Abstract

Plasmids are commonly used for various purposes and are a vital tool in the hands of a synthetic biologist. We have met with different stakeholders to understand the difficulties they experience when working with plasmids. Among others we met researchers from various institutes around the world, teams from biotech companies and bio-foundries, and entrepreneurs.

Through those meetings, we have learned that the ability to control plasmid copy number is very limited even for model organisms such as E. coli and could be valuable for various purposes.

We adapted our initial idea based on the academy and industry experts' feedback. Throughout the project, we constantly reached out to new companies and researchers to expand our understanding and improve our solutions.

Additionally, we have filed a provisional patent application related to our model.

Introduction

As part of our Human Practices, we recognized the importance of seeking expert advice. We reached out to experts from various faculties at our university, Tel Aviv University (TAU), including Life Sciences, Engineering, and Medical Science. Among them our steering committee that includes prof. Martin Kupiec, prof. Itai Benhar, prof. Avigdor Eldar and prof. Uri Gopna.

We also engaged with industry experts with extensive experience working on projects involving plasmid copy numbers.

During our consultations, we engaged in scientific discussions with researchers who possess expertise in plasmids, specifically focusing on copy number. These discussions allowed us to delve into the intricacies of our project and gain valuable insights.

Furthermore, we actively engaged with business experts and entrepreneurs to assist us in developing a realistic and practical business model, conducting market studies, and exploring potential opportunities for the successful launch of our product into the market. Through these interactions, we aimed to gather a wide range of perspectives and expertise, ensuring that our project considers both scientific and business aspects in a well-informed and practical manner.

Entrepreneurship workshop

Learning how to properly engage with stakeholders

In the early stages of our project, we understood that simply engaging with stakeholders isn’t enough. We must first learn how to do it properly. Otherwise, we might arrive at wrong conclusions about what is desired. To better understand how to communicate our project to different communities and various stakeholders and how to listen and learn from their feedback, we first met with Yair M. Sakov, founder and managing director of Tel-Aviv University Entrepreneurship Center (figure 1).

*Figure 1: Yair Sakov, founder and managing director of Tel-Aviv University Entrepreneurship Center.*

To get Yair acquainted with our project and with the field of synthetic biology, we conducted two preliminary meetings in which we elaborated on our project, presented our findings regarding the potential impact of our project as we see it, raised certain assumptions that we based our idea around and much more (figure 2). Following our two meetings, Yair was able to understand exactly what are the things that would be beneficial for us to learn before heading out and communicating our project to the outside world.

The main subjects we discussed were value proposition and how to conduct a client interview. In entrepreneurship, the value proposition is a concise statement of the benefits a company delivers to customers who buy its products or services. The same principle remains true when thinking about our iGEM project. Yair explained that for our project to be successful, we must first understand what exactly it is that we are offering.

He also taught us how to do a client interview taking into account insights from the “mom’s test” guide by Rob Fitzpatrick, which talks about ways to tell whether a business idea is good by assessing the opinions of different stakeholders and not believing everything they say just to make one feel good- “It boils down to this: you aren’t allowed to tell them what their problem is, and in return, they aren’t allowed to tell you what to build. They own the problem, you own the solution.” With this understanding we were able to articulate a set of questions to ask potential clients. The knowledge we've acquired and the set of questions we've assembled helped us communicate in our next meetings with several companies that could be potential clients in the future.

*Figure 2: Our team members at the entrepreneurship center.*

Provisional Patent Application

We have worked with Ramot, the tech transfer center of Tel Aviv University, in order to file a provisional patent application on our models. We learned a lot from the process on intellectual property, tech transfer, and patent law (figure 3).

The Industry Perspective

We have reached out to several companies to better understand the SynBio landscape and the challenges they face and asked for honest feedback and advice on our ideas and assumptions.

Alagene

We have reached out to Alagene - the first bio-foundry in Israel. Alagene offers End-to-End SynBio solutions in FoodTech, AgTech, Environment, Pharma, Cosmetics, Textile, Chemicals, BioFuels, Diagnostics and more. Alagene serves customers by offering an end-to-end development pipeline (figure 4) that includes:

A computational design module that employs data science, structural biology, and bioinformatics to generate data and AI-driven predictions.
A construct & host engineering module that utilizes genetic engineering procedures and automation to streamline the construction of genetic libraries.
An optimization by automation and analytics module that makes use of automation to screen numerous variants quickly, and analytic methods determine analyte structure and quantity.
A precision fermentation, pre-production, and POC module that is taking the genetic optimization effort towards commercialization by complementing superior genetics with process development and completing a POC.

Alagene’s pipeline — *Figure 4 Alagene’s end-to-end pipeline.*

Their vast knowledge made them a great source of knowledge for us on industrial SynBio projects and pipelines and also for the challenges in SynBio today, and where our solution can fit in. We had several meetings with Alagene.

