No project exists in isolation, and all actions we take will have an impact on the world around us. This is, in fact, desirable as our team, brash young souls that we are, hopes to change the world for the better through Coup Dy’état. However, it is wise to temper our exuberance by responsibly and thoughtfully evaluating whether our work will indeed be good for the world. To us, the Human Practices aspect of iGEM is a serious undertaking, and we attempted to critically examine our work from as many perspectives as possible. Each voice was given due consideration and used to shape our project throughout our iGEM journey.
This section charts how we identified and investigated one or more Human Practices issues in the context of our project, thus fulfilling iGEM's requirements for Human Practices.
Respecting the rights and opinions of others is important, and we consider it a cornerstone of the spirit in which Integrated Human Practices should be conducted. Before carrying out any Human Practices activities, all our team members took an online course offered by our university on the Personal Data and Protection Act, a piece of legislation which establishes a general data protection regime for Singapore. This was so we could learn how to responsibly handle personal or privileged information shared by participants in our Human Practices activities. In addition, a number of our team members took a communication module, ES2331: Communicating Engineering, also offered by our university, which covered interview techniques as well as national and institutional guidelines for conducting this kind of social science research. Our activities were also vetted and sanctioned by our principal investigator, A/Prof Poh Chueh Loo.
After familiarizing ourselves with the relevant policies, we created a Human Practices Guide to ensure that our work would be reproducible, and always comply with national and institutional standards. This protocol comprises a workflow and four templates - interview request email, thank you email, statement of informed consent for face-to-face interviews, and statement of informed consent for email interviews. Our guide is available for perusal here. A caveat: This protocol was written when our team was looking into the feasibility of a project based on MDROs, hence our Workflow makes references to that. Please make your own appropriate modifications and substitutions.
Additionally, once we confirmed our project’s objective, we worked closely with the iGEM Safety Committee. Despite iGEM’s Do Not Release Policy, we believed that it was not just possible, but imperative that we bring our product out into the world, but we needed to consider biosafety and security. We thus developed a protocol for safe extraction, proposed it to the Safety Committee, and obtained their approval. After demonstrating our protocol’s effectiveness to the Committee, we invited potential users, such as local fashion designers, to give feedback on our dyes. Please visit our Safety page for more details. There, you will also find our Safety Form, which shows how our team thoroughly assessed the risks and implications of our project.
At the beginning, based on our extensive literature review, we proposed several problems we were interested in solving, and brainstormed synthetic biology solutions. To investigate the viability of our ideas and demarcate the frontiers of current research, we interviewed experts in the relevant fields as the insights we required could not be obtained from literature alone.
After finalizing our problem statement and approach, we identified groups our project would affect, and Human Practices issues. Interviews were conducted at critical stages of our project as tests for our design iterations, following which we redesigned and rebuilt our prototypes.
These groups are synthetic dye manufacturers, natural dye manufacturers, fashion designers, ordinary consumers, and people suffering because of synthetic dye pollution. The most straightforward way to find out how our project would affect these groups is to ask their representatives directly, and we have tried our best to do so. We considered using surveys to determine general sentiments, but after further thought, found this endeavour impossible. Our methods and rationales are explained below.
We conducted interviews with high-ranking Singapore-based representatives of major synthetic dye companies.
Unfortunately, natural dye manufacturers are not based in Singapore, and our attempts to contact them were unsuccessful. We thus interviewed experts in biomanufacturing instead, to determine how disruptive our technology could be in contrast to conventional manufacturing methods.
Local fashion designers who used natural dyes were also interviewed. Because there are too few of them, surveys would not be statistically significant. Moreover, having conversations instead would allow us to explore the issue more deeply. Views of international fashion designers were gleaned from secondary sources.
Singapore does not have a textile and dyeing industry. The problem we wanted to solve is far removed from the ordinary consumer here, and so surveying them would yield little useful information. We interviewed a “super consumer” instead and sought her advice on how to make ordinary consumers aware of systemic problems in fashion. We also initiated public engagement efforts via social media.
As implied earlier, such people are not available in Singapore, and are difficult to meaningfully contact. To substitute, we drew on secondary sources such as newspapers, paying special attention to direct quotes from the people affected.
