Team:TAU Israel/Human Practices
Human Practices
Human Practice
Advances in synthetic biology have led to an arsenal of proof-of-principle genetic circuits that can be leveraged for applications, ranging from therapeutics to bioproduction. A unifying challenge for most applications is the presence of strong selective pressure that will lead to an unstable evolutionary genetic construct and undeniably cease to work in a short period of time (Liao et al 2019). This predicament is hindering any major advances in biosynthetic engineering or more importantly, its implementation.
As our multidisciplinary team formed (find out more about our team members), we invested a lot of thought and effort in choosing the problem we would like to address. It was important to us that its solution would succeed in creating real change and that the work required will be fitted to the abilities of the group members. We started by learning and researching about synthetic biology and its applications. One of our team members noticed a common problem that recurred in many of the applications we learned about - genomic instability. As we began to explore more on the subject, we realized the immense power of Evolution, which was our inspiration for a solution. Visit our Project description page to find out more on our inspiration.
Before we decided to proceed with developing a proper solution, it was important for us to grasp on the extent of the problem and try to understand what the impact of the solution will be. The key for apropriate answers to these questions is comprehensive Human Practice work.
And so, began our HP work that included many hours of research which made us fully understand what our goal was and how to properly plan our vision. We read about the topic on the iGEM website and made a presentation on how to succeed in HP (you can find it here). We then explored successful projects from previous years and received advice from last year's iGEM group of Tel Aviv University. We spent the first month building a work-model that will guide us on the way to finding the solution to this complex problem.
As Einstein once said: “If I had an hour to solve a problem, I'd spend 55 minutes thinking about the problem and 5 minutes thinking about solutions.
We came up with a unique workflow, dividing our work into 6 main planes:
We called it – “ICE-PCR” (figure 1). In addition, we ensured each step of our research is done safely and responsibly by using the AREA framework (Figure 2), listed on RRI website. This framework helped us maintaining our focus always in the right direction. It helped us understand what the most fundamental questions are and how to optimally integrate the answers into our project. you can find out more about the AREA framework on our integrated HP page.
Slowly but surely, we began to have a vision - we saw how responsible research and innovation integrates into each of our areas of work and how it fits with our inspiration – Evolution. For instance, consulting with experts will improve our solution so that it is better tailored to the needs of the user; Comprehensive public engagement will help us reach as many segments of society as possible and increase awareness of synthetic biology and Equality in research.
In other words, responsible and pre-planned work in each of the areas we have defined, will help us constantly Evolve. Evolve as an individual, evolve as a team, evolve as a society.
1. industry- In order to understand the necessity of our solution in the real world, as well as the best way of implement it, we had serval meetings with some of the leading pharma & biotech companies in Israel and around the world, such as Lonza and Teva Pharmaceuticals. These meetings helped us understand better the needs of the industry. Find out more on our Integrated HP page!
2. Consult - we refined our product again and again thanks to dozens of academic consultants we have met. Find out more on our Integrated HP page!
3. Entrepreneurship- In order to protect our idea, we decided to file a patent application. For that purpose, we needed to convince our university that our idea is original and could be profitable to develop and invest in it. We wrote a short paper describing the issue that we are addressing with our idea, its importance, and the lack of any other good solutions for it. We also addressed the originality of our idea and its feasibility.
Finally, we presented our idea to experts in our university, in order to receive the investment of time and money needed to fill the patent application. With their help, we wrote a more detailed paper describing: the problem, the current solutions, and our solution including an outline of our plan. In the end a team of lawyers from our university filed the patent for us.
A picture of the approval of the patent application:
In addition to filing the patent, we decided to write a business plan in order to leverage our product even after the competition end with the understanding and feedback we received that our solution is fundamentally relevant in the market. We wrote the business plan together with Lonza, which gave us consultation during the time of the competition.
For a detailed document of our business plan click here =>
4. Public engagement- We presented our project in the scientific community at conferences in order to receive feedback from the environment and reviews regarding the safety of the project. We also held lectures to raise awareness of synthetic biology to the public and decided to take the initiative and raise awareness on the lack of representation of disadvantaged groups in Israeli society in science in general and in synthetic biology in particular. Here are some of the activities we took part in:
- 25/2/20- ISMBE conference
We participated in the annual meeting of the Israel Society for Medical Engineering (ISMBE) in Haifa’s Congress center. This conference provides a unique meeting place for the leading biomedical engineering scientists, medical doctors and industry leaders, who present their latest scientific and technological advances.
