann has grown up in Styria on an Apple and Wine farm. He studied Food and biotechnology and is currently doing his masters degree at the TU Graz. On the one hand, he grew up with this background and is now integrated into the family business, and on the other hand, he is also well versed in the field of molecular biology and was, therefore, a very interesting interview partner to us.

Some years ago he changed his business to organic farming, not because of the governmental support for organic farming, but because of his conviction. According to him, there is still a long way to go. One of the most important points for him is soil safety. He does not shy away from a possible additional expenditure if thereby pesticides can be avoided. Unfortunately not all measures are possible due to financial and geographical circumstances.

He had a harvest loss of about 90% in 2016 and about 85% in 2017. That was a pretty intense loss, but luckily in 2018, it looks to be a good harvest. The reason for these big losses was late frost. At the beginning of April it got warm so flowers of the trees sprouted, but at the end of April a late frost occurred and the flowers died due to that. There are some technological possibilities to prevent that. Firstly, you can light a fire and lead the smoke through the fields. Secondly, you can spray water and due to the heat of crystallisation, the trees remain in bloom. Furthermore, there are other possibilities like wrapping the blossom in blankets and similar strategies.

The chances to save the blossom with our toggle switch seems interesting, but there are still a lot of difficulties. Firstly, it is not legal to release such crops in Austria yet and secondly, for him as an organic farmer, he has to be aware of customer requests. As long as GMOs have such a bad reputation the customers will cut it off. If one day GMOs were legal, he would decide from situation to situation whether to use them or not. Obviously, there are many advantages, but if there is a chance for a solution with fewer disadvantages the decision would be easier.

From his scientist’s point of view, our project is very interesting to him and he mentioned that our Toggle Switch could not only be used in agriculture. Another point of our Project was very interesting for him. The usage of a drone in agriculture has many advantages. The space between the tree array could be reduced because there would be no need for a tractor to pass. Which in turn affects soil health in a positive way. An automotive drone would make his work much easier.

After 2 hours of interesting and inspiring conversation, we took a look at his field. He showed us how his trees are arranged and how he tests if the apples are ripe. In the field, chickens are allowed to run around and we were allowed to try the fruits.

With respect to our project, we got an insight into the challenges of farming and its complexity of environmental circumstances. It was refreshing to talk to a farmer with this scientific background, who knows and understands both sides of the GMO discussion.

He is building bridges, combining different points of view and for this reason, he was the perfect person to talk about this topic.

Controlling native genes

On the 13th of August we had a meeting with the Austrian company Biomin, which specialises in animal health and nutrition with a focus on providing solutions for eliminating mycotoxins from animal feed. They are committed to supporting the research endeavours of aspiring scientists and were interested in sponsoring our project. They requested that we give a presentation at their R&D headquarters in Tulln, to give them a better idea of what our project is about. During the following discussion, they made a very interesting point. They suggested that in order to control native genes it would be quite complicated and invasive to replace the whole genes with genes controllable by our toggle switch and that instead we should look into having our guide RNA directly bind and thereby block the unaltered native genes. Afterwards we started brainstorming how this could be achieved. We concluded that it would be possible to do so if the promoter that controls the native gene is unique enough to allow the selective binding of our specially designed gRNA. However this still leaves the problem that according to our model multiple binding sites for the dCas9/gRNA complex would be needed to efficiently block a gene. There are a few solutions to this problem. One is to use multiple toggle switch cassettes to control the gene, which would significantly increase the design and cloning effort, though. Another possible solution is the fusion of a transcriptional repressor domain to dCas9 which has been shown to increase repressor strength [2]. A solution that is more invasive, and would therefore probably take more effort to engineer and fine tune, would be to replace the native promoter with an engineered one of the same strength that contains the appropriate gRNA binding sites.