Human Practices Issues Identified
Our project required the additional investigation of these Human Practices issues: environmental impact, philosophy/ethics, public engagement/dialogue, product design, public policy/legislation. Safety, security, and risk assessment are addressed on our Safety page.
See our Design page for more on how we compared our environmental impact against other dye manufacturing methods.
We considered three basic types of first-order ethical theories as frameworks to reason if our project is moral, i.e., if it would have an overall positive impact on the world - duty-based theories, consequentialist theories, and virtue-based theories. Duty-based theories are not useful because it does not have sufficient explanatory power to help us resolve what we see as the main quandary - the choice between the duty to reduce the harm caused by pollution, and the duty to ensure others’ livelihoods in the face of disruptive technologies. Virtue theory was discarded as its focus is too personal for our purpose. It seems fundamentally misguided to evaluate how our project affects others by emphasizing our own character. In contrast, although consequentialist theories have their flaws, it is clear that the main thrust of Human Practices is to weigh our actions against alternatives, and intuitively this is the most appropriate type of theory in the context of iGEM.
However, the “utility calculus” was challenging to implement for this complex issue. We estimated, to the best of our abilities, that if our product is scaled up to industrial levels and widely adopted, we would improve more lives by eliminating the pollution caused by synthetic dyes, than harm by threatening others’ employment in dye manufacturing companies. At the very least, our project at this stage, being a prototype of a novel biomanufacturing process, is of some scientific value and therefore has a positive impact on the world. We thus decided to go ahead with our project.
Additional ethical considerations have been evaluated in our Safety Form.
Ordinary consumers are not directly affected by synthetic dye pollution. In fact, students in Singapore, i.e. future consumers or perhaps even already prolific consumers, are mostly ignorant about synthetic biology and its potential as a tool to solve such problems. To raise awareness, not merely about the focus of our project but also of synthetic biology itself, we strategically targeted post-secondary students in our outreach events and facilitated meaningful dialogues on both topics. Read more on our Education and Engagement page.
See our Integrated Human Practices section below for our discussions with experts on how to improve our product design.
Biomanufacturing and biomanufacturing research is generously supported in Singapore, as shown by the establishment of NUS SynCTI and the Biotransformation Innovation Platform at A*STAR, a statutory board supporting research aligned to areas of competitive advantage and national needs for Singapore. We have also been supported by NUS Synthetic Biology for Clinical and Technological Innovation, a highly interdisciplinary research program for synthetic biology. Thankfully, public policy and legislation was not a hurdle we had to clear.
Integrated Human Practices
This section shows how our investigation of Human Practices issues in the previous section has been integrated into the purpose, design, and execution of our project, thus fulfilling iGEM's requirements for Integrated Human Practices.
Our project's purpose was influenced by experts in various fields, including industry experts, researchers at universities and research institutes (e.g. A*STAR), the Yong Loo Lin School of Medicine, and also medical staff at the National University Hospital. Talking to people as knowledgeable as Dr. Foo Jee Loon, for example, helped us eliminate three other problems we were considering to focus on fashion, namely, tropical diseases, wound healing, and diagnosing multi-drug resistant organisms.
Our project's design changed after we used expert interviews to forecast our potential impact. At first, we wanted to produce many colours of dyes in a single cell - orange, pink, and brown. However, after conversations with major synthetic dye manufacturers and fashion designers, we understood the market better. Based on the advice of experts such as Mr. Holger Schlaefke and Miss Leong Minyi, we decided to focus on synthesizing luteolin, a yellow dye.
Our hardware also went through several design iterations based on user feedback. For example, our characterization tool, PDF-LA! developed a functional symmetry following discussions with Dr. Teh Ai Ying, a research fellow in NUS. Please visit our page on PDF-LA! to find out more!
For our project's execution, we consulted experts on biomanufacturing like Dr. Nic Lindley and Dr. Yvonne Chow to find out how to optimize our production through bioreactor design. They helped to validate our project's design choices, such as the choice to utilize xylose in our feedstock and the use of optogenetics instead of chemical inducers. We used their suggestions to consider a futuristic bioreactor design.
To help our project engage a wider audience, we sought out a fashion studies scholar, Miss Angelene Wong, to identify which parts of our project would appeal to others, and understand how we could better communicate to the ordinary consumer. Her insights have informed how we chose to present our project on social media and this wiki.