Our team presented the sTAUbility project idea. We were delighted to see that the lecture had aroused curiosity among the general public and attracted interest from leading scientists in the field.
Link to TAU's ISMBE presentation
- 26/2/20- Meitzar Lecture
Meitzar is a pre military college for leadership and social responsibility. The college’s vision is to promote a wide view that combines learning life and practice. The pre military course lasts 10 months, during which the youth experience spiritual and value growth, which will help them fulfill in the future a life of achievements and leadership. We wanted to reach these young people and expose them to the world of synthetic biology. We lectured them on the principles of synthetic biology and its possible applications.
- 3/3/20- Schulich Lecture
Schulich Leaders Scholarship is a program that provides scholarships to undergraduate students in Israel. The scholarship is awarded to selected students in the fields of science, engineering, technology and mathematics. The program aims to encourage young people to study these professions and to become the next pioneers in the academy and in technological entrepreneurship. We wanted to expose these students to the world of synthetic biology which is an advanced and groundbreaking field. We lectured them on the principles of synthetic biology and its possible applications.
- 18/3/20- Iasa Lecture
Iasa is a high school and boarding school for gifted, outstanding and knowledge-hungry children from around the country. The school was established to provide an intellectual, social and emotional response to children with high abilities in one of the fields of knowledge: science, music, humanities, art, etc. We wanted to expose these children to the world of synthetic biology and introduce them to this field which they can be a part of. We lectured them on the principles of synthetic biology and its possible applications.
- 7/5/20- Synthetic Biology Online Meet-up of SBA-Sydney
We participated in the event of synthetic biology Australasia (SBA) that took place on the zoom platform. This meet-up aims to further strengthen Sydney’s SynBio community by providing a platform that promotes engagement, collaboration and impact. SBA operates as a community hub to develop collaborations within academia and between academia and industry, to engage in public outreach, education, and training in synthetic biology. We enjoyed the lectures that were given by senior lecturers of different universities in Australia and even got the chance to win a prize of 1000$!.
- 15/6/20- Let the Animals Live
“Let the Animals Live” is a non-profit organization, that aims to improve the protection of animals, promote their rights and raise public awareness of the importance of preventing animal abuse.
As part of the effort, the organization decided to deliver a series of lectures to students on the subject of “groundbreaking science – without the use of animals”. We had the honor of participating in this initiative and lecturing on the “Breakthroughs of Synthetic Biology”. The lecture was delivered through zoom by our group member Matan Arbel and the audience were about 30 students from a variety of fields and faculties in Israel.
- 18/06/20 – Hosting the second Israeli iGEMer’s meetup
We invited Israeli iGEM groups to hold a meet up in order to present the projects of each group and look for potential collaboration. We gathered to think of an idea on how to raise the awareness of underrepresented groups in Israel and encourage their integration in science. Together we created the Israeli HP team and we collaborate to create inspirational video series which we published on YouTube and social media.
Find out more on our Diversity and Inclusion page.
- 5/7/20- German’s iGEM groups Meeting
We had the honor of taking an active part in the annual German meetup. As preparation for the meeting, we sent the organizers a sketch for a workshop we prepared on the subject of "genomic stability of engineered microorganisms." Our workshop was chosen and on the day of the meeting we were privileged to present it through zoom to about 30 participants from all over the world! The workshop had 2 parts - a short lecture on genomic stability and practical work with instructors in small groups. As part of the workshop, participants were required to improve a plasmid prepared by us in advance, using the tools learned in the lecture and using the Benchling platform. In addition, participants experimented with another useful tool - EFM Calculator. Their purpose was to "fix" the plasmid so that it would express the target protein for the longest period of time and at the highest level of expression.
Our instructors went through with the participants step by step and helped them acquire some basic and important skills for maintaining the genomic stability of engineered constructs. We hope and believe that the information provided as part of the workshop contributed to the participants and to the groups they represent.
- 14/10/2020 - Participating in the online Fifteenth Retreat of the Edmond J. Safra Center for Bioinformatics.
We registered for the online fifteenth retreat of the Edmond J. Safra center for bioinformatics in order to present our project to people involved in bioinformatics and to look for investors. During the online retreat we presented a prerecorded short presentation of our poster. The idea was to attract people to visit our poster online after the conference and discuss it with us.