The first meeting was with Dr. Or Rotem, the CTO of Alagene. We have interviewed him on the SynBio landscape in Israel and the challenges in the field. We learned a lot from the interview and wanted to spread this information to a wider audience, so we created, in collaboration with Alagene, an interview paper that we published.

Interview with Alagene

We also had a meeting in which we asked Alagene about their challenges and insights on plasmid copy numbers and introduced our project to get feedback. The meeting was in their offices, where we saw interesting devices to automate biological experiments.
During our meeting, we learned that our solution is relevant for bioproduction, biosensors, and bioremediation. They also pointed out that E.coli with puc19 plasmid is used a lot for POC purposes and encouraged us to expand our tool to ColE-like plasmids (figure 5).

The meeting provided valuable insights for enhancing our project in the future. It became evident that to improve the validation of our model, we must incorporate a more extensive set of biological data. We recognized the importance of gathering diverse sequences and analyzing them for model validation. After this meeting, we decided to collect and analyze plasmid sequence from various organisms to understand the potential hosts of ColE-like plasmids In addition, this meeting showed us the significance of robust model validation, which has subsequently influenced our approach to data analysis (see more details in the Model page).

Phibro Animal health

We contacted Phibro Animal Health, a leading company dedicated to advancing animal health and nutrition. With a focus on livestock, poultry, and aquaculture industries, Phibro is committed to developing innovative solutions that enhance the well-being and productivity of animals. Through years of expertise, Phibro has established itself as a trusted partner for those in the agricultural sector, providing essential tools to support the health and performance of diverse animal populations.

We had the privilege of meeting with Dr. Katya Sodolevsky, an R&D scientist at Phibro (figure 6). Our meeting was structured into two parts, with the initial phase consisting of a client interview. During this session, our objective was to delve deeper into Phibro's operations, gaining insights into the challenges they encounter in working with plasmids, understanding the pivotal role plasmids play in their products, and exploring additional facets of their processes.

We wanted to know whether E.coli is used as a chassis in a Phibro’s line of products and if not why? Katya explained to us that E.coli is a great chassis, due to the abundance of knowledge and tools available to those who wish to work with it, for this reason, working with E.coli is often considered the default approach, however in recent years Yeast and Fungi are proving to be better suited for certain procedures, with Fungi enhancing the yield of certain processes substantially. With that being said, E.coli is still highly used for both research and proof of concept purposes.

We also inquired about the usage of plasmids versus editing the chromosome and inserting target genes. In her response, Katya laid out the benefits of working with plasmids. The two major aspects she mentioned were the simplicity of using a plasmid instead of editing the chromosome and the added level of control that comes with working with a plasmid, in other words the ability to alter the plasmid to suit their specific needs. This response didn’t surprise us, however we wondered if all those benefits are even relevant to the production of vaccines. Our main concern was the fact that retaining a plasmid usually requires antibiotics and we assumed that this would prove problematic for meeting the desired safety and regulation criteria. We were surprised to learn that purifying the antibiotic from the product is pretty simple and doesn’t take away a significant portion of the yield, however the main risk regulators are afraid of is actually remnants of the plasmid. The reason for this is that it could result in an uncontrolled spreading of antibiotic resistance genes, which were used as selection markers for the plasmids. To avoid this, manufacturers should show significant evidence that proves that their final product doesn’t contain any remnants of plasmid and bacterial DNA, but with all that being said, Katya still emphasized that most of their manufacturing process involve plasmids, meaning that the benefits greatly outweigh the drawbacks. Thus, following this meeting we added ‘vaccine development’ as one of the major field related to our platform.

In the second part of the meeting, we presented our project. Our goal was to receive feedback both on the idea itself and on our presenting skills, with the clarity at which we deliver our project being the most important metric for us. Katya was impressed with our presentation, she mentioned that even though she lacks training in AI models, she was able to understand the core principles of the way our model works. In terms of the project itself, she commented that the ability to control the plasmid copy number, simply by modifying the plasmid is indeed intriguing and could be extremely beneficial in avoiding cell toxicity while still maintaining a decent level of expression. She also pointed out that showing the potential of such regulation in E. coli could lead to the need to develop similar tools for different plasmids and different hosts. This has deepened our realization that to enhance our project's efficacy, we must aim to broaden its applicability to plasmids and host organisms beyond ColE1 and E. coli. This objective now stands as a central goal for our future efforts and was verified through an evolutionary perspective as detailed below (see more details in the Model page)..

Twist Bioscience

Twist Bioscience is a public biotechnology company based in South San Francisco that manufactures synthetic DNA and DNA products for customers in a wide range of industries, as can be shown from their website below (figure 7).