For more details, do consider spending time browsing through our expert interviews writeups!
Bringing our dye to a natural dye designer
Coup Dy’etat is a project that is committed to integrating user feedback into every aspect of our project, from project direction to execution. One artist in particular, Miss Leong Minyi, was with us at the beginning of our project. Her feedback was instrumental to our decision to produce yellow dye. Now, after having successfully produced luteolin, amongst other achievements (see our demonstrate page), we returned to seek her comments on our last ever batch of luteolin, thus finally completing a full revolution of the design-build-test-learn cycle of engineering.
Our conversation with her was, as usual, fruitful as we learnt much about dyes that we had not previously considered. We gained insights as to why our naturally-produced dye did not behave as expected - she raised the possibility that the fabric we dyed was turning brown due to the possibility of tannins being present, or it could also have been due to oxidation of the dye upon exposure to air. Another interesting point she raised was regarding the application of mordants to the textiles to facilitate the binding of the natural dye to the fabric - the most common mordants are aluminium, which brings out the vibrancy of the yellow colour, and iron, which tones down certain colours. As mordants are currently necessary for the successful dyeing of textiles using natural dyes, the holy grail would still be creating a natural dye that does not require mordanting, a problem synthetic dyes evade.
We also asked her to share her method of extracting dyes so that we could compare it against our own methods. Our team tried DMSO and methanol separately, and it was interesting to find out that she simply used water as a solvent, but performed repeated rounds of extraction by heat. It is important to note here that Ms Leong Minyi specialises in extracting dyes from natural sources, especially from leaves and roots, whereas our project focuses on bacteria-produced dyes, which could account for the differences in methods of extraction.
Lastly, she advised us once again that we should keep the consumers’ needs for light-fastness, colour-fastness etc in mind, and ultimately aim to have our dye’s properties evaluated through professional testing. Also, when the time comes to market our dye, we should consider that consumers may still not have come to terms with biomanufacturing as a concept. Thus, although we may be excited to share synthetic biology with the world, the world may not be ready for us.
How we integrated her comments into our project
It was extremely heartening to have been able to meet a major source of inspiration for our project before we left for the Giant Jamboree. Due to time constraints, we were unfortunately unable to incorporate her comments into the design of our project. However, her insights have set the stage for the next iteration of a design-test-build-learn cycle. We were able to use the production of natural dyes as a case study to learn how to better optimise our bio-manufacturing platform, as the principles discussed were broad.
From working on the way bacterial dyes are perceived, to producing “super” natural dyes that do not require mordants - the possibilities for our project are plentiful. With the support of inspirational creatives such as Ms Leong, the world of natural dye making is our oyster!
Write-ups for each interview summarizing the stakeholder/Human Practice issue involved, what we learned, and how we subsequently acted on this feedback are presented below. These write-ups have been approved by their subjects prior to their upload on our wiki.
Click on each photo in the gallery to enjoy its corresponding write-up. We have over 10 interviews!
25 May 2018 - Dr. Foo Jee Loon
Dr. Foo Jee Loon is a senior research fellow from the Department of Biochemistry at the NUS Yong Yoo Lin School of Medicine. We approached him for his expertise in engineering microbes for biochemical production. He reviewed the metabolic pathway that we were proposing and determined that controlling the flux of intermediates was key to allowing our engineered cells to switch between different product pathways.
He noted that the project would be complex due to the large number of enzymes involved. Because of this, he warned us of the large amount of characterization work that would be required if the functional data on these enzymes were unavailable. He advised us to start by engineering a bacterium that could perform the second half of the pathway using one the intermediates as feedstock. At the same time, we could design an efficient and sensitive method to control the expression of key genes to influence the flux of intermediates.
Some of the other tips he gave us included the use of monoculture rather than polyculture due to better mass transfer characteristics. He also introduced us to the concept of bioremediation, which would further enhance the environmental impact of our project.
25 May 2018 - NUH Staff
We contacted one of the NUH System’s Assistance Directors, who is an expert on infectious diseases as a part of our team’s final efforts to select a project. She gave us a pleasant surprise when she invited a senior consultant of the NUH's Department of Laboratory Medicine to participate in the interview as well. We hoped to find out if our idea for a better detector for CP-CRE was feasible, and if not, what else could we attempt in the three precious months before the Giant Jamboree.
