Team:TAU Israel/Human Practices

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Integrated Human Practices

Introduction

Our project this year is what we consider a “foundational advance”, meaning we came up with a general solution to what we felt was a bottleneck in the field of microbiome engineering. Of course, we needed to better understand where our project actually fits into the research and industrial world of genetic engineering. Therefore, we focused our human practices work on speaking with many stakeholders, which were mainly researchers in various microbiome fields, and businesspeople familiar with those fields.

Below, we have compiled how our original concept and idea was shaped and influenced by our discussions with these stakeholders.


Figure 1: People we talked with

At the beginning of our iGEM season, we designated a few team members as the “HP team”, which would be in charge of all HP activities. Very early on, we had a meeting with Omer Edgar, who was the HP team leader of iGEM TAU 2020. He helped introduce us to the idea of HP, told us about the successes and challenges of their team, and gave us useful tips on how to start working.

We began our work by surveying previous’ teams’ HP and presenting it to one another, summarizing the highlights that would help us plan our own approach. We particularly looked over teams that had won HP prizes in the past. In addition, we looked over teams that were also of the foundational advance track, which is a bit more general and therefore harder to assess stakeholders for and define a direct impact. This helped us see the possibility for incorporating integrated HP into our project and set us on our way.

After the research over previous HP iGEM team winners, we understood we need to decide on a model that will guide us through our work this year. Inspired by the model of Exeter 2019 and TAU 2020 iGEM teams, we decided to work according to the AREA framework for Responsible Innovation, which was originally created by Professor Richard Owen [6].


Figure 2: The AREA Framework

We implement this model by the following guiding questions:

Reflect: What did we want to know?

Engage: Who did we turn to?

Act: What did he/she tell us?

Anticipate: How did it affect our project?

We believe that working according to this framework will help us organize our goals, be prepared well for our meetings and ask the right questions. 

Soon after we had our idea, we started brainstorming with the whole team on possible implementation for our project. We came up first with the main microbiome fields our team was familiar with - the human microbiome, food microbiomes, soil and root microbiome - and did an initial investigation of the possibilities in each for using our software and beneficial genetic engineering.

At the time, there was a massive oil spill crisis ongoing on the shores of Tel Aviv [9], which made us reflect on how our project could be used to alleviate a problem of this importance in times of unprecedented ecological disasters. After further research on the topic, we realized here too that our project could be useful in oil degradation and bioremediation by microbiomes in the water.

Once we had our ideas in place, we began to define for ourselves clear goals for our future HP work, which were as follows:

  1. Better understand the practical needs and uses for our project
  2. Establish a connection between the design of our project with practical needs
  3. Gain insight on possible risks and our responsibility to manage them

In late March, we took our ideas to the Ignite Coller Startup Hackathon, where we had the opportunity to discuss our ideas and develop business plans and models together with local leaders in innovation, venture capitalists, start-up leaders, and more. It was here that we realized that industrially, our product may be most useful in the agrotech field, and in some aspects of our project, we focused on that as our “lowest hanging fruit”. For example, we ended up speaking with many researchers in the agricultural research community, we were able to find a lot of genomic data in the field, and once our software was near completion, we also did analyses on relevant genes. With the help of Hackathon mentors and organizers Eyal Benjamin, Or Kashi, Erez Gavish, and Ilanit Kabessa Cohen, we got started on a stakeholder analysis and definition of end-users. You can read more about this in our description of the Hackathon.

This, together with our goals, helped us come up with the big questions we wanted to answer about our project and where it fits in the real world. To answer these questions, we evaluated which local researchers and industry leaders we could talk to, and in the next months we met with many of them and integrated their feedback into our developing project.

Below you’ll find our big questions and where we took them.

The process of sharing genetic information between different organisms, also known as Horizontal Gene Transfer (HGT), is problematic when we use genetic engineering in order to change the behaviour of a bacterial community. Although it is usually believed that HGT happens only between bacteria, genetic information can be transferred between different types of organisms - fungi, animals, and plants can act as recipients [2,4]. When we engineer bacteria genetically, there is a possibility that HGT will happen and a new gene will be introduced to an unwanted organism, causing unpredictable side effects. These effects range from suboptimal essential gene expression to alteration of the evolutionary driving force by affecting the regulatory and physiological systems within cells [1].


Figure 3: Horizontal Gene Transfer in a transformed plasmid

In order to solve the issue, we thought about two possible methods: 

  1. Preventing HGT from happening
  2. Not preventing the HGT, but controlling the gene expression in the recipient organism

We chose to focus on the second method. However, before settling with the specific approach, we looked at the previous work that has been done by researchers to control gene expression at a microbial community level. 

