Difference between revisions of "Team:NUS Singapore-A/shadow/Hardware"

 
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<h1>Introduction</h1>
 
<h1>Introduction</h1>
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<p>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.</p><br>
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<br>
  
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<h1>Policy Compliance</h1>
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<p>Our hardware team developed two sets of hardware to address two problems in synthetic biology, and complement the work of the wet lab team to complete our optogenetic biomanufacturing platform. </p>
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<p>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. <br>
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<br>
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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. <br>
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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.</p><br>    
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<h1>Methodology</h1>
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<h2>Problem #1</h2>
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<p>In the beginning, based on 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.<br>  
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<br>
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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.</p><br>
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<p>The first problem is that while there is a rapidly-growing interest in using optogenetics for biomanufacturing, development of custom tools to support the research of optogenetic circuits cannot match this pace, and is insufficient to meet user needs<sup>[2]</sup>. An example of the most current hardware tools available is a modified Tecan microplate reader, which provides controlled illumination on top of its usual measurement capabilities<sup>[3]</sup>. Such an approach is costly and requires specialized knowledge of the microplate reader model. Another example would be the open-source light exposure tool constructed for a 24-well plate<sup>[4]</sup>. To our team, it seemed that scaling-up in optogenetic research (Figure 1) was not well-supported by current hardware solutions, which only cater to microwell plates. </p>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 1</b>. Scaling-up in optogenetics research - from the microplate to small-scale bioreactor</figcaption>
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</figure>
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<br>
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<p>Yet, the biomanufacturing industry is expected to deliver products to the market, in high volumes, at high quality, and at competitive prices<sup>[5]</sup>. If we are ever to bring our optogenetic biomanufacturing platform to an industrial scale, it is necessary to bridge the gap between the microplate and the industrial bioreactor, and adapt our cells for actual large-scale bioreactor conditions. We thus designed a suite of three devices, called <i>PDF-LA!</i>, which enables the characterization of optogenetic circuits at different scales - 12-well <b><u>P</u></b>late, petri <b><u>D</u></b>ish, and conical <b><u>F</u></b>lask. We also created a bench-top optogenetic bioreactor, <i>Light Wait</i>. It is our vision that these devices will empower optogenetic researchers to make great leaps forward in their research, although we acknowledge that there is a still-greater leap between our humble bioreactor and an industrial bioreactor (Figure 2). For now, it is enough for us to have taken the first few steps.</p>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b>. The components in Figure 1 (bottom right-hand corner) are still dwarfed by an industrial bioreactor.</figcaption>
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</figure>
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<br>
  
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<h2>Groups Identified</h2>
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<h2>Problem #2</h2>
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<p>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.</p>
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<p>The second problem is that although a proof-of-concept already exists for optogenetic biomanufacturing, the process can be further optimized to bring the vision of an industrial-scale optogenetic bioreactor closer to reality. </p>
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<br>
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<p>For some background, Zhao et al. have increased yield of isobutanol from yeast by using a blue light repressible system in a simple bioreactor, showing the potential of optogenetics in biomanufacturing<sup>[6]</sup>. However, they did not optimize the duration or intensity of blue light, instead shining blue light periodically. We discovered that dynamic regulation is a good method for optimizing biomanufacturing, because prioritization of growth and production can be achieved simultaneously. We distilled this observation from both literature<sup>[7]</sup> and our <a href="#">Human Practice</a> activities. Dynamic regulation can be achieved through computer-assisted feedback control, and we found that Argeitis et. al developed automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth<sup>[8]</sup>. So far this is the most recent and sophisticated feedback system for optogenetics. However after examining his method, we found that while his feedback control system was closed-loop, his physical system was open. Measurement samples were discarded as waste. This is not advantageous to biomanufacturing, as this will lead to much product being wasted, lowering effective yield.</p>
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<br>
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<p>
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To solve this, we combined the insights and design features from these two systems (Zhao and Argeitis) to create an automated, closed-loop feedback control system for <i>Light Wait</i>.</p>
  
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<h3>Synthetic Dye Manufacturers</h3>
 
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<p>We conducted interviews with high-ranking Singapore-based representatives of major synthetic dye companies. </p>
 
  
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<h3>Natural Dye Manufacturers</h3>
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<h3>PDF-LA!</h3>
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<p>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. </p>
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<br>
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<h4>Function</h4>
 +
<br>
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<p>Plate-Dish-Flask Light Apparatus (<i>PDF-LA!</i>) supports optogenetic research by allowing researchers to investigate cells cultured in 12-well plates, petri dishes, and Erlenmeyer flasks.</p>
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<br>
  
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<h3>Fashion Designers</h3>
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<h4>Product Demonstration</h4>
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<p>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.</p>
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<h3>Ordinary Consumers</h3>
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<br>
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<p>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. </p>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b>. gif in progress</figcaption>
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 1</b>. Showcase of PDF-LA!</figcaption>
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</figure>
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<br>
  