5. Collaborations- We hosted the second annual meeting of all iGEM groups in Israel. We helped groups on various topics and collaborated with groups from Israel and abroad to improve our project and theirs. Find out more on our Collaboration page!
6. Responsible research-Inspired by the work of previous iGEM teams, we utilized the AREA framework and simplified it, making it easier to implement. This way, we ensured that each step of our research was done safely and responsibly. Find out more on our Integrated HP page.
Integrated Human Practices
In our Project Description Page, we explained the inspiration for our idea and how we came up with our proposed solution for genetic stability. At this section we will describe our journey to find answers to the most primary and essential question of the Human practice work- How does our project affects society and how does society influence the direction of our project?
Our journey includes several meetings with some of the leading pharma & biotech companies in Israel and around the world. These meetings helped us understand better who are our main consumers, what are their needs and what should be done to better adapt our product to these needs. In addition, we refined our product again and again thanks to dozens of academic consultants we met.
We documented each end every meeting in our Timeline. Check it out!(at the bottom of the page)
The AREA Framework
As described on our HP page, building the model prior to our HP work, allowed us to plan in advance how we would handle and organize the massive amount of information we would gather. Our approach to HP and the model we have created were inspired by the Exeter iGEM team of 2019. The team beautifully integrated the AREA framework into their project (Figure 1). With some adjustments, we thought this framework would fit perfectly into our project.
What is the AREA framework?
As described on Exeter 2019 Wiki page -The AREA Framework for Responsible Innovation, created by Professor Richard Owen [1] “seeks to promote creativity and opportunities for science and innovation that are socially desirable and undertaken in the public interest” [2] and is used by many research councils to encourage a responsible approach to innovation.
Consisting of the following 4 steps:
Anticipate –
Describing and analyzing the impacts, intended or otherwise, (for example economic, social, environmental) that might arise. This does not seek to predict but rather to support an exploration of possible impacts and implications that may otherwise remain uncovered and little discussed.Reflect –
Reflecting on the purposes of motivations and potential implications of the research and the associated uncertainties, areas of ignorance, assumptions, framings, questions, dilemmas and social transformations that may bring.Engage –
Opening up such visions, impacts and questioning to broader deliberation, dialogue, engagement and debate in an inclusive way.Act –
Using these processes to influence the direction and trajectory of the research and innovation process itself.
How did we implement it?
Although we were convinced that this framework would suit our needs, we felt it was a bit complex to understand as it is not intuitive. Therefore, for simplicity we changed the order of the steps and rewrote each of them as a simple question:
Reflect →
What is the problem we are facing with? What do we want to achieve?Engage →
What did we do to achieve what we wanted? What happened during the conversation?ACT→
What did we learn? how we would apply it in our project?Anticipate→
what is our next step? What implications does it arises?
How did it help us?
The implementation of this framework helped us maintaining our focus always in the right direction. It led us to the most fundamental questions we needed to ask and served as a guide to integrate the answers into our project:
- What do we offer, a product or a service?
- Where our solution is most relevant?
- Who are our proposed end users?
- How can we improve our suitability for real world application?
- What are the safety aspects we must consider?
Visit our Proposed Implementation page to see the summary of the answers we got and how we integrated them into our project. However, if you want to explore the full story of our journey, check out the Timeline below!
TIME LINE
Note: click on the dates bellow to see the full content!
Industry
Reflect:
We wanted to understand from the point of view of the representative in a big biotechnology company, how the industry deals with the genomic stability problem today, what does he think about our solution, and whether it can improve services of optimization of protein expression that GenScript offers.
Engage:
We met with Mr. Felix Fu via online meeting. Mr. Fu is the sales account manager from GenScript for Israel. In the beginning, we presented him a presentation explaining the problem and solution we offered. Later we interviewed him with questions especially about the possibility of a collaboration with GenScript in the field of its service for optimization of protein expression and the effect of genomic instability on the industry. Fu was impressed by our idea, and said it has potential. He also offered us to send him more details about our project so he would connect us to the R&D team for future collaboration.
In another context, he admitted that genomic instability is not a significant problem that affects GenScript’s services such as optimization of protein expression. Nevertheless, he noted that in his opinion our idea can be applicable for companies which produce large amounts of proteins from microorganisms.
Act:
We realized that our focus should be on contacting more companies and especially companies which produce large amounts of proteins from microorganisms.
Anticipate:
Continue the collaboration with GenScript and at the same time contact more companies that produce synthetic proteins via bacteria and yeasts, with the aim of finding experts to help us better understand the industry.