As Prof. Itzik Mizrahi told us, researchers have been working on plasmid systems for organism-specific expression since the 1980s, but all of the solutions suggested are community-specific, so that there are no such systems which can fit different cases.

In addition, at our meeting with Dr. Jonathan Friedman, we learned about another successful approach in the field. Scientists suggested recoding the gene using synthetic nucleotides, which can be read only by specific transcription machinery. The approach is based on simultaneous transfer of a recoded gene together with the DNA polymerase which can read it. That way, even if the gene is transferred horizontally, no other organism can express it due to the lack of the transcription tools.

We understood that suggesting an alternative solution for gene expression control could be very useful - from one point of view, it is more general than community-specific plasmid systems, although it is more accessible than nucleotide recoding. Therefore, we decided to develop the idea of a software tool which encodes a gene of interest in order to optimize the gene expression in one group of organisms, and to deoptimize it in the other.

We understand the risks and biosafety hazards involved in engineering an environment directly (read more about this in Safety). Therefore, we found it important to understand that our project, which is designed to be used in situ, as well as in vitro, can be responsibly implemented. 

When engineering bacteria, we want to have as many safety nets as possible, which is why we decided to engineer plasmids, to which a kill-switch can be added to, rather than the chromosome directly, which is a more permanent edit.

When speaking with Dr. Yuval Dorfan, who works with soil and water microbiomes, he agreed that a solution which would incorporate a guarantee to stop the plasmids function would be useful in products such as fertilizers that would be spread directly in the environment.

Another one of the fears of GMO’s is the long-term and unknown effects of them in someone’s body or in the environment [12]. Therefore, we aimed for a project that doesn’t necessarily directly alter the genome of say, an edible plant, but rather the surrounding microbes which affect plant growth.

Implementation: With the advice of Dov Greenbaum (read more below in the Ethics section), we included the SafetyNet feature to our software, which scans inputted sequences for any possible pathogenic or toxic genes. You can read more about it on our Safety and Software pages.

As our approach ensures optimization as well as deoptimization of protein production, one of the possible research implementations is gene production optimization for just one specific bacteria. Dr. Yael Helman suggested us to build a test case on her research. In the lab, they work with a specific bacteria which they’re having difficulties transforming genes into. She claimed that our robust computation optimization approach could be a useful tool for them.

From the meeting with Dr. Nadav Kashtan we found out that one issue of researching live microbial interactions is not having imaging methods which can be used on live organisms under the microscope. Our tool can enable researchers to transfer a plasmid with a fluorescent protein into a microbiome sample, and to optimize its expression to one organism only – this way, only one type of organism will be colored inside the plate full of many different bacteria. This can be done simultaneously with 3-4 different plasmids each carrying a different fluorescent gene optimized for one of the species. 

From the same meeting (with Nadav), another idea came up. Nadav and his research group are working on a synthetic bacterial community. When it comes to fine-tuning the metabolic pathways of the community, there is a need for a tool which can make a local change, without affecting the surrounding bacteria. The above was suggested by Nadav as another test-case for Communique.

Implementation: After consulting with a bunch of researchers in the microbiome field, we understood that our project could prove useful to scientists researching and engineering bacterial consortia (read more about the meeting with Prof. Itzik Mizrahi). Scientists need a more selective method for engineering specific species while preventing expression in others, and while keeping conditions as close as possible to a natural microbiome. This selective engineering could, for example, allow for more efficient consumption of resources within species we want, while preventing consumption by other species. It would allow us to study the effects of different genetic modifications on different species within a microbial community, and use the learned knowledge to inform genetic modification decisions within microbiomes in the future. We believe that using our project would also ensure safer and more exact practices, by preventing the effects of horizontal gene transfer and even possible contamination.

Our technology of microbiome engineering fits in the pipeline after the stage of data analysis (read more in question pipeline), therefore the existence of data is critical for wide-scale use.

Natural microbial communities consist of tens or hundreds of bacteria. These communities are unique and vary between individuals, therefore data analysis should be performed case-by-case. In some cases, the identification of the microbiome composition is challenging, and there is not enough data about the microorganisms' genomes.

The main natural communities we chose to focus on for our proposed implementationare agriculture and the human microbiome. Data analysis in the agriculture field is quite developed in respect to the other fields, and there are sequenced genomes of many microorganisms in different microbial communities (roots, leaves, soil etc.)[5]. However, human microbiome analysis is more complicated; there are companies that are involved with this analysis, mainly of the gut microbiome, but this data is not available for external use due to ethical issues (read more in question H).

Unlike natural microbiomes, in synthetic microbial communities we know the composition of the community, and probably have enough available data. This makes food tech a good field for using our technology.

Moreover, we do not have information about interactions inside the microbiome, and there is no way to know theoretically how genetic change is going to affect the community. The only way to check that is to perform lab experiments.

