Human Practices

While reviewing papers on the mechanisms of the NLRP3/caspase-1 pathway, we thought that it would be beneficial to reach out to an expert who does research on NLRP3 to further understand how pterostilbene directly interferes with this pathway, expanding what we had gathered from the literature. Some papers we came across were written by Dr Coll and we found that her work is focused on the NLRP3/caspase-1 pathway, with Dr Coll leading the biological characterisation of a small molecule inhibitor of the NLRP3 inflammasome. We thought she would be helpful in answering our queries and provide insight. We discussed the mechanism of the NLRP3 pathway cascade including its connection to NfkappaB, oligomerisation of NLRP3 inflammasome with caspase-1 and adapter protein ASC, production of IL-1, etc. Known molecular targets discussed include toll-like receptor 4, adapter protein ASC, gasdermin-d, NLRP3 inflammasome, and NfkappaB kinases. We were given examples of inhibitors of each of the targets. Lastly, we have discussed the potential of this pathway as a target in other diseases. Not only in AD, but diseases such as Covid-19 and chronic kidney disease, have shown potential to have a therapeutic effect. Similar to our meeting with Prof. Wendy Noble, the discussion clarified our purpose and the direction of our project. This discussion aided our understanding of the pathway in much closer detail, guiding our research and presentation for our project design page. We also reviewed currently studied molecular targets of NLRP3 and their inhibitors to compare the benefits of pterostilbene in comparison, as shown in our project design page. Its potential in other diseases also supported part of our proposed implementation as well.

In our research, we found a challenge in which research papers showed that LDL cholesterol is increased with pterostilbene monotherapy, which we have detailed on our proposed implementation page. We then reached out to Dr Charles Brenner who wrote the most recent paper on this we could find. He is also the chief scientific advisor of Chromadex, a company that produced 99% pure pterostilbene to be used as an ingredient in supplements. It has now been discontinued due to the issue of LDL increase. As he has worked closely with pterostilbene, we valued his insights greatly. While heavily critical, he thoroughly explained the flaws in its research and usage as a therapeutic. He explained that in his pterostilbene and polyphenols in general have limited potential therapeutically and that we cannot claim to cure any disease with these compounds. This was based on criticism of the literature in this area of research, how their purpose, methods, and claims can be questionable. When presenting our project, we made sure that we carefully use our language, without making any explicit claims that pterostilbene would be a definitive cure for AD, nor any disease -- our aim is to produce a large yield of pterostilbene through improved synthetic biology methods as a potential therapeutic to delay progression of AD. We also considered the issue of LDL increase which may be an unwanted side-effect of pterostilbene use --thought of ways to address this issue and not cause harm to future patients if we were to take Symemco Therapeutics to the next level in the future as shown in our proposed implementation page. Lastly, the discovery of this issue helped us recognise that we might come across unexpected limitations. It is a matter of how transparent we are to our audiences, discussing limitations as part of our human practices.

Following our discussions with Dr Brenner and Dr Amorim, we spoke with Dr Rodriguez-Mateos to discuss polyphenols in a therapeutic context. As with Prof. Wendy Noble, we reached out to Dr Rodriguez-Mateos after finding that her research focus in our institution was relevant to our project. Her focuses include polyphenols, their bioavailability and cardiovascular health benefits. While her research is not related to neurodegenerative diseases, the compounds she researches and their application to human health were still relevant. We asked about pterostilbene's interest and potential in the field and how important specificity is. A significant note she made was how the non-specific actions of polyphenols can be beneficial. For example, the improvement of vascular function by a polyphenol would improve blood flow and in turn improve one’s cognition. We also asked about her views on side effects due to multiple targets of polyphenols (specifically, LDL increase with pterostilbene intake). She explained how especially with pterostilbene, the research is still ongoing. Only LDL increase is an observed side effect so far, more clinical trials would need to be undertaken. Furthermore, we were told that in her view, in vitro and in vivo studies in literature supporting pterostilbene’s therapeutic potential are sufficient to keep moving forward.