Reflect
Once we understood the problem and the need that arises from it, and after consulting with a number of academics about a possible solution, we felt ready to consult with experts from the industry . Our goal was to better understand how the pharmaceutical industry uses transgenic microorganisms and whether the need to maintain genomic stability arises. Furthermore, we wanted to understand whether the solution we offer adequately meets the need, and what can be done to improve it.
Engage
We needed experienced professional people who deeply know the industry. That is why we contacted Teva, one of the largest pharmaceutical companies in the world. We met with two experts from the Biosimilars department- Mrs. Orit Aharonovich and Mrs. Ganit Yarden. We began by introducing our project. Then Orit and Ganit gave us a short presentation on the development and production of proteins from engineered microorganisms with a focus on their area of expertise- mammalian CHO cells. We learned that in the production phase there are 3 possible approaches for manufacturing proteins:
1. Batch culture- BioReactor in which all reactants and solvents are introduced prior to setting the reaction’s conditions (temperature, pressure, etc.). Products are only taken from the reactor upon conclusion of the reaction process.
2. Fed-batch culture- a culture in which a base medium supports initial cell culture and a feed medium is added to prevent nutrient depletion. Fed-batch culture is superior to conventional batch culture when controlling concentrations of a nutrient.
3. Perfusion processes - an upstream processing which retains cells inside the bioreactor while continually removing cell waste products and media depleted of nutrients by cell metabolism. Fresh media is provided to the cells at the same rate as the spent media is removed.
The experts explained that genomic stability is indeed an important factor in the process, but this problem is addressed before reaching the production stage, in the development process. Therefore, a cell line that has reached the production stage in the fed batch process will encounter other limiting factors before the formation of the mutations as a result of genomic instability. The primary limiting factor in these processes is the media of the culture. In perfusion processes however, the need to maintain genomic stability over time is much more significant.Therefore, the experts argued that our product is highly relevant in two cases-
1. Development stage - At this stage, the most genetically stable cell line is selected and has the most efficient phenotype, i.e. it produces the highest amount of the target protein. Our solution may ensure successful cell line selection easily and quickly.
2. Production processes using the perfusion method - genomic stability has high importance in these processes. Our solution can increase production time while maintaining constant expression levels.
The experts added that the level of expression of the protein produced is the most important and critical thing, both in the development process and in the production process. A solution that manages to raise the expression levels or alternatively keeps them constant over time, while maintaining the integrity and quality of the protein produced, will be of enormous economic importance.
ACT
We learned several things from this meeting-
1. The development process and the production process must be separated. Our product may be of high importance in the development processes of all cell-produced products, as it shortens the time and effort required to maintain a stable cell line. However, this will not significantly extend the production time with methods like pad batch.
2. We should focus on processes that use the perfusion method, since the importance of genomic stability over time is higher in these processes.
3. A solution that manages to maintain genomic stability while raising the initial levels of expression - is the Holy Grail in this field.
4. The most important thing we understood thanks to the meeting - our idea has the potential to help with a real world issue! The problem of genomic instability exists, and is well known by experts from the industry. Further clarification regarding microorganisms is required since the experts we met understood mainly in the field of mammalian cells.
Anticipate
1. Learn more about the development processes of products produced by cells. We should seek advice from biotechnology companies that provide consulting and development services to pharma companies.
2. Learn more about perfusion processes and find experts that specialize in manufacturing using this method.
3. We started exploring ways that would allow us to control expression levels. One solution that caught our eye the most was Pseudoknot (secondary RNA structure that allows the co-expression of 2 reading frames in one sequence, using a -1 frameshift). One frameshift will be the essential gene, and the other- a target gene. Theoretically, using this type of linker would allow us to control the production levels of the target protein.
4. We are required to characterize the problem and the relevance of our solution also for microorganisms. Therefore we need to consult with experts in this field.
Reflect
We wanted to understand from a big company’s expert, what kind of method and procedures are being used to produce proteins from microorganisms in her department and whether the genomic stability of engineered constructs is taken into account in their work
Engage
We have contacted an expert from Teva Pharmaceuticals company in Lithuania, who works in a site that performs manufacturing in E.coli. We sent a document explaining our project and asked her some questions via email. The expert told us that the genomic stability issue is essential for them because sometimes genetic instability limits their work, which raises the relevance of our project.