Our meeting with Esteban Toro, Senior Director of R&D at Twist Bioscience, proved to be significant for our project (figure 8). Mr. Toro talked about the different services that Twist bioscience offer that are related to plasmids, and potentially to copy number control. His interest in our work and his insights into the preferences of Twist Bioscience's clients were interesting. He mentioned that clients usually tend to prefer high copy numbers for their applications. This realization has provided our project with a valuable direction for the future, focusing on the control and optimization of plasmid copy numbers within the higher range. This brought us, while analyzing our results, focus on the aspects and influences of the high copy number plasmids on protein levels and growth rates (see more details in the Results page). Furthermore, Mr. Toro's mention of Twist Bioscience's services related to plasmids has opened up opportunities for collaboration and access to additional resources that can further advance our project's development and impact. Overall, our interaction with Twist Bioscience has significantly enriched our project's perspective and potential.

Lonza

Lonza is a global partner for pharmaceutical, biotech, and nutrition industries, dedicated to advancing treatments that promote health and wellness. They leverage scientific innovation and manufacturing technology to empower their customers in delivering solutions for patients and consumers.

we conducted a zoom meeting with lonza representatives around the world -among them Frida Grynspan, Head of Lonza Collaborative Innovation Center in Israel, Armin Baumschlager, Synthetic biologist in zurich switzerland, Bernie Sweeney, group leader in england and Karl Heinz, head microbial in Bern switzerland (figure 9). In the meeting we presented our project while answering great questions of the participants regarding the replication system and the model training. A question that caught us in particular was whether we can control the copy number on the fly, i.e. in dynamic systems. We explained that currently the system is static, but in the future it will be interesting to think about such mechanisms. It has inspired us to explore plasmid copy numbers while taking into account dynamic scenarios. In addition, we had a discussion about the fact that copy number measurement is particularly heterogeneous since it requires the analysis of a population and not a single cell. We talked about the fact that there is variability within the population in certain ranges, and that this is something we would like to learn more about in the future and be able to control possibly in collaboration with Lonza. At this point, we decided to plots graphs that includes the variability of some of the measurements for various copy numbers. We were very happy to hear that Lonza were also very impressed by our project and donated money to support us. This support contributes significantly to our project.
Thus, this interaction with Lonza representatives has contributed significantly to our project's evolution.

Academy Perspective

We have reached out to Manel Camps, Shay Tal, and Miles Rouches experts in plasmid copy number research (figure 10) and asked them for advice and insights. The meetings were highly interesting, and we learned a lot about the complexity of the biological mechanism. Besides a lot of meetings with professors from our university, we have reached out to world-wide experts in plasmid copy number regulation.
Here we highlight few of our meetings:

Dr. Shay Tal, Israel Oceanographic & Limnological Research

We met with Dr. Shay Ta from Ben-Gurion University of the Negev at the early stages of our project. Our main goals were to understand which model organism is feasible for a POC, and to learn more about the research on copy number regulation mechanisms.

Dr. Shay Tal has a lot of experience with plasmids. In his lab(https://lifewp.bgu.ac.il/wp/talshay/) , he characterized the plasmid population (plasmidome) at different marine locations, analyzed the functional contribution of the plasmids to the ecosystem and study the dynamics of the plasmidome under various conditions.

We consulted with Dr. Shay Tal about our initial idea and consulted with him which model organism to choose for our project. Initially we considered E. coli, Bacillus and S. cerevisiae. He explained to us that using E. coli is more feasible for tuning copy numbers because there is much less understanding of the mechanism that governs the copy number in the other model organisms. He also kindly explained to us the mechanisms that control the plasmid copy number and their possible implications on the cell. He also mentioned that estimating the protein abundance correlation with the copy number might be very important and have a large impact on biology. The talk with Dr. Tal played a crucial role in our project, assisting us in selecting E. coli as the host for our proof of concept.

Doctor Manel Camps, University of California at Santa Cruz, Department of Microbiology and Environmental Toxicology

The biology team made a Zoom meeting with Manel Camps, Professor of Microbiology and Environmental Toxicology at the University of California in Santa Cruz, who published on the mechanisms of ColE1 plasmid replication, and on determinants of ColE1 plasmid stability. During the meeting, Manel talked about his article's results and about his current research that focuses on plasmid stability. Manel explained that while plasmid replication rate is indeed an important factor in plasmid stability, there are many other factors that affect it, for example - degradation and spatial structure. Manel showed interest in our group idea and referred us to several articles relating to similar issues. To conclude the meeting, Manel recommended exploring other plasmids besides pUC19 and talked about the industrial importance of plasmid stability. Manel explained that using antibiotics to increase plasmid stability, while very effective, is a costly and problematic process when done on industrial scales. Therefore, increasing plasmid stability without the usage of antibiotics would greatly benefit the industry. This realization led us to over-consideration on gene expression during data analysis. We realized that the antibiotic resistance gene may affect parameters such as growth rate and protein expression levels besides the plasmid copy number. Indeed, our analysis of the biological results support this conclusion and based on this meeting we were able to provide explanation to our results(see more details in the Results page).