Legal Considerations

Drones

<a href="" data-toggle="lightbox"> <img src="" class="w-50" alt="Human Practice"> </a>

Within our Project we designed a drone as well but due to financial and time constraints we decided against the construction of a prototype. Nevertheless, we wanted to talk about the legal issues surrounding drones. Therefore we spoke to Dr. Iris Eisenbauer, an expert on agricultural and drone law. She introduced us to the legal classification of drones:

class 1:

• Flight in direct visual contact without technical aids and within a radius of more than 500 metres and/or within a radius of operations for remuneration or commercial or non-commercial purposes other than for the purpose of the flight itself
• An operating licence is required.
• the technical and operational certification of unmanned aerial vehicles with an operating mass exceeding 150 kilograms falls within the scope of Union law.

class2:

• blind flight
• These drones are subject to the aviation regulations applicable to manned civil aircraft, such as in particular the required airworthiness certifications, registration requirements, pilot licence for the pilot, compliance with air traffic regulations and much more.
• the technical and operational certification of unmanned aerial vehicles with an operating mass exceeding 150 kg falls within the scope of Union law.

Impact on our project

If you want to use drones in agriculture, they must fall into either category 1 or category 2. However, our drone does not fall into either category because it is supposed to act autonomously. Apparently, a major legislative gap exists here regarding our special case (i.e. an autonomous drone). Either a corresponding legal text or a precedental case has to be waited for, to fill this legislative gap. A possible solution for this issue would be a modification towards a new category for special agricultural utilization or application. This is a problem in the legislation which concerns not only autonomous drones but also autonomous vehicles and will therefore, hopefully, be regulated in the next few years. The law finding regarding the autonomous drone is probably easier. Another important point is the environmental question. Drones can monitor the ground from the air and thus have no direct influence on ground safety. Furthermore, drones can scan the ground, the field, the weather, etc. and can therefore transmit warnings more quickly. If the drone is also equipped with a camera to scan the ground, there will be another problem, namely data protection. A legal framework would also have to be created for this.

Spraying agents in Agriculture

Of course we had to deal with legal issues as well. To deal with the spraying issues we called the ministry of agriculture but they forwarded us to the AGES, the austrian agency for food safety. We had an very interesting conversation on the telephone and he sent us some documents about the licensing procedure. That was important because only copper is an the list for legal spraying agents in agriculture. <a href="https://psmregister.baes.gv.at/psmregister/faces/main?_afrLoop=2100600944118707&_afrWindowMode=0&_adf.ctrl-state=ksl01x0di_4">Here</a> you can find the list. In general the regulation (EG) Nr. 1107/2009 shall apply that the european parliament and the european council forms the legal basis for the placing on market and the control of plant protection products and their active substances. This regulation shall apply in all EU-states and does not need to be changed in national law. You can find those legislative texts here. The prerequisite for approval is that all active substances have already been approved in the eu. In the pesticide database you can find all information for approving active substances, the link is <a href="http://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/public/?event=activesubstance.selection&language=DE">here</a> .

If your active substance is build up by another manufacturer you have to do the so called equivalence test. This evaluation is an EU-procedure but the assessment must only be carried out by the Member State in which the application was made. If the review shows that the active substance is equivalent to the original active substance that has already been approved, they can access the active substance dossier of the approved active substance. If the review shows that the active substance is not equivalent to the original active substance, an application for authorisation of a plant protection product containing that active substance is not possible.

Application for authorisation of a plant protection product Article 33 of the regulation (EG) Nr. 1107/2009. Thereby the EU is split up in 3 zones, where the plant protection product evaluation is carried out by a zonal reporting Member State, which subsequently forms the basis for the subsequent specific evaluation and authorisation by the Member States. However, an application for authorisation must be submitted in each Member State in which the plant protection product is to be placed on the market or used. There are two ways to do this:

• In all states where a marketing is planned, the application must be made simultaneously. The applicant proposes a Member State to carry out the assessment for the zone. The other member states must wait until this application has been examined and approved. From that date, Member States may examine the national requirements and other requirements. The deadlines for the first evaluation are one year and for the national evaluation 120 days.
• In this case, this Member State is, of course, the zonal applicant. After authorisation has been granted, an application for mutual recognition may be made in the other Member States in accordance with Article 40 of Regulation (EC) No 1107/2009. Classification of the Zones:
• Central zone: Belgium, Germany, Ireland, Luxembourg, Netherlands, Austria, Poland, Romania, Slovakia, Slovenia, Czech Republic, Hungary, United Kingdom
• Northern Zone: Denmark, Estonia, Finland, Latvia, Lithuania and Sweden
• Southern Zone: Bulgaria, Croatia, Cyprus, France, Greece, Italy, Malta, Portugal, Spain, Croatia For field applications (excluding seed treatment products), a rapporteur Member State must be selected for each zone to carry out the assessment for that zone.