In order to cope with these problems, they perform biosynthetic processes that keep the bacteria away from the limiting point (time limited). In her opinion, our solution is relevant to the strain and product development stages, but it is too complex to implement for the production stage
ACT
The conversation has further clarified for us the previous conversation with Teva regarding the advice that we should focus on the development processes and not on the production processes. Thanks to her answers we realized that this is also true for E.coli, and not just for human cells as we learned in conversation with Teva.
Anticipate
Looking for more companies that perform manufacturing in bacterial cells and microorganisms to understand whether our solution can be integrated with their work.
Reflect
At this time we already had a basic understanding of the relevance of our product to the pharmaceutical industry, but we wanted to discover more ways in which we could better adapt our product to the needs of the industry. As part of what we learned from the conversation with Teva, this time we wanted to consult with experts who are well acquainted with the development stages of cell-produced products.
Engage
We contacted Dr. Frida Grynspan, the Head of Lonza Collaborative Innovation Center, Israel and briefly introduced her to our idea. Dr. Grynspan kindly accepted to set a zoom session With some of Lonza's most influential experts -
•Dr. Andy Racher- Director, R&D IP Strategy
•Dr. Alex Gutteridge, Head Of Bioinformatics
•Dr. Peter O'Callaghan, Senior Scientific Group Leader (R&D)
•Dr. Christoph Kiziak- head of the Microbial Research & Technology Team
•Dr. Bernie Sweeney- Director R&T
•Dr. Iris Geffen Gloor -Head of Business and Commercial Development
Although previous encounters were highly significant, , this encounter was without a doubt the most significant for us. The excitement in the group ahead of the meeting was high, we knew this is a life-time opportunity - receiving advice and support from the greatest experts of one of the most successful biotechnology companies in the world..
We held two sessions - in the first we briefly presented our product and in the second we went into the depths of the details and revealed our future plans. In both sessions we received feedback from the experts on our product and we had the opportunity to ask questions. The core of the discussion was the relevance of our product, its economic importance and how these two can be improved. During the conversation we received many feedbacks and recommendations, here are the main ones:
•Our product addresses an issue of high importance in the biotechnology industry. As Dr. Kiziak said it- “5 out of 5 for addressing this topic!”
•The solutions that are suitable for microorganisms will not necessarily be suitable for mammalian cells. A thorough separation between the two cell types and a separate examination of the solutions for each type is required. For example: in mammalian cells there are Multigene families. Those are groups of genes from the same organism that encode proteins with similar sequences over all or part of the sequence. In order to use one gene as an essential gene in our system, we will have to disable the whole gene family in the genome and not just a single gene.
•The GFP and promoter that we used in our experiments do not constitute enough measure for a solid POC because they do not create a large metabolic burden on the yeast cells (explore more on our experiments page). However, the results of the experiments do show an interesting trend. It is very likely that the connection of the genes in the way we performed the experiment, does indeed contribute significantly to the genomic stability. More in-depth research is needed with additional proteins.
•The experts asked if our solution is suitable for secreted proteins. We revealed to them our initial solution for this type of protein - Super Linker. This linker uses a pseudoknot in combination with a signal peptide, in order to provide a solution for secreted proteins (discover more in our ___ page link). There was agreement among the experts that such a solution, at least on a theoretical level, could work.
•We need to take into account that even a slight change in the amino acid sequence of the target protein Is out of the question in the pharmaceutical industry. Solution that involves a change in the amino acid sequence is unlikely to be approved by the FDA.
ACT
After the two sessions, Lonza generously offered us a $ 10,000 grant to further develop our product. In addition, the company offered to provide us with consulting services for the entire duration of the competition. Following the feedback, we implemented a few things:
1.At this point, our best efforts and focus will be on microorganisms. The application of the solution to mammalian cells is more complex.
2.After the time frame of the competition we will start another round of experiments with RFP under a strong constitutive promoter, which creates a higher metabolic burden on the cell.
3.Due to the approval of the experts, we decided to continue developing our Super Linker. After the competition we will start experimenting in the wet lab.
4.A change in the amino acid sequence of the target protein is not possible in products in the pharmaceutical industry. Therefore any solution that does not explicitly maintain the sequence is not relevant to our product. (See for example the kind offer we received from our friends from the Go Paris Team).
Anticipate
We will continue the cooperation with Lonza and examine the feasibility of receiving its support for a government grant from the Israel Innovation Authority. This grant is worth about $ 150,000 and could help us continue to develop the product even after the iGEM competition. More about the grant at this link.