We consulted Gemma Singleton, a Research and Ethics officer from our institution to discuss the ethics of our project in two parts. We reached out to her not only because of her relevance to this area of the project, but because taking our project to higher levels in the future, it would be done on behalf of our institution and she would be one of the first points of contact. First, we explained our process of yielding pterostilbene in wet lab and predicting the ideal plasmid copy number to use and justifying our promoter strength choices in dry lab. Our activities were confirmed to be minimal risk and no further ethical clearance would be needed, shown in Safety Considerations, showing how we are progressing on this project safely and responsibly. However, if we were to go further and test the efficacy of this therapeutic for example, we were told to consult with our institution’s ethics board, then the Health Research Authority (HRA), and obtain ethical clearance for using NHS services. We used this as advice one of our guidelines in developing the proposed implementation.

We reached out to Amir Bozorgzadeh, CEO and co-founder of Virtuleap, through LinkedIn. We found a LinkedIn post of Virtuleaps’ which outlined their use of virtual reality (VR) to improve cognitive impairment which we thought was relevant to our project. In the meeting with Amir Bozorgzadeh, we asked for advice and insight on how to successfully build a startup company. “Virtuleap is a health and education VR startup aiming to elevate the cognitive assessment and training industry with the help of emerging technologies such as virtual reality and artificial intelligence” (Virtuleap, 2022). We inquired about the difficulties in starting a company in healthcare, a competitive sector. Amir mentioned that having the right allies and several clinical partners is of great importance when emerging into a big sector such as healthcare as it is greatly competitive, therefore we aim to have a clear aim and unique selling point when we reach out to them. It was also highlighted that we should be open to change and embracing ideas that come with the progression of our project, which we are implementing through our human practices, understanding that our initial vision might not be what the end vision is; this is all part of the company’s lifecycle. Standing out to investors was a topic that we discussed as we were in the beginning stages of writing our business plan and pitch deck. Amir highlighted the importance of how founder-passion fit as something investors look for when we approach companies and investors. Making sure we choose a team with passion for our cause will inspire people to invest in us, collaboratively driving our company forward, an emphasis we made sure to implement throughout our pitch deck. Through discussing the realities of starting a company in the field of biotechnology, we took on Amir’s advice and altered our pitch to focus more on our team and our goals rather than our product. We were also able to take inspiration from how Amir and his co-founder promoted the business through networking which also inspired our networking guide. We utilised the connections we make through iGEM to gain a more established network and promote our idea as a start-up to help us create a successful business.

We realised our pitch deck was missing some key components and sought advice on pitching from Adrian Signell from The Creator Fund, whom we know is a student at our institution as well. We knew of his role within the Creator Fund and thought to gain an insight into investments, specifically for university start-ups.Adrian explained the essential components of the pitch that we needed to include based on our current business stage and agreed to schedule a future meeting where we could present our new iteration of the pitch for further feedback. After receiving invaluable feedback and advice from Adrian Signell on our adapted pitch deck we were able to further refine our pitch with the vision for it to be presented to potential investors. We also developed a rough pitch deck script that we used to verbally aid the pitch slides.

We reached out to BIOINX® after coming across them on LinkedIn. They are a spin off project from Ghent University specialising in commercialising materials and bioinks for 3D bioprinting and biofabrication. Their work and business practices, we thought, were quite relevant to the type of advice we sought. We spoke to the CEO of BIOINX®, Jasper Van Hoorick, about the journey transitioning from a university project to a research-based startup company and the entrepreneurial and synthetic biology aspects of their company. Dr Van Hoorick outlined the challenges surrounding commercialising a university project due to companies and PhD programs having different end goals. As we are currently focusing on research and development of our product, we needed to create future protocols addressing batch to batch reproducibility that comes with commercialising the product. Another area of interest was how they navigated obtaining intellectual property coming from a university background, as we do. Dr Van Hoorick noted we must consider whether it is in our best interest to file a patent through the university or later once the company is established in its own right. This has helped our team develop our patenting strategy accordingly, in which we will apply for a patent to protect our product after establishing the company to avoid transferring the patent and having to negotiate with the university. Moreover, one of the members at BIOINX® was developing a 3D printed blood brain barrier (BBB). We identified that this was an avenue in which we could potentially partner with BIOINX®, by using materials supplied by them to create a BBB model to test the permeability of our drug. This would accelerate the process of testing, allowing us to reach the market faster as well as avoid animal testing. We also discussed a potential additional revenue stream through commercialising the BBB model alongside our therapeutic development. As part of Dr Van Hoorick’s feedback, he suggested we participate in pitching competitions to help us gain experience and receive feedback on our pitch to investors. This will allow us to refine our pitch to increase the chances of investors wanting to partner with us and promote Symemco Therapeutics. Overall, the meeting gave us insight on how our team aims to proceed with the project beyond iGEM, tackling challenges such as IP, finding investors and potential revenue streams through partnership.