In order to evaluate how relevant our technology is for the industry, we scheduled some meetings with researchers and stakeholders in the field.

Nissim Mashiach said that our project sounds very impressive and innovative. As a businessman, he recommended us to examine which companies in the field of microbiome engineering have succeeded and which haven’t, and what were the reasons for each. He said that without understanding the business environment, and why certain clinical trials failed/succeeded, it would be hard to know if a project is worth pursuing. He also shared with us his experience of dealing with gene editing regulation procedures with the FDA, and gave us more things to take into consideration with our final product.

Prof. Dov Greenbaum recommended for us to think more deeply about our product and what we might market it as (for example, are we releasing the plasmid or the software alone?). This would drastically change the regulations we would face and the road to a final released product. He brought up the question of what exact service we would be providing and suggested that we focus our energy on the responsibilities and regulations specific to that product or service. He also encouraged us to think about what exactly we would and should guarantee with our product (such as “100% efficiency”).

As we saw, there is not quite enough data about genomes within all the microbiomes in the world, and sometimes even the identification of the microbiome composition itself is challenging (read more in question F). Based on the feedback we got, we believe that once microbiome analysis will be more developed, our technology will be more valuable and relevant for industry as well. As of now, our technology can be implemented and useful in research and for some synthetic communities first. For example, Dr. Yael Helman suggested a collaboration, where she will give us relevant data of a plant and we will try to optimize a GFP to be transformed efficiently into its microbial community.

Moreover, in the meantime, we’ve managed to prove our project works only for a community composed of two microorganisms, and further scaling up is necessary for a natural microbiome, which can be composed of tens to hundreds of bacterial species. Prof. Elhanan Bornstein suggested several strategies of algorithm improvement, such as producing several optimized sequences instead of one, and recommended for us to add a tuning parameter that would give weight to the optimization degree in relation to deoptimization.

Implementation: After discussing with Prof. Dov Greenbaum, we narrowed down our idea to just a software, rather than a product that also includes sequencing analyses of microbiomes and producing plasmids ourselves.

We took into consideration Elhanan's comments, and added to the algorithm an option of optimization tuning parameter that is determined by the user. Our future plan is to continue improving the software according to the other comments in order to get a more optimized solution.

Since we do not intend to analyze microbial communities, and might still be distant from industrial uses, we also shifted our focus to making this a tool for mainly researchers in the near future.

The process that a customer goes through in order to make use of our solution in a real-world application is comprised of a few major steps:

  1. Sampling the bacterial community that they are looking to engineer
  2. Sequencing the bacterial community and identifying the species that make up that community.
  3. Identifying the species that they would like to engineer the vector into.
  4. Optimizing the vector according to the results found previously using our tool.
  5. Acquiring the optimized vector.
  6. Transforming the vector into the community.


Figure 4: Flow Chart of the Pipeline

There are a multitude of regulatory issues that arise from handling genetic material directly, making the infrastructure required for providing step 2 and step 5 quite cumbersome to set up and operate. Also, considering the fact that there are already existing companies that provide community sequencing services and others that provide DNA synthesis services, it is quite illogical to try and set up the infrastructure for those processes in-house.

Stemming from the reasons stated above, we believe that Communique’s role in the pipeline (outlined above) should be to provide the customer with consultation services about which species to engineer in the community and providing the optimized sequence to the customer (steps 3 and 4), leaving the sampling and sequencing along with the synthesis of the vector itself to other entities, which are already specialized in those fields.

Our meeting with Prof. Dov Greenbaum helped us ask ourselves and answer many questions related to ethics. The first is surrounding the responsibility that comes with creating any gene editing tool - what responsibility do we, as the creators, have to make sure our product can’t be taken advantage of and used to do harm? Cyber-bio hacking is becoming an increasingly concerning issue as we delve further into the progression of gene editing and computational abilities [7]. He suggested that we consider adding some sort of additional check into our software that might prevent perverse use of it.

Dov also encouraged us to think about where we’re getting our data from and whether or how we’re keeping it, who has access to it, and what type of responsibility we have with that data. As our algorithms are built on analysing data inserted by the user, there could be an ethical issue regarding confidentiality and privacy if we were to keep the data.

In addition, he suggested we think about who the software might be available to, and who should it be available to? Balancing between the accessibility and restrictions will lead to a safer usage of our technology. 

Implementation: After speaking with Dov, we understood that considering ethical questions is a big part of almost every synthetic biology product, and even more so in our project, which lies on a borderline with genetic modification.

We realized that it was critical to add some kind of safety check into our software. Soon after, we found out about iGEM Heidelberg 2017’s SafetyNet software[11]. We set up a meeting with Julius Valten from the Heidelberg team, who helped us implement the safety scan in our model to ensure that no pathogenic sequences would be optimized by our software. You can read more about how we integrated and updated their work on our software page.