While they acknowledged the importance of improving MDRO detection methods, they counselled that given our circumstances, it would be challenging to produce a working prototype. They suggested that our efforts could be better spent elsewhere, such as developing a test for active TB to assist clinicians with the differential diagnosis. We discussed potential methods and biomarkers, and also learned that to improve on existing tests, we needed to prioritize patient care and create an affordable point-of-care test.
From the interview, we gained an overview of current solutions to MDRO and TB detection, and the Drs. made us aware of the stiff competition we would face, from other university research teams, to pharmaceutical conglomerates, all of whom are investing a staggering amount of time and money into solving the same problem we were considering. We concluded that for either CP-CRE or TB detection, it was unlikely that our team would produce a solution to outperform current or developing ones, and the resources so generously granted to us could be better spent solving another problem. This avenue of inquiry was thus drawn to a close.
As a footnote to the conclusion of our MDRO adventures, we must mention that the Drs selflessly sacrificed their lunch hour for the interview! We are very, very grateful for their help.
*The doctors have declined to be named, and hence the anonymity throughout the write-up*
31 May 2018 - Dr Melissa Fernandez
Dr. Melissa Fernandez is a Senior Research Fellow at the Institute of Medical Biology, A*STAR. We spoke with Dr. Melissa and her team to gain a better understanding on one of our initial ideas – promoting wound healing of chronic wounds in the tropical setting. She explained to us that developing wound dressings with biosensors would be extremely difficult, and would not be a feasible project to accomplish within three months.
She advised us about the complexities of chronic wounds – namely the different types of chronic wounds, from diabetic ulcers to venous leg ulcers. On top of the types of wounds, patients will have a lot of comorbidities, which very heavily influence the wound healing factor. This makes wound healing very complex, whereby the underlying pathogenesis is hard to elicit. Focusing on one pathway may capture only 10% of the patients.
She gave us a thorough walk-through of the wound healing process, and identified problems we had not anticipated; for example - testing for the other substances that the bacteria produce and their effects on the wounds will take very long. These might have unforeseen effects, such as binding to enzymes and growth factors produced by our bodies, making them inactive or even pathogenic.
14 June 2018 - Mr. Holger Schlaefke
Mr Holger Schlaefke is the Global Marketing Manager for Cellulosic Dyes at Huntsman Textiles Effects. With an impressive 21 years of experience in the dyeing and textile industry under his belt, he was well equipped for us to approach to find out more about what we should consider when designing a dye. It helped that Mr Schlaefke was warmly hospitable and accommodating, and extremely forthcoming with his knowledge about dyes.
The interview validated the need for more sustainable dyeing technologies to reduce the water pollution caused by the textile industry, and helped us affirm the key aspects of the problem. We also found the answers we sought! From an industry perspective, we now know that we should take into account how dye manufacturers need to be agile in response to the fashion industry’s ever-changing demands for the trendiest colours of the season, and the requirements our dye must fulfill to be considered eco-friendly, among others.
On the topic of natural versus sustainable dyes, we learned that the industry is keener on using synthetic dyes rather than natural dyes even though natural dyes are considered more environmentally friendly. This is because of the industry’s perception that firstly, synthetic dyes are superior to natural dyes in terms of wear resistance, secondly, producing synthetic dyes is less complicated and time consuming, and finally, companies producing synthetic dyes would have to completely change their machinery and infrastructure. This was why he foresees that it will be difficult to persuade textile producers and other major stakeholders to adopt new, potentially industry-disrupting solutions involving natural dyes.
We then shared our vision of producing natural dyes biosynthetically, to which he listed important challenges and obstacles to anticipate and overcome should we decide to continue on this path. Furthermore, he suggested that producing primary colours or brighter and bolder colours would be more impressive and would make our solution more attractive to our stakeholders.
At the end of the interview, Mr Holger encouraged us to never give up, and even jestingly reminded us that whatever we do, there was one thing we should never forget - to get a patent for our project!