In addition, we arranged a meeting with Dr. Iris Geffen Gloor, the head of Business and Commercial Development at Lonza, to promote the construction of an initial business plan.
Reflect
After the meeting with Lonza we decided to focus on microorganisms so we wanted further advice from experts from the industry in this field.
Engage
We contacted an Israeli pharmaceutical company called BTG. BTG is a manufacturer of human health care products derived from genetic engineering and biotechnology processes. The Swiss based Ferring group acquired the company along with several of the therapeutic protein products BTG had successfully produced.
We set a 1 hour zoom session with two experts specialized in microorganisms:
•Dr. Daniel Plaksin,Head of Biologics and Technology Innovation at Ferring Pharmaceuticals
•Dr. Elinor Erez, AVP Biotech Development
We presented our product to the experts and then continued with feedback and questions. Here are some key points:
1.The relevance of our product to the production process of proteins from microorganisms is low, this is because the problem of genomic stability is not the limiting factor in the prevailing production method - Fed Batch. However, our product is highly relevant in the development processes as it saves time and resources to find the most genetically stable cell line.
2.Dr.plaskin argued that in the developmental stages the most significant thing is the expression levels of the target protein. Second in importance is the quality of the protein produced, and third is the genetic stability of the engineered construct.
3.As we learned from previous conversations, a change in the target protein at the level of amino acids is not possible because of regulatory problems (FDA).
4.Dr. Erez argued that our product may be beneficial in raising the expression levels of proteins with low expression levels in the first place. For example - toxic proteins.
5.Development processes of a new drug in BTG and similar pharma companies rarely take place.
Therefore, our main consumers should be biotechnology companies that provide consulting and development services to the pharma industry. For example: Lonza (which we have contacted before) and Catalent.
ACT
The advice of the experts made it clear that the conclusions we have drawn from previous discussions about mammalian cells are also relevant for microorganisms. In addition, it reinforced our understanding that the Holy Grail of this field is increasing the expression levels of the target protein.
Anticipate
Continue to focus on microorganisms. In addition, we contacted Catelent company.
Consult (Academic experts)
Reflect
We wanted to perform the 10 genes and co-culture experiments. We were looking for libraries created by other labs that have yeast’s genes connected to a fluorescent gene in order to conduct our experiments.
Engage
After explaining to Maya and her team our goals, they were happy to share their studies and their libraries with us free of charge. The libraries contain yeast genes connected to either RFP or GFP at the N terminal.
ACT
We used their libraries for our experiments. We learned that we need negative controls that were missing from the libraries and therefore we had to create them ourselves.
Anticipate
The next step was to understand how we can separate the different genetically engineered yeast out from each other once the experiment is done. After consulting we developed a solution with the help of restriction enzymes
Reflect
We wanted to create an algorithm that will find one or two restriction enzymes that are suitable for a big number of sequences and gives us the desirable product in as many sequences as possible.
Engage
We introduced Prof. Tuler the algorithm we used to solve the problem in order to consult with him whether our solution is good enough. Prof. Tuler noticed that we did not refer to the complement sequence of the restoration enzymes. He suggested we check the genes that are cut down by less than 25 nucleotides and see whether the remaining sequence is unique or not, and if it is possible to work with very short sequences and succeed in identifying the genes. He further suggested using a single restriction enzyme model.
ACT
We upgraded the algorithm according to Prof. Tuler advices and used the single restriction enzyme model.
Anticipate
Making the tool useful for other iGEM groups in the future.
Reflect
We wanted to understand what an expert in the field of microorganism evolution thinks about our idea and the way we will implement it. In addition, we wanted to get an opinion on the experiments we had planned, before entering the wet lab, in order to anticipate difficulties and faults that could happen along the way and try to prepare for them in advance.
Engage
With that goal in mind, we met with Prof. Uri Gophna via Zoom. Prof. Gophna is a Microbiology and Biotechnology expert, specifically in the field of evolution of microorganisms. We presented the professor with a presentation explaining the problem and solution we offered. Later we interviewed the professor with questions targeted specifically for his knowledge. Prof. Gophna was impressed by our idea, approving the POC experiments we offered. He pointed out that we might have a problem separating the fused protein after the corresponding folding, and he advised further investigation in that matter.
ACT
we realized that our focus should be on designing the solution in a way that is best suited for industrial purposes. In addition, we began to research literature on possible ways of separating proteins after co-folding.