Figure 5: SafetyNet software created by the Heidelberg iGEM team 2017

We should consider including cybersecurity algorithms into the software, which will decrease the possible hijacking and usage for the worse. Although the data should be saved locally in order to perform an analysis, we can consider deleting the user’s data right after finishing the calculations. In terms of accessibility, we could give access to universities and Research and Development groups in the industry, rather than making it open-source for all. In order to make the process even more open and safer, using preregistration of the experiments should be considered.

It is important to find a right balance between business considerations and ethical questions. Only this way we will drive science forward without being afraid of usage for the worse.

Our team is based in Israel, which adopted a mixture of the American and European lines into its regulations. The Israeli Health Ministry generally allows any growth of genetically modified organisms inside the lab. However, the regulations for the commercial growth and distribution of GMOs, including microorganisms, are way harsher [3].

In order to commercially grow and distribute products modified by our software in Israel, any new product line containing modified genetic or protein material must go through a long, expansive list of safety checks. Products approved by the committee of new food of the health ministry and the grand committee of modified plants of the agriculture committee may distribute their products without a special mark.

We spoke with Nissim Mashiach of Nubiyota, who told us about some of his challenges passing gene modification products in the US. He told us that as the field is still emerging, rules and regulations are being passed as new problems come up.

That being said, while we are certainly dealing in the realm of GMO’s, our product is not per se GMO, as we are not the facilitators or sequencers of the genes themselves, but rather provide the tools. This would suggest quite different and probably less strict regulations. In addition, we would argue that engineering the root and soil microbiome of crops would not be considered as “GMO crops”, as the plants themselves aren’t modified.

There is plenty of prejudice regarding genetic engineering. A lot of people perceive genetic modification as a threat only and not as a helpful tool [8,10]. By exposing more groups of people to possibilities of genetic modification, we were able to better educate, answer questions, and increase awareness of the positive outcomes of genetic engineering. Today, speaking of GMO’s in a positive way is a kind of taboo, so one of our goals was to give another perspective on GMO’s and to present it as something less threatening than how it is perceived. We have met with different groups of a wide community, from youth to elderly, and explained to them about the scientific background of genetic modification in general and our project in particular. It was important for us what these groups think about our project and what concerns they have regarding it. For an in-depth description of our educational activity, look in the Educationsection.

Other ways to spread our project and ideas to the community were our Instagram and Facebook pages. We kept our followers updated with our progress and the experiments we made. We also followed other iGEM teams and searched for collaborations via social media. 

  1. D. A. Baltrus, “Exploring the costs of horizontal gene transfer,” Trends in Ecology & Evolution, vol. 28, no. 8, pp. 489–495, 2013.
  2. G. Ã. L. Rosano and E. A. Ceccarelli, “Recombinant protein expression in escherichia coli: Advances and challenges,” Frontiers in Microbiology, vol. 5, 2014.
  3. Health.gov.il. [Online]. Available: https://www.health.gov.il/UnitsOffice/HD/PH/FCS/NovelFood/Pages/EngFood.aspx. [Accessed: 19-Oct-2021].
  4. M. E. Inda, E. Broset, T. K. Lu, and C. de la Fuente-Nunez, “Emerging frontiers in microbiome engineering,” Trends in Immunology, vol. 40, no. 10, pp. 952–973, 2019.
  5. P. Hunter, “Plant microbiomes and Sustainable Agriculture,” EMBO reports, vol. 17, no. 12, pp. 1696–1699, 2016.
  6. R. Owen, J. Stilgoe, P. Macnaghten, M. Gorman, E. Fisher, and D. Guston, “A framework for responsible innovation,” Responsible Innovation, pp. 27–50, 2013.
  7. R. Puzis, D. Farbiash, O. Brodt, Y. Elovici, and D. Greenbaum, “Increased cyber-biosecurity for DNA synthesis,” Nature Biotechnology, vol. 38, no. 12, pp. 1379–1381, 2020.
  8. S. Blancke, “Why people oppose gmos even though science says they are safe,” Scientific American, 18-Aug-2015. [Online]. Available: https://www.scientificamerican.com/article/why-people-oppose-gmos-even-though-science-says-they-are-safe/. [Accessed: 19-Oct-2021].
  9. S. Paget, “Oil spill leads Israel to close beaches as it faces one of its 'most severe ecological disasters',” CNN, 22-Feb-2021. [Online]. Available: https://edition.cnn.com/2021/02/22/middleeast/israel-oil-spill-ecological-disaster-intl-hnk/index.html. [Accessed: 19-Oct-2021].
  10. T. C. Wallace, “Opinion: It's time to end fake news about GMO foods being unsafe,” Journal, 26-Dec-2019. [Online]. Available: https://www.courier-journal.com/story/opinion/2019/12/26/gmo-vs-organic-consumers-being-fed-fake-news-food-safety/2708255001/. [Accessed: 19-Oct-2021].
  11. Team:heidelberg/software/safetynet. [Online]. Available: https://2017.igem.org/Team:Heidelberg/Software/SafetyNet. [Accessed: 19-Oct-2021].
  12. “Will gmos hurt my body? the public's concerns and how scientists have addressed them,” Science in the News, 17-Jan-2021. [Online]. Available: https://sitn.hms.harvard.edu/flash/2015/will-gmos-hurt-my-body/. [Accessed: 19-Oct-2021].