BetaSense is a start-up focused on producing a diagnostic tool to measure protein misfolding associated with neurodegenerative diseases, including AD, using infrared technology. We found BetaSense through their posts on LinkedIn that highlighted the development of AD diagnostic tools which were relevant to our project. We reached out to BetaSense to further understand how AD is diagnosed and its pathophysiology, the new technologies evolving regarding AD diagnostics and what it is like to be a start-up within the AD diagnostics field. We met with the head of finances, Felix Oeding-Erdel, in which we asked for advice on beginning a start-up in university. He highlighted that it is wise to develop research within the university as they provide laboratory equipment for free allowing cheaper research development. As we are an undergraduate university team, we face limitations with laboratory resources as opposed to postgraduate students, therefore our team plans to focus on a proof of concept to be commercialised post-iGEM. We also asked how to appeal to investors with the technology that we are developing. To appeal to investors, Felix suggested creating an extensive business plan that is problem oriented in order to present a viable solution, while also focussing on gaining investors as part of our near-term company plan. This is a point that we have taken on by creating a detailed business plan highlighting various aspects of our business and our plans for our company in the short-term and long-term. Felix also suggested doing a global analysis for which diagnostic treatments are available. He emphasised that without diagnostics, treatments cannot be given at the correct time, therefore it's important to mention how we can use our product in conjunction with diagnostics. Therefore, we suggested the idea of partnering up in the future by using their diagnostic tools to track the progress of our therapeutic as well as further understand the pathophysiology of AD and how the diagnostic tool can be further modified for improvement. Overall, this meeting opened up our scope of opportunities by sparking our interest in diagnostic tools to integrate into our future collaboration plans.

We reached out to Dr Sara Holland alongside her trainee Isobel McLeod at Potter Clarkson, as suggested by a mentor of the team and whom several team members knew of prior to starting this project. Dr Holland is a patent attorney specialising in synthetic biology and biotechnology. She was able to guide us through protecting intellectual property (IP) whilst participating in iGEM, an open-source competition. Dr. Holland outlined how ownership plays an important role when creating IP under King’s College London and in what instances the College would have ownership of the IP. Therefore, we identified aspects that would allow us to obtain ownership of our IP, such as being a student-led team and not employee-led, a criterion that is mentioned for the College to gain IP rights. Dr. Holland and her trainee also addressed pre- and post-patent processes in which she highlighted that the length of gaining rights to an IP is 4-6 years. She emphasised that any information disclosed to the public about changing scientific aspects of our project to make it more novel could be argued against gaining our IP rights as it is in the public domain. We will ensure our product information is kept private during this time to avoid issues. Additionally, Dr. Holland explained that granted patents are not valid in every country posing a risk when partnering with companies and supplying and distributing our product internationally. Therefore we now aim to apply for patents in countries relating to the research and development, distribution and use of our product. Overall, patent attorney Dr. Sara Holland and her trainee Isobel McLeod gave us clear insight on the process of protecting our IP in the scope of iGEM and beyond.

The study “Enhanced Production of Pterostilbene in Escherichia coli Through Directed Evolution and Host Strain Engineering” conducted by Yan et al. (2021) served as a foundation for much of our idea and protocol development, as it presented the highest pterostilbene yield reported in the literature. Therefore, contacting Dr Yan was an obvious and necessary step towards justifying the different aspects of our protocol and further understanding how the production of pterostilbene could be improved. We came across this paper during the earlier stages of consolidating our project idea and protocol. There were a variety of fundamental scientific aspects that had to be considered for our production of pterostilbene, such as bacterial strains, plasmids used, expression systems, transfection approaches, and quantification methods, among others related to the biosynthetic pathway. Firstly, Dr Yan provided crucial insight into the importance of low copy number plasmids in minimising the metabolic burden on the bacteria. Through more recent experience, Dr Yan also suggested a co-transformation of two plasmids rather than all four genes in a single plasmid, as well as additional supplementation recommended for a greater pterostilbene production, specifically in aiding the last step of the pathway. Finally, Dr Yan assured HPLC (High-performance liquid chromatography) to be sufficient for an accurate identification and quantification of our metabolites without the need for further mass spectrometry analysis. He further recommended a cultivation period of 24 hours before pterostilbene quantification, which we further extended with another timepoint at 48 hours in our protocol to allow direct comparison with his study. Following this discussion we have implemented these suggestions into our protocol. Additionally, it helped us justify our use of HPLC and choices made such as going forth with low copy number plasmids.