Miles Rouches, Biophysicist from Cornell University

Our Zoom meeting with Miles Rouches, the author of the article that provided the data for our model, was a pivotal moment in our project's development (figure 11). At the meeting we presented him with our idea and asked him questions about the way he analyzed the data. From this conversation we learned some important things that influenced our project later on. First, we realized that the data of RNAi is much lower than RNAp since they have overlapping frames. This insight helped us better understand the data and avoid potential redundancy in our analysis, ensuring that our model would focus on relevant information. Secondly, we realized that the negative copy number values found in his article indicate plasmids in bacteria that failed to grow, and we decided to ignore them. The most valuable lesson from the meeting was the importance of training the model with the appropriate data. We discovered a hidden step between Miles' biological results and the published copy numbers. His measurements were relative and correlated with actual copy numbers. He used a polynomial fit to convert these measurements. This knowledge helped us to improve our model by transforming relative copy numbers into actual ones. We added more data for better fitting, and a power log relationship emerged, leading us to use linear regression for transformation. This enhanced our model's accuracy and performance, showing a strong correlation with validation data. Our meeting with Miles Rouches provided us with a deeper understanding of the data, helped us refine our dataset, and shaped our approach to model training, ultimately improving the accuracy and effectiveness of our work.

Venture Capital Perspective

We met with a Grove Ventures VC. To prepare for the meeting, we have created a one-pager and a deck that illustrates our project (figure 12).

We asked Grove Ventures VC for feedback on our project and consulted with them on approaching companies and design partners to validate our solution. Through their feedback and guidance, we gained valuable insights into how to effectively approach potential companies. Their advice, particularly the reference to the "Mom's test guide" and the emphasis on considering market size and impact, reshaped our project's strategy.

Learning from Grove Ventures VC, we realized the importance of not only developing a technically sound solution but also ensuring its practicality and resonance with end-users (figure 13).

Conferences

We took part in different conferences, including Israel’s SynBio conference, Ilanit - the Experimental Biology Conference(https://conf.fiseb.org/), and Tuller’s lab conference(https://www.cs.tau.ac.il/~tamirtul/), and presented posters on our project at Israel’s SynBio conference and Tuller’s lab conference. It allowed us to engage with stakeholders and reach new insights.

Our posters attracted a lot of interest. We have learned a lot from interacting with people and got good feedback. For instance, in the SynBio conference in Israel that was located at Ben Gurion University, we were asked to identify Col-E1 in non-model systems because it could help the industry, academy, and future iGEM teams that wish to work on systems with a desired copy number for different optimizations. This insight highlighted the broader implications of our work and the need to expand its applicability. In response to this understanding, we made the decision to create a bioinformatics data analysis pipeline for ColE1-like plasmids. This pipeline would enable us to gain fresh insights into the key regulators of plasmid copy number, including RNAp, RNAi, and their respective promoters, from an evolutionary perspective. By understanding the evolutionary dynamics of these modulators, we aimed to develop the capability to control plasmid copy numbers in a wider range of host organisms in the future (see more details in the Results page).

We have actively tried to get feedback and improve our solution, by creating a survey that we gave to different participants in the Ilanit conference (figure 14). This survey showed us that people are interested in our project and support our idea.

Israel iGEM Meeting

Our collaborative meeting with the iGEM team from Technion University was a great opportunity for knowledge exchange and skill development. During this meeting, we made thoughtful discussions about our project ideas and explored various significant aspects of synthetic biology. One of the most valuable aspects of this interaction was the feedback we received on our project presentation and ideas. From this meeting we gained insights into how to refine and enhance our project presentation, making it more engaging and informative. We learned how to respond to questions confidently and comprehensively, ensuring that our project's key points were effectively communicated. On that day, we conducted a cross-border Zoom meeting with a representative from the iGEM group in Abu Dhabi, where he shared insights into their ongoing project and objectives.

IsraGem

We had the opportunity to present our project to high school students and their biology teachers as part of our IsraGem initiative, which aims to promote synthetic biology in high schools through a competition. Presenting our project to a high school audience allowed us to simplify complex scientific concepts and terminology to make our presentation accessible and engaging for students who might not have an in-depth background in biology. This exercise helped us learn how to effectively convey our ideas to a broader audience, which is a valuable skill in science communication.