Anticipate
We approached companies who produce synthetic proteins via bacteria and yeasts, with the aim of finding experts to help us better understand the industry. We also arranged a meeting with a protein expression expert - Professor Itai Benhar.
Reflect
We wanted to understand what an expert in the field of protein expression thinks about our idea and the way we implement it. In addition we wanted to hear about an effective way to separate the target protein from the essential protein after the translation.
Engage
With that goal in mind, we met with Prof. Martin Kupiec via Zoom. Prof. Kupeic is a Molecular Microbiology and Biotechnology expert, specifically in yeasts. We presented the professor the problem and solution we offered. Later we interviewed the professor with questions targeted specifically for his knowledge. Prof. Kupeic thought that our solution seems to be a good idea, and gave us helpful tips regarding restriction enzymes. In addition, he recommended to use a site-specific TEV protease that cuts amino acids, in order to separate the target protein from the essential protein after the translation. However, he pointed out that we might have a problem using the Chi-bio system, since some mutant yeast can develop the ability of adhesion to the glass vessels. This could lead eventually to biases in the surviving yeast strains population.
ACT
We realized that our focus should be on understanding the problem from the industry perspective and designing the solution in a way that is best suited for industrial purposes. In addition, we began a literature review about the prevalence of developing the mutant ability of adhesion to the glass vessels of the Chi- bio system, and also about possible ways to avoid this phenomenon.
Anticipate
We approached companies that produce synthetic proteins via bacteria and yeasts, with the aim of finding experts to help us better understand the industry. We also contacted an expert in the field of genomic stability in the industry. In addition, we contacted representatives from GenScript Biotech company and also experts in the field of biosimilars from the company TEVA in order to understand more on their methods to deal with genetic stability.
Reflect
We wanted to understand what an expert in the field of protein expression thinks about our idea and the way we implement it. In addition, we wanted to get an opinion on the experiments we had planned, before entering the wet lab, in order to anticipate difficulties and mistakes that could happen along the way and try to prepare for them in advance.
Engage
When we first presented Itai with our idea he told us that in the industry there is a method of implementing a target gene with the help of an essential gene which is called stable transfection. However, he said that in contrast to our idea, this method does not take in account the accumulation of mutations in the target gene and as soon as the target gene is no longer functional, the cells are removed and a new batch is set as a replacement. He also pointed out that our focus should be on the separation of the two proteins from each other. In order for our idea to be relevant to the industry we must find a way to develop a linker with some protease recognition site. He advised us to perform an RNA sequencing to find out the expression level of the target genes because a main key is to keep the expression levels high and not only the stability of the gene. He concluded that at least tenfold change in the stability is needed for us to make our idea impressive enough.
ACT
We realized that our focus should be on the industry, understanding the problem from the industry perspective and designing the solution in a way that is best suited for industrial purposes. In addition, we began to research in the literature for possible ways of separating proteins after co-folding and about stable transfection.
Anticipate
We approached companies who produce synthetic proteins via bacteria and yeasts, with the aim of finding experts that can help us better understand the industry. We also contacted an expert in the field of genomic stability in the industry. In addition, we contacted experts in the field of biosimilars from the company TEVA in order to understand more on their methods to deal with genetic stability.
Reflect
We wanted to consult with an expert in the field of engineering and machine learning algorithms about the implementation of fluorescence prediction models, since the performances of the initial configuration that we tried to implement were poorly accurate and very over-fitted. We wanted to consult with Prof. Shinar about the following points: feature selection, feature normalization, filling missing values and algorithms for the prediction of fluoresce.
Engage
We met with Prof. Shinar via zoom. We presented him with our project and the fluorescence prediction model and its limitations. He suggested many options for normalization of our data, such as independent normalization (per feature), gaussian normalization, normalization to a specific range. As for filling missing values, he suggested we remove them. He presented us with many algorithms for feature selection and different models. He also noted that we may have an issue of overfitting since we use so many features in our model.
ACT
We implanted Prof. Shinar’s ideas and recommendations in our fluorescence prediction model. We tried many of the algorithms of feature selection he suggested in order to overcome the issue of overfitting. We manage to highly improve our model due to this meeting.
Anticipate
Continue consulting with Prof. Shinar in the future if needed.
Reflect
We wanted to consult with someone experienced with FACS of yeast colonies. The Consultation was about FACS sample preparation and validation of the FACS protocol we found.