In considering specific enzymes to use for our synthesis of pterostilbene, our research led us to Dr Hererra’s paper “Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene” (Hererra et al., 2021). As resveratrol is only one enzymatic reaction away from pterostilbene, this informative article on the enzyme Vitis vinifera Resveratrol O-methyltransferase (VvROMT) and the potential of mutagenesis to improve ROMT activity has been very helpful in designing our project. Dr Hererra’s expertise on the most appropriate choice of promoter type, specific ROMT variant, production of soluble protein and specific reagents to produce the maximum yield of pterostilbene has helped inform our protocols. Dr Hererra’s suggestion of using HPLC-UV due to the ease with which it can be standardised compared to liquid chromatography Mass spectrometry was useful for the planning of our protocols. Furthermore, her suggestion to grow our E. coli culture in the dark to minimise stilbene trans-cis isomerisation was a valuable insight. Dr Herrera importantly advised us to consider that the fusion of a solubility tag to the VvROMT homodimer could affect its activity, which we would have to ensure beforehand if we were to pursue this idea.

A crucial part of wet lab was understanding the different requirements and recommendations in constructing BioBrick parts, as well as achieving various medal criteria through the contribution to and improvement of existing parts. For this purpose, Marko became an essential point of contact for the considerations and analyses required to prepare our sequences for synthesis, especially on RFC (replication factor C) assembly standards and compatibility. We initially contacted Marko based on a suggestion made by our PI and after knowing of his work and experience, decided that discussions would be insightful. As well as informing us about the RFC10 and 1000 assembly types, Marko detailed the considerations we should have when preparing our sequences for RFC10, which guided us in subsequent analyses and changes to our sequences. Regarding our protocol, Marko provided suggestions on how we could further control the expression of our genes and thus pterostilbene yield through experimenting with a range of different promoter strengths, emphasising the importance of thoroughly characterising these enzymes to allow future teams to effectively build from our work. His knowledge on the contribution of new information from the literature to existing parts further solidified this approach that would help us achieve the corresponding bronze medal criterion. With an understanding of Joint Universal Modular Plasmids (JUMP) type IIS assembly, Marko reviewed our JUMP guide we made as a contribution for future iGEM teams to design and carry out this recent assembly method for a variety of applications in synthetic biology. With his feedback, we improved our guides for our wet lab contribution, making it more succint, adaptable to a wider audience, and of a quality that would be appreciated by future iGEM teams.Marko also importantly clarified certain details in our designed protocol for improving existing BioBrick parts, such as the sufficient and expected number of replicates that would allow us to accurately demonstrate that we successfully improved the existing part, as well as supporting potential modeling and molecular docking approaches for these improvements. This enabled us to further refine our assays.

We decided to contact Dr Marcos Valenzuela after our first attempt to assemble our gene constructs using the newly added pJUMP plasmids designed by Dr Valenzuela found in the 2022 registry kit. Given this assembly system is very novel we had a series of questions we had regarding this type of assembly. He was very helpful in clarifying the advantage of using particular restriction enzymes, how fusion sites can be reused and how particular restriction enzymes enable successful assembly from level 0 to level 1 and 2. Dr. Marcos also confirmed what flanking sequences are necessary to make the pJUMP parts compatible with PhytoBiobrick standards. Furthermore, he provided insight on differences between the sequences found on the registry and of his own paper and how this would affect our assembly design. Dr. Marcos additionally provided us with an updated version of his protocol that we now have access to use for the assembly protocol design for our project. Consequently, we successfully re-attempted our plasmid assembly design with the new insights. Our new assembly design followed the suggestions of using BsmB1 restriction enzymes for level 2 assembly using the version of his plasmids available in the 2022 distribution kit as he confirmed they contained sfGFP reporters originally described in his paper.