18 June 2018 - Miss Leong Minyi
Miss Leong Minyi, a Fashion Design graduate of the Nanyang Academy of Fine Arts, is the founder of Mai Textile Studio. Her beautiful art and clothes are created using indigo dye and traditional Japanese techniques such as shibori and katazome. As she currently works directly with natural dyes and textiles, we felt that her perspective on our problem would be invaluable.
Having come fresh from a team meeting about the salient points gained from our interview with a representative from the synthetic dye industry, it was interesting to see which were supported by Miss Leong, and on which points did their views diverge. For example, both parties agreed that natural dyes appealed to a niche market in Singapore, and for the most part, consumers are not concerned with the origin of the dye, but rather how it looks on their clothes as well as their bank account statements.
However, Miss Leong was more skeptical about the representative’s recommendations on how natural dyes could be made more appealing to a wider market, so she gave us some suggestions of her own.Miss Leong mentioned that natural dyes could be made more appealing through branding and marketing. Having a narrative behind the brand can evoke emotions in consumers. Furthermore, colour play, cutting and modernization of designs are also essential to appealing to a wider market. For greater public acceptance of bio-manufactured dyes, a narrative on its environmental sustainability is required, in addition to informing consumers on important dye performance indicators (stability, lightfastnest, colurfastness, reliability). She also stated that removing biased preconceptions against “bacteria” products would be helpful.
Miss Leong cautioned us that yellow was not a popular locally worn colour, but was a non-issue compared to the replacement of synthetic dyes. Miss Leong also recommended to focus on the production of synthetic dyes, achieve good colour mixing and produce an array of colours.
Drawing on her experience of working with natural dyes, she taught us much about the different plants we could consider extracting dyes from and creative techniques such as infusing cellulose-based textiles with proteins or tannins to increase the the fabric ability to absorb dyes. We even touched on her deeply moving experience of meeting her idol, the late legendary experimental textile designer Junichi Arai. From this interview, we became aware of even more factors to consider when designing our dye, such as the ratio of water to dye to the weight of the fabric, or how much dye is required to get a specific intensity.
Her artisanal approach has introduced our team to a whole new paradigm, where the inherent flaws of natural dyes are valuable precisely because of their imperfect nature. One criticism of natural dyes is that they are dull and muted. However, during the interview, we learned a secret - because of this, all natural dyes match well with each other. In contrast, synthetic dyes would appear garish.
Miss Leong’s interview was incredibly helpful, yet our task ahead has become even more difficult, because we now have to consider the problem on a more visceral, aesthetic level. But we believe that makes our problem all the more worthwhile to solve.
28 June 2018 - Mr. Gerard Talhoff
Mr Gerald is the Global Manufacturing and Supply Chain Vice President of the DyStar Group. His current responsibilities range from managing global manufacturing footprint to supply chain management and even to corporate sustainability. With more than 20 years of experience in the dye industry under his belt, Mr. Gerald was able to impart pearls of wisdom gleaned from his many years of experience to our inquisitive young minds.
During our interview with Mr Gerald, he introduced to us the core tenets driving DyStar’s sustainability initiatives. Central to their thrust was a three-fold approach - reducing the production carbon footprint, ensuring consumer safety by keeping hazardous chemicals out of textiles and dyes used, and striving for biodegradable textiles and materials used for dyeing.
Mr Gerald validated our proposed design, pointing out that balancing environmental friendliness and commercial feasibility would no longer be unfeasible. Instead, co-opting sustainable practices in textile dyeing would become a competitive advantage; governmental agencies around the world are taking tougher actions against environmentally-unfriendly practices and pollution.
When discussing our potential solutions, he anticipated a critical hurdle we would have to overcome: our solution must attain significant yield for it to have a significant impact on the dye market. From his experience, past attempts at producing bio-engineered dyes have failed to become commercially-viable due to their failure to achieve significant yield. This is a potentially disastrous pitfall that we must seek to circumvent.
Besides our solution of producing microbial dyes, Mr Gerald warned us against the production of natural dyes using agricultural biomass as feedstock. The resulting competition between food production and natural dye production would indeed be very unfavourable for. In addition, disposal of used biomass would exacerbate the problem of resource wastage. Adding to his previous point, Mr Gerald taught us that it was imperative to evaluate the entire production process for its eco-friendliness, taking into account energy and water consumption, waste generation, to name a few.