Engage
We met with Dr. Dana Laor via online meeting. Dr. Laor is well experienced with FACS of yeast colonies. We presented her with the FACS protocol we found on the Heidelberg's university hospital website. According to the protocol, the buffer needs to consist of several materials and we wanted to validate this with Dr. Laor. She said that only one buffer material is needed (PBS). In addition she gave us recommendations on how to operate the device.
ACT
After consulting with Dr. Laor we understand better how to use the FACS device and how to handle the FACS sample preparation.
Anticipate
Continue consulting with Dr. Laor for further guidance in the future if needed.
Reflect
One of our modelling tasks was to model and optimize the mRNA local folding energy. This parameter affects the translation efficiency and therefore is significant for stability of the construct with the target gene. There is a signal for weak folding at beginning of the sequence, and thus making it easy for the ribosome and other translation factors to engage the initiation site. In order to model this phenomenon and optimize the folding energy we wanted to consult with an expert in the field of bioinformatics and mRNA signals.
Engage
We met with Zohar Zafrir via online meeting. Dr. Zohar Zafrir, who is a bioinformatic and an expert in the field of understanding mRNA signals in eukaryotes. We presented him our project and the purpose of the meeting. We told him we are trying to create a model that will engineer our sequence (target gene – linker – essential gene) so that in the beginning of the sequence the folding energy signal will be weak. He suggested that we may use the linker’s length as input and then provide accordingly the right linker. We should note that the linker’s length divides in 3 and has no stop codon. In addition, he explained that around the ATG there is a sequence named Kozak sequence that allows normal folding of the translated protein. Therefore we should look for this sequence in order to understand how to engineer the beginning of the coded area. He also presented us with several models for mRNA folding we can use such as ViennaRNA. Furthermore, he suggested we optimize the mRNA folding energy at the beginning of the essential gene and also at the beginning of the target gene in order to separate the translation of these genes.
ACT
We realized that we can use ViennaRNA for calculation of the mRNA secondary structure of a sequence and for calculation of the mRNA folding energy of this structure. Thanks to Zohar Zafrir we understood we can use ViennaRNA via python. We calculated the mRNA folding energy of the beginning of the sequence using ViennaRNA and we optimize it in order to receive a weak folding that will lead to high gene expression levels. In addition we took his advice of using the linker’s length as an input and implanted it in another project.
Anticipate
Continue the research about optimization of the folding energy in order to specifically tailor parameters to improve our model.
Reflect
We were looking for a tool that will help us predict a linker’s folding. In our construct a linker is binding the essential and target genes. Therefore, in order to optimize the construct’s stability the linker should be unfolded. One of the tools we found is FoldIndex that returns a folding score for a specific amino acids sequence. However, we didn’t understand the meaning of the output score. In addition, we wanted to combine FoldInex in our software and didn’t understand how to properly do so.
Engage
We emailed Jaime Prilusky, one of FoldIndex’s developers regarding our problems. Jamie kindly explained how to run a web query with our desired output. He further said that the FoldIndex score is in the range of -1 to 1. Values greater than zero indicate areas with folding potential. The more positive the value, the greater the probability that it will fold.
ACT
We wrote a code that extracts a sequence’s score from FoldIndex webtool and we were able to rank the potential linkers using FoldIndex’s score.
Anticipate
We wanted to check how we should determine the whole construct folding and not only the linker’s one. For that purpose we later consulted Prof. Tuller.
Reflect
We wanted to know more on Nanopore sequencing and what is the best way to prepare our samples for the sequencing with this method. In addition, we needed help conducting our Nanopore sequencing since the COVID19 crisis prevented us from doing it in Israel.
Engage
We consulted with Dr.Marcozzi (former IGEM alumni), a world expert in the field. He pointed out a flaw in Nanopore sequencing that could be problematic to our project, and that is the high error rate when sequencing with Nanopore (around 10%) which means it would be extremely hard to distinguish mutation in our sequence from errors from the sequencing itself. In addition, Dr. Marcozzi kindly agreed to be a sponsor of our IGEM team and do the sequencing for free in their facilities.
ACT
We decided to take Dr. Marcozzi’s advice and incorporate RCA (rolling circle amplification) as another step in the preparation of the samples before sending them for sequencing, this would help us distinguish between mutations and errors of the sequencing method.
Anticipate
Our next step is to send the samples for sequencing at Cyclomics.
references
[1]R. Owen et al (2013) Responsible Innovation: Managing the Responsible Emergence of Science and Innovation in Society Wiley. pp.27-34