Dr. Jonathan Friedman

September 14, 2021

1. What did we want to know:

- Which scientific considerations need to be taken into account with a project like this? - What regulations are associated with GMOs/genetic engineering?

2. Who did we turn to:

Dr. Jonathan Friedman, Postdoc Center for Physics of Living Systems, MIT; PhD Computational and Systems Biology, MIT; BSc Physics and Biology, Tel-Aviv University. His main area of interest and expertise is microbial community dynamics.

3. What did he tell us:

- Dr. Friedman is a big fan of iGEM and was fascinated to hear about our project. He mentioned that we should consider changing a bit the presentation of the project, and redefine the problem we want to solve. - He told us that GMO regulations in Europe are generally not clear.

4. How did it affect our project:

We took into consideration his advice regarding the presentation, and changed it accordingly.

Dr. Yael Helman

September 1, 2021

1. What did we want to know:

- How much is known about/what kind of data exists for the type of microbiomes she works with? - How hard/easy is it to elucidate the composition of a certain microbiome? - What kind of examples could there be for engineering specific species within the microbiome? - Key aspects to look out for in agro-tech engineering? - Regulation of agro-tech microbiome engineering? - What GMO regulations should we take into account?

2. Who did we turn to:

Dr. Yael Helman is the principal investigator of a lab that investigates microbial interactions in the rhizosphere, biological control of plant pathogens, and mechanisms of bacterial stress responses at the Faculty of Agriculture at Hebrew University. We spoke to Yael about possible uses for our project and what kind of data exists for the type of microbiomes she works with.

3. What did he tell us:

Optimization of expression levels of plasmids is already very important in industry, so she thinks that this project could be very important in industry as well. She also says that inserting DNA into plasmid, even GFP, is not always so easy and that there are a lot of researchers who try and don’t manage. She thinks that if we manage to make genes transformable into bacteria that have a hard time with transformation, it’s already a good use for researchers. In addition, Yael was happy to give us the genome to try as a use-case, and offered to try our software in her lab! She also raised a few concerns: In our checks, we did gram-negative and gram-positive bacterias, which are very different from each other. In nature, even in synthetic environments, the similarity between the different species is much larger and she doesn’t know whether the codon usage and even the will be different enough as we show in our model.

4. How did it affect our project:

We did an analysis of our software and model to check how an increasing phylogenetic distance affects the optimization abilities. You can read more about this in our Software section.

Dr. Yuval Dorfan

August 24, 2021

1. What did we want to know:

- How can we move the project from the lab into the industry? - What regulations are associated with GMOs/genetic engineering in Israel? - Who could we talk to that could help us with further design, use-case, regulations, bioethics, etc. of this?

2. Who did we turn to:

Dr. Yuval Dorfan from the founding director of the Israeli SynBio Institute (ISI), and a lecturer at the new Innovation Center at the Interdisciplinary Center (IDC), Herzliya, Israel. We turned to Yuval in order to understand better how our project can fit into the industry and in which ways it can help companies. In addition, Yuval is also a research fellow at IDC’s Zvi Meitar Institute (ZMI), where he deals with regulation and bioethics questions relating to new SynBio applications. He referred us to Professor Dov Greenbaum, his colleague, for consultation in this area.

3. What did he tell us:

He connected us with his colleague, Professor Dov Greenbaum, to talk about GMO/bioethics. He said that the project is interesting from the research point of view, however, practical applications would possibly come in the long term. Yuval was interested in how our project might be used for biosensing and was interested in collaborating in the future.

4. How did it affect our project:

Thanks to Yuval, we eventually met with Prof. Dov Greenbaum and got to talk to him about regulations and GMOs. In addition, we scheduled a visit to the ISI at Reichman University at the end of October, in order to see and hear more about their projects and ideas, and understand better where our project can fit in.