An interesting twist to the end of the interview, Mr Gerald raised the possibility for the obsolescence of dyes in future, as textiles could coloured by virtue of its physical properties, or perhaps the invention of new materials that are not amenable to current dyeing methods. To tie the interview up, before continuing on our journey with synthetic biology, he encouraged us to evaluate bioengineered products around the world critically.
11 July 2018 - Mr. Vinod Agnihotri
Mr Vinod Agnihotri manages LANXESS operations in Singapore, as well as its Material Protection Products Business Unit for the Asia Pacific region. While LANXESS is no longer involved in textile dye production, Mr Agnihotri himself has a degree in Chemistry, and another in Textile Chemistry and Fibre Technology. As Singapore does not have a textile industry, we felt very fortunate to have found such an expert so close to home.
We started the interview by discussing fashion’s environmental impact. Once again, we were told that the textile and dye industries were not environmentally-friendly in all aspects. He confirmed our problem statement by identifying the most pollutive part of fashion - the production of the dye itself. The water quality of water bodies near dye factories are an especially big concern, and the pollutive impact of the chemicals depends on the method of production and the yield.
After hearing what we planned to do to solve this, Mr Agnihotri told us that to be competitive, our dyes need to achieve industry fastness levels, and we have to produce many shades with high reproducibility, while remaining economic. In short, while natural dyes could be relatively better for the environment compared to synthetic dyes, our natural dyes still need to meet performance criteria demanded by consumers.
To wrap up our interview, we went through the perceptions of different stakeholders. Firstly, as consumers are getting more savvy, there is a growing demand for higher-quality, less pollutive dyes. Secondly, he pointed out that the educated layman, as a consumer, may be repulsed by the idea of bacteria having previously been in the dye, even if we claim that all the bacteria has been removed from the dye. This was valuable to us because our team initially believed that consumers and designers would be more interested in our dyes if they knew it had been made using synthetic biology. It indicates that our next step for Human Practices should be to find out how many other people share his opinion. Thirdly, eco-friendly dyes are something chemical companies would welcome, because the average consumer associates “chemical” with “harmful”. Lastly, he suggested that to become commercially successful, we could collaborate with prominent brands in the fashion industry who are willing to experiment with natural dyes.
03 August 2018 - Miss Angelene Wong
Ms Angelene Wong is many things - a dancer, a writer, and a fashion studies scholar. She currently studies at Parsons Paris, and is en route to completing her Masters in Fashion Studies in May 2019. Her passion for fashion is fueled by her understanding of fashion as a cultural phenomenon - a lens through which we could better understand society, a veritable avenue for artistry and creativity, and an undeniable amalgamation of culture, history, and identity.
07 September 2018 - Dr. Nic Lindley
Dr Nic Lindley is the Strategic Director of the Biotransformation Innovation Platform (BIP) at the Agency for Science, Technology and Research (A*STAR) Singapore. BIP is a research initiative to discover novel sustainable biotechnology to produce high value-added specialty chemical ingredients for use in food, nutrition and consumer care. This spans the screening of natural and synthetic biodiversity to identify new target molecules which harness the natural or engineered metabolic capacity of microbial platforms adapted to the constraints of industrial scale fermentation. They hope to provide sustainable biotechnology solutions which satisfy consumer desires to see natural ingredients and clean labels. As their research objectives seemed highly similar to ours, we sought Dr Lindley’s expertise and experience to understand how we could refine our own system, and increase our luteolin and naringenin yields.
We explained our project to him, and he validated our approach, saying that the devastating effects of chemical synthesis on the environment provides us a compelling reason for favouring biological synthesis instead, though it remains important to keep organic synthesis cost-effective and high-yield. He also approved of key aspects of our project, namely, our use of xylose in our feedstock, and our optogenetic circuit. Although we initially chose to utilize xylose just to give empty fruit bunches a new lease of life and make our process more environmentally friendly, Dr. Lindley was enthusiastic about our use of xylose due to the abundance of cheap lignocellulosic waste sources, amongst other advantages. However, he reminded us that as xylose is a hemicellulose molecule, hemicellulose degradation produces other pentose sugars which could have compatibility issues with our substrates, and thus it is important to retain the cells’ ability to metabolize glucose. As for our blue light-repressible circuit, he said that it was good to induce enzyme expression by the absence of blue light, as light is more cost-effective than chemical inducers. Another advantage of using a repressible system is that light penetration would become limited anyway as cell densities increase. He anticipated that using light as a control would become a problem once we try to scale up our project to the industrial level. Once again, light penetration would be the crux, but perhaps we could consider maintaining light distribution throughout the medium using fibre optics.