In particular, the following guidelines must be observed for the zonal authorisation procedure: Zonal Evaluation and mutual recognition: <a href="http://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_ppp_app-proc_guide_doss_reg-report-draft.zip">http://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_ppp_app-proc_guide_doss_reg-report-draft.zip</a>

Format template for the draft assessment report (= summary of the assessment): <a href="http://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_ppp_app-proc_guide_doss_reg-report-draft.zip">http://ec.europa.eu/food/sites/food/files/plant/docs/pesticides_ppp_app-proc_guide_doss_reg-report-draft.zip</a>

<a href="https://ec.europa.eu/food/plant/pesticides/approval_active_substances/guidance_documents_en">The Commission's homepage</a> , DG Health and Consumer Protection, provides information and guidelines on the approval procedure for active ingredients and the approval procedure for plant protection products.

Impact on our project

It was clear to us very early on in our planning that we would not be able to span the gap between using environmentally friendly signal molecules that are legal as spraying agents and the limitations that are set on us regarding time constraints. Therefore we had to settle for signal molecules that have already been shown to work, even though most of them would not be allowed in an agricultural application, instead of designing our own receptor pathway. This is why we made sure to make not just the output of our system flexible, but also the input, to make it easier for others to build on our work and improve it through the use of signal molecules that would be applicable in the real world.

<a href="" data-toggle="lightbox"> <img src="" class="w-50" alt="Human Practice"> </a>

General considerations regarding synthetic biology and GMO crops

Focus Group discussion with Christoph Strasser about iGEM and responsible research and innovation.

As Christoph Strasser was writing his Bachelor Thesis about responsible research he contacted us and our PI Prof. Diethard Mattanovich with the request for a focus group discussion to get to know us and our project a bit more. Christoph’s special focus was on our work in Human Practices and how we plan on integrating it into our project design and how important it was to us and iGEM in general. He wrote the following statement about us and our project. :

I wrote my bachelor thesis “The iGEM Competition in Context of the concepts Authentic Learning and Responsible Research and Innovation” from March to July 2018. In addition to a literature research I decided to investigate the research questions of my work with a case study approach. I interviewed the PI of iGEM Vienna @ BOKU, Prof. Diethard Mattanovich and did focus group discussions with the iGEM-Team of 2017 (Project: “D.I.V.E.R.T.”) and the iGEM-Team of 2018 (Project: “Robocrop”). The discussion with the Robocrop-Team took place on May, 2nd 2018. It was ascertained how the team and the project topic came about, which work steps had already been carried out and which considerations had been incorporated in the topic selection. It was thus possible to deduce which authentic learning experiences have already been made and in what form the principles of RRI have already been included. To do so, I oriented my research to the four questions on RRI, that SYNENERGENE suggested to eight iGEM teams in 2014 [3] and to the four dimensions of Authentic Learning, RULE [4] stated. On the one hand the participation of the iGEM Team of 2018 in the focus group discussion helped me to increase the practical relevance of my bachelor thesis. As I was informed a while after the discussion, on the other hand the participation also helped the team, to be more sensitive to the questions of RRI, thus taking a step towards achieving the "Integrated Human Practices".

Interview with Timo Küntzle

29th of August evening at the Siebenstern Cafe in Vienna. Meeting with Timo Küntzle talking about GMO, modern agriculture and its public debate about rising technologies and finally about the political evaluation of these new concepts.