Dr. Lianet Noda

October 2, 2021

1. What did we want to know:

- How much is known about/what kind of data exists for the type of microbiomes she works with? - How hard/easy is it to elucidate the composition of a certain microbiome? - What kind of examples could there be for engineering specific species within the microbiome? - User control over our software - Appropriate analysis and use cases

2. Who did we turn to:

Dr. Lianet Noda-García is the principal investigator of a lab that investigates biofilm formation using directed and experimental evolution of bacteria in the Faculty of Agriculture at Hebrew University. We spoke to Lianet about our software analysis use case, both in terms of the microbiome itself and the selected gene for it.

3. What did he tell us:

Similar to Dr. Yael Helman, Lianet stated that optimization of expression levels of plasmids is already very important in industry, so she thinks that this project could be very important in industry as well. However, she did highlight the importance of optimization of the origin of replication in our future versions of the software. In addition, she said that the use of soil microbiome was a good idea and offered us the following options for genes to optimize: * anti-phage defence genes * genes in biochemical pathways unique to the organisms in our community * enzymes related to phosphorus and nitrogen recycling

4. How did it affect our project:

We decided to optimize the ZorA anti-phage defence system in order to test the performance of our software on a reasonable and potentially useful study case. You can read more about this analysis on the Software page.

Dr. Eyal Benjamin

March 3, 2021

1. What did we want to know:

How do we find an industrial application of the project with the highest market potential?

2. Who did we turn to:

Dr. Eyal Benjamin from Tel Aviv University is a 5X serial entrepreneur, founder of automotive ventures and ICT, with a venture capital investments background. He holds a B.Sc. in engineering from TAU, Tel-Aviv Israel; Masters of Entrepreneurship and Innovation; and a PhD in new-venture-strategy both from SUT, Melbourne, Australia.

3. What did he tell us:

After talking about possible applications for the project that we considered, Dr. Benjamin told us that, in his opinion, the agrotech industry is the “lowest hanging fruit” for us to achieve. Given the huge size of the agrotech market, even a small improvement of a plant’s growth rate might drastically increase the yields and the financial outcome. During the talk, we settled up with the idea of “personalised agro-therapy”: using the information about the microbial composition of the plant, we are able to develop a bio-fertilizer, which contains a plasmid specific for the plant microbiome. Proper usage of this technology might lead to the enhancement of a plant’s growth rate.

4. How did it affect our project:

Understanding that our project has a big potential in the agrotech industry helped us focus our subsequent HP work on the agricultural field: we turned to many researchers in the agricultural microbiology, and we set a goal to test our software on real data from this field.

Ilanit Kabessa-Cohen

March 3, 2021

1. What did we want to know:

We turned to Ilanit during the Hackathon, right after settling up with the agrotech industry as a preferred field for a use-case. We discussed with her the following: - How do we define our end-users? - Where exactly in the personalised biofertilization pipeline does our project stand?

2. Who did we turn to:

Ilanit Kabessa-Cohen is a venture partner in NovoProteins by Novozymes and Agri-food technology advisor in DAF, Dole Asia Holdings Pte., Ltd.

3. What did he tell us:

Ilanit helped us understand that defining farmers as our end-users is not practical. Therefore we discussed the possibilities of working with companies, which produce biofertilizers, instead, and define these companies as our end-users. In addition, during the talk we understood that there are two different areas in the biofertilization industry: microbiome analysis and microbiome engineering. It was important for us to figure out that our project is relevant for the engineering area only, so we should use the data about microbiome composition from outside. This way, our place in the biofertilizer production was formed: we bridge the gap between the analysing companies and the companies which produce the fertilizer itself.

4. How did it affect our project:

We understood that our project should use information about microbiome composition. Therefore, we focused on finding databases in the field and contacting researchers which could have analysed such information themselves, in order to test our software.

Liam Kimel

June 4, 2021

1. What did we want to know:

Wanted to understand what our business looked like from the perspective of someone in the biotech field, as well as feedback on our presentation skills and ideas on how to move forward the business side.

2. Who did we turn to:

Liam Kimel of Sanara Ventures.

3. What did he tell us:

Liam asked us many valuable questions throughout our presentation, which gave us a better idea of what we were missing in our presentation or in our overall thought process of the project. Because he has a background in biotechnology, he also asked practical questions about the functionality of the software itself. In addition, he gave us ideas for the implementation of our product, as he has much experience with researching technology which is available (or yet unavailable) in the field. He suggested some business moves we could make to take forward our ideas, such as how to find end-users, how to do a landscape analysis, growth projection, minimal viable product, and recommended we add an “action points” slide to our presentation as well.

4. How did it affect our project:

After discussing with Liam, our initial product presentation under went a lot of change due to his feedback. Being a person that has been presented to many times, we really appreciated his input and added as much as we could. We also followed some of the leads he gave us for both sponsorship as well as Human Practices. Liam also brought up the biosensing idea for the first time, which we added to our Implementation section after further research and discussion. Mostly, Liam helped us take a better business-minded approach to our project.