Of course, our project at that stage was far from perfect. When we shared our problems with Dr. Lindley, he gave us suggestions on how to increase our yield and expounded on bioreactor design theories. One key principle would be to strike a balance between growth and production. This can be achieved by maintaining a very minimal vital growth phase, as once cells stop growing, they inevitably lose biological activity. Getting the right feeding would be essential to achieving that fine balance, but trying to limit growth by restricting sugar supply would actually cause cells to favour growth over production instead, as they are inclined to prioritize their own survival. This illustrates the possibility of manipulating the feedstock to control metabolic flux. Turns out xylose has more utility than we previously thought! Another insight he offered was for us to select an organic layer and incorporate it into our growth medium, so that our products could move into the layer and stop inhibiting cell growth, which is basically the operating principle of biphasic bioreactors.
Dr. Lindley gave us feedback on other components of our system as well, in particular, the stress sensor. While he felt that our idea was of value, he was unsure if it would continue to work as expected once our system migrates to an industrial bioreactor. As cells move around inside a bioreactor due to mixing, they will encounter different physical and chemical environments and will thus frequently experience different stresses, for example, stress due to heat shock response, oxygen limitation, and carbon limitation. It would be challenging to determine the predominant stress in such a complex system. However, our current system is small enough that it is reasonable to assume uniform mixing, and thus a homogeneous physical and chemical environment throughout. The predominant stress is then most likely the stress from recombinant protein expression, and our stress sensor’s detection scope is sufficient for our current purposes.
Throughout the interview, Dr. Lindley strove to help us envision our project beyond iGEM by sharing the problems faced by industrial-scale bioreactors, relating these to the challenges we were facing, and proposing ways for us to circumvent or alleviate these issues. Since we learned how the industry is particularly conservative when it comes to bioreactor design, we decided to challenge ourselves. Using Dr. Lindley’s valuable input, we radically rethought the bioreactor and designed one that would synergize with our optogenetic circuit.
Take a look at our futuristic bioreactor here!
11 September 2018 - Yvonne Chow
Dr. Yvonne Chow is a research scientist at the A*STAR Biotransformation Innovation Platform. Her work mainly revolves around bioprocess engineering and fermentation technology; it was with this in mind that we interviewed Dr. Yvonne to find out more about the bioreactors and their associated challenges. During our interview, we took the opportunity to share our project design and methodology, and sought her opinion about our team progress.
After sharing more about our proposed novel method for the bioproduction of luteolin, a natural flavonoid dye, through using xylose as a feedstock, she endorsed our efforts to increase xylose uptake in bacteria, as xylose is often formed during lignocellulosic breakdown together with glucose. However, glucose is normally the favoured product that bacteria use, leaving xylose underutilised and regarded as “waste”. Dr Yvonne commented that better utilisation of xylose in our cells would definitely enhance our narrative of using natural fruit products as our feedstock material.
On bioreactors, Dr. Yvonne cautioned us on the need for a good stirring method and equal distribution of light to each cell, as well as take into consideration the amount of time required for our promoter to be exposed to the light for the repression system to work effectively. Using a conventional magnetic stirring method could result in cells exposure to blue light repression on the exterior of the bioreactor to be limited to at most 5 minutes. She then recommended for us to consider internal lights in the bioreactor, or even attach the lights onto the stirring mechanism.
As we discussed cell-stress and cell toxicity, Dr. Yvonne suggested to include an in situ extraction of the products of the bioreactor, as this could help moderate cell toxicity levels. Dr. Yvonne also validated the benefits of having a automated feedback regulation, as it could result variability to the bioreactor as much as possible. To end off, she advised us to test our laboratory constructs in a scaled down bioreactor, as cellular behaviour could change in a constantly stirring environment.