Timo Küntzle has grown up in south-western Germany in an agriculturally influenced society. Because of such a youth, it is no surprise he focused his studies on agricultural qualification. After finishing his studies he decided to join Journalism. Because of a documentation about the worldwide use of GMO crops produced this year he was the perfect interview partner for us.

He did a great job in showing the actual usage of GMOs in different regions all over the world with big efforts to present the pros and cons of GMOs as objectively as possible. The documentary called “Im Kontext: Schöne Neue Gentechnik - Revolution in der Landwirtschaft” was part of a bigger series about GMOs by the journalism-collective “Addendum”, which was published leading up to the CRISPR ruling by the ECJ (European Court of Justice). Furthermore, the docu was publicly shown on “Servus TV”.

On his journey, he had the chance to get in touch with farmers and other stakeholders who are experienced with GMO-agriculture or directly affected by their local regulations. Very interesting is the fact that he met a wide range of different kinds of farmers, showcasing the impact GMO crops have on different communities.

We were positively surprised by his open-minded and optimistic argumentation about GMO in agriculture, while at the same time being absolutely aware of all the risk and problems involved in their practical implementation at the same time. He told us about poor Bangladeshi farmers, who received free “Monsanto” seeds. Based on these expressions he was able to draw the bigger picture about a sustainable agriculture not solitarily affected by economic exploitation by global companies. After carefully assessing all the factors it was obvious to him that in many cases the chances for a sustainable, self-determined agriculture often outweighed the risks involved in the use of GMO crops. On the basis of his Twitter Timeline, we already knew that he is aware of his public impact as a journalist and he is very confident in his tweeting. In the course of the interview, we quickly noticed our similar beliefs regarding the possible utilization of GMO crops in the future.

It was evident that the differentiation between economic factors and technology-based factors is crucial in both today's and future debates about the chances and risks involved in the implementation of GMOs.

As long as politicians are not able to dissociate these two a general legislation and regulation that serves the community is not possible and purposeful, because a commercial launch is too expensive for anyone but the ”big players”, who are often not interested in providing individual solutions that serve individual communities.

After being briefed about our “ROBOCROP”- concept we were happy to see Timo be curious about its impact and further possibilities. Interestingly for us, we found out that his family who is still actively running a farm already uses drones to bring out so-called ichneumon wasps as an organic pesticide.

Regarding the debate and possible usage of GM-tech in Europe his conviction is that in the relatively near future GMOs will become more common. According to him, the international pressure and technological progress will inevitably push the European society to the point of a final debate about GMO implementations and regulations.

After 3 hours of inspiring discussion, we had covered a wide range of topics connected to our project and we are very thankful for this motivational evening. We got to know him as a kind person who has been around the world, is well informed and not afraid of speaking up.

With respect to our project, we got new insights into stakeholder related topics. It was refreshing to get in touch with an insider and his special relationship to farmers and their opinions about GMOs, respectively.

Although he is not an expert in genetic engineering it was very compelling to hear his opinions about said topics.

More Information about <a href="http://kuentzle.net/">Timo Küntzle</a>

[1] Unterberger C, Brunner L, Nabernegg S, et al. Spring frost risk for regional apple production under a warmer climate. Delzon S, ed. PLoS ONE. 2018;13(7):e0200201. doi:10.1371/journal.pone.0200201.

[2] Agnieszka Piatek, Zahir Ali, Hatoon Baazim, Lixin Li, Aala Abulfaraj, Sahar Al-Shareef, Mustapha Aouida, Magdy M. Mahfouz; RNA-guided transcriptional regulation in planta via synthetic dCas9-based transcription factors; Plant Biotechnology Journal (2015) 13, pp. 578–589; doi: 10.1111/pbi.12284

[3] STEMERDING, D. (2015). iGEM as Laboratory in Responsible Research and Innovation. Journal of Responsible Innovation, Vol. 2, Issue 1, pp. 140-142, January 2015. [4] RULE, A.C. (2006). Editorial: The Components of Authentic Learning, Journal of Authentic Learning, Vol. 3, No. 1, pp 1-10