Dr. Nadav Kashtan

September 12, 2021

1. What did we want to know:

How can the project be implemented into microbiome research? In respect to the model, how can we improve the optimization? Can we overexpress a gene and see what changed in the microbiome? Maybe it can tell us about some interaction pathways in which this gene plays a role. (knock-in or knock-out with transposase).

2. Who did we turn to:

Dr. Nadav Kashtan from The Hebrew University of Jerusalem uses systems biology to study the ecology and the evolution of microbes. We consulted with Dr. Kashtan about the scope of our project and heard his feedback about our POC.

3. What did he tell us:

This project has many options on the horizon, as it is an elegant solution and a relevant project to work on. Regarding agrotech, the plant microbiome is less known than the human microbiome. There is a need to understand exactly what and how different dysbiosis affect the plant microbiome.

4. How did it affect our project:

We came up with the idea to use our product to study the interaction of genes in a microbiome (like knocking out gene in order to study its function in a pathway, but within a microbiome). Nadav also sent us some articles including genomes of plant microbial species, which we ended up using for our software analysis.

Omer Edgar

February 2, 2021

1. What did we want to know:

How to present to companies and improve our professionalism?

2. Who did we turn to:

Omer Edgar, IGEM TAU 2020 Human Practice team leader

3. What did he tell us:

Omer shared with us some insights from his work in the TAU 2020 team. He recommended us to use AREA and ICE-PCR frameworks, in order to establish our attitude to different aspects of the projects. Moreover, Omer gave us some efficient tips on documentation and presentation processes.

4. How did it affect our project:

With Omer’s input, we were able to kickstart our Integrated HP work and understand what iGEM was looking for, as well as what Human Practices is exactly.

Nissim Mashiach

September 5, 2021

1. What did we want to know:

- How does a project like this move from the lab to industry? - What regulations are associated with GMOs/genetic engineering, especially with human microbiome engineering? - Which scientific considerations need to be taken into account with a project like this?

2. Who did we turn to:

Mr. Nissim Mashiach, CEO of NuBiyota - a microbiome therapeutics company. He holds an MBA from the University of Manchester, an MPharmSc from the Hebrew University of Jerusalem and a B.Sc, Chemical Engineering from the Technion-Israel Institute of Technology in Haifa, Israel.

3. What did he tell us:

- Nissim said that our project sounds very impressive and innovative. As a businessman he recommended us to examine which companies in the field of microbiome engineering have succeeded and which haven’t, and what were the reasons for each. He said that without understanding of the business environment, and why certain clinical trials failed/succeeded, it would be hard to know if a project is worth pursuing. - He said that the whole microbiome field is difficult because it’s very new, regulators haven’t yet managed to specify clear rules, and it’s still being developed. In his company, they interact with the regulators and ask for feedback in every stage of the progress. The FDA is sensitive to consistency and reproducibility which are key values in pharmacy and medical products, and they want to see 'microbial purity'. This is what makes personalized medicine very expensive and complex, and that's why he thinks that as long as there is a generic, alternative solution, they would prefer it. - The method NuBiyota uses to engineer the microbiome is different from our computational method. Nissim doesn’t consider himself as an expert on the computational aspect, so he offered to connect us with other genetic experts/bioinformatics that he works with. He thought we should talk to them once we get further, in order to get more specific feedback.

4. How did it affect our project:

Nissim helped us think about our project from biotech business aspects and consider which regulations we might face and how.

Professor Avigdor Eldar

February 2, 2021

1. What did we want to know:

We wanted to present the general concept of our idea and get some practical and technical feedback from experts: - How is working with B. subtilis and E. Coli? - What other bacterial species might be relevant for us? - Which plasmid could work well for both? - What other selective factors could be used? - Ask for protocols. - What implementation might this have?

2. Who did we turn to:

Professor Avigdor Eldar of the Faculty of Life Sciences at Tel Aviv University. His lab studies quorum sensing and the design principles of cooperative behavior in bacteria. Avigdor also has a background in mathematics, astrophysics, and systems biology.

3. What did he tell us:

Avigdor was happy with our idea, and turned us to several other researchers he knew who could give us good feedback. He was mainly worried with how phylogenetic distance could decrease the optimization abilities, and how we could get high selectivity. He came up with the idea for using CRISPR and miRNA as highly selective features that could increase the specificity, and we developed many ideas together in this first meeting.

4. How did it affect our project:

Avigdor and his students became constant supporters of our project; they provided us with the bacterial strains and plasmids we used, as well as materials and supplies when we were out. While we decided that we shouldn’t change our whole project to focus on CRISPR and miRNA, we did incorporate them into our future plans as additions we would like to develop at some point.

Professor Dov Greenbaum

September 19, 2021

1. What did we want to know:

- Which ethical considerations should we take into account? - Different applications mean different regulation policies. What path is the easiest one? Is one application better for our project in terms of regulation? - What is considered a product? Just a plasmid or the program itself? What do we need to approve? - GMO regulations - is microbial engineering considered GMO, even without changing the chromosomal genes?

2. Who did we turn to:

Prof. Dov Greenbaum directs the Zvi Meitar Institute for Legal Implications of Emerging Technologies, he is a licensed attorney before the State of California and the United States Patent and Trademark Office, and is a Certified Information Privacy Professional. Following the conversation with Dr. Yuval Dorfan, we talked to Prof. Greenbaum about GMO and ethical regulations. We tried to better understand how to define our project- a plasmid or a software, what regulations we should take into account in each application of our project.

3. What did he tell us:

Dov said we should think about how to define our project- the software or the plasmid? A lot of jurisdictions don’t have regulations, meaning one person could say it’s not GMO but there’s a huge GMO component that’s being used to create it, so someone else could decide to label it as GMO anyways. He also came up with another issue - when you work with these algorithms, there’s a whole fear of bio/cyber security; there’s an interesting aspect when developing DNA algorithms; someone could hijack the algorithms and use it to mask something else that they’re putting in there. He raised the following questions about the data we will get from our software: - What information do you keep? - Where are we keeping the data? - Who is the data available to? Should it be accessible by everyone, or should it be limited? Who should have the tools to play with DNA? - If you’re collecting all the data on the backend, how are you protecting it?

4. How did it affect our project:

We integrated the SafetyNet idea and DeepProtein model into our software, so we can scan the edited gene and prevent optimization of potential pathogenic genes and toxins. That way, we can make our software more safe to use. Read more about this in our Software section.

Professor Elhanan Borenstein

September 9, 2021

1. What did we want to know:

What are the possible implementations of our project in the microbiome field? How much data is available on the human microbiome? What are the ethical concerns?

2. Who did we turn to:

Prof. Elhanan Borenstein from Tel Aviv University. Prof. Borenstein focuses on computational study of the human microbiome and of other complex microbial ecosystems.

3. What did he tell us:

First, There is a huge gap between our POC (testing only two species separately) and our vision of dealing with a whole microbium, mainly because we don’t know the connection between two species within a microbiome. He also said that computationally, he has more worries about the scaling up (for example bringing up 10 spesies instead of 2). Regarding the models, he said we could do some tuning parameters, and add something that would differentiate between monoculture and coculture. Besides, he thinks we should define microbiome interactions, as well as the experimental side, and show that the prediction of the algorithm is relatively similar to the experimental results. As to the software, he thinks we can try and think of a way to insert some type of interaction or information with the user. In regard to regulations, he pointed out that our solution might be slightly problematic, because we need to plan a plasmid per patient per microbiome species. The ability that we’d be allowed to regulatory acceptance and approval to make a different plasmid per patient are very low.

4. How did it affect our project:

We integrated letting the user add other preferences in the software, besides the genome which is required.We also added the tuning parameter, which allows users to choose whether they prefer optimization or deoptimization. Furthermore, the analyses looked at group sizes and phylogenetic differences, and we came up with a future plan for integrating plasmid families into the software.

Professor Itzik Mizrahi

April 29, 2021

1. What did we want to know:

What work has been previously done in the field?

2. Who did we turn to:

Prof. Itzik Mizrahi, Principal Investigator of the lab of microbial ecogenomics.

3. What did he tell us:

Scientists have been working on this idea since 1980’s, but up to date there is no robust solution available. One of the most important problems was an inability to deliver a plasmid to a wanted host. There were some system-specific methods invented, but none of them can work with any other system. So, there is an urgent need in developing a “one-fit for all” tool. Another suggestion of Prof. Mizrahi’s was to take the phylogenetic distance between organisms into consideration - how close to each other the bacteria in the POC are going to be. Two points of view were proposed: - If the organisms are very distant one from the other, the POC will not carry enough information, because the reducing the similarity of an organism reduces the probability of the plasmid expression in both organisms at the same time - If the organisms are very close to each other, we could come to the resolution which is too small for the method

4. How did it affect our project:

Overall, Prof. Mizrahi gave us a confirmation of our idea’s relevance for academia and industry. Moreover, we took into account the phylogenetic resolution of the POC, but nevertheless we decided to stay with the initial plan, and to show the effect on E.Coli and Bacillus first, with a future goal to perform experiments on different types of bacteria, considering the phylogenetic distance.

Communique

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igem.tau.21@gmail.com

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