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<h3>People Suffering from Synthetic Dye Pollution</h3>
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<video src="#" width="300"> uploaded to drive </video>
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<p>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.</p><br>
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<figure><figcaption>Video 1. With PDF-LA!, you’ll be light-years ahead of the competition! At the very least, you can program your own snazzy light show and be the envy of other optogenetics researchers.</figcaption></figure>
  
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<h2>Human Practices Issues Identified</h2>
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<p>The utility and functionality of <i>PDF-LA!</i> was validated by user feedback. We also used it when characterizing the behaviour of EL222 in repressible and inducible systems, thus producing what you see on our Results page.</p>
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<p>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.</p>
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<br>
  
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<h3>Environmental Impact</h3>
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<h3>How it Works</h3>
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<p>See our Design page for more on how we compared our environmental impact against other dye manufacturing methods. </p>
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<br>
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<h4>Operation</h4>
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<br>
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<p>This operation guide assumes that all electronics have been assembled and programmed. Ensure that this has been completed before operation, else results may vary.  Instructions may be found on our dedicated page for <a href="#"><i>PDF-LA!</i></a>. </p>
 +
<br>
 +
<ol>
 +
<li>Place your container into the required holder. If using an Erlenmeyer flask, first rest the flask on <i>D-LA!</i>, then place the flask adapter over the flask to form <i>F-LA!</i>. Keeping a firm grip on <i>F-LA!</i>, pull the flask upwards sharply to ensure a tight fit.</li>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b>GIF of plate, dish, flask going into each container</figcaption>
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</figure>
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<br>
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<li>Connect the AC adapter to the Arduino and wall socket.</li>
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<br>
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<figure class="figures">
 +
  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b>arduino, AC adapter picture, wall socket picture, arrows to indicate</figcaption>
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</figure>
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<br>
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<li>Turn on the wall switch controlling the AC adapter. </li>
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<br>
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</ol>
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<br>
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<p>The devices should light up as shown in the product demonstration video above.</p>
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<br>
  
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<h3>Philosophy/Ethics</h3>
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<h4>Possible Configurations</h4>
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<p>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 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.<br>
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<br>
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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. <br>
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<p><i>DF-LA!</i> was designed with modularity and flexibility as fundamental guiding principles. Many configurations are possible, enabling researchers to customize their experimental setups to a greater degree. While P-LA! was designed separately and thus does not have this functionality, a final solution, <a href="#"><i>PDF-LA! 2.0</i></a>, to provide a truly integrated solution was designed and can be found on our dedicated page for <a href="#"><i>PDF-LA!</i></a>. Unfortunately, while we could not actualize this solution due to time constraints and limits on our 3D printing equipment, it is our hope that future iGEM teams may be able to experience, test, and improve <i>PDF-LA! 2.0</i>’s utility and functionality.</p>
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Additional ethical considerations have been evaluated in our Safety Form.</p>
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<br>
  
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<h3>Public Engagement/Dialogue</h3>
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<p>Examples of the possible configurations can be found below.</p>
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<p>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.</p>
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<br>
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<ul>
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<li>1 x D-LA!, bottom illu, bottom and top illu</li>
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<li>1 x F-LA!, bottom illu, bottom and top illu</li>
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<li>N x DF-LA!, bottom-bottom and bottom and top illu</li>
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</ul>
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<br>
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<h4>Components</h4>
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<br>
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<p><i>P-LA!</i> comprises a tech holder and a lighting plate</p>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b>picture of tech holder, picture of lighting plate, GIF of collapsed assembled <i>P-LA!</i></figcaption>
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</figure>
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<br>
  
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<h3>Product Design</h3>
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<p>Collectively, a single unit of <i>DF-LA!</i> comprises a tech holder, a petri dish illumination column, and a flask adapter. </p>
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<p>See our Results section below for our discussions with experts on how to improve our product design.</p>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b>picture of tech holder, a petri dish illumination column, and a flask adapter, GIF of collapsed assembled DF-LA!</figcaption>
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</figure>
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<br>
  
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<h3>Public Policy/Legislation</h3>
 
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<p>Biomanufacturing and biomanufacturing research is generously supported in Singapore, as shown by the establishment of the Biotransfomation Innovation Platform at A*STAR, a statutory board supporting research aligned to areas of competitive advantage and national needs for Singapore. Thankfully, this was not a hurdle for us.</p><br>
 
  
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<h1>Results</h1>
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<p>Presenting, <i>PDF-LA!</i> Click <a href="#">here</a> for its dedicated page.</p>
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<p>Write-ups for each interview summarizing the stakeholder/Human Practice issue involved, what we learned, and how our project was subsequently informed by this feedback are presented below. These write-ups have been approved by their subjects prior to their upload on our wiki.<br>
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While we cannot release full interview transcripts or recordings, the questions we asked our interviewees are available for downloading as .pdf files at the end of each write-up. This is to make our process more transparent. We hope that the reasons why we asked each interviewee the questions we did will be self-evident. We also believe that the depth and breadth of the questions we asked will show how our team attempted to creatively, meticulously, and exhaustively understand the problem, our solution, and our impact.<br><br>
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Click on each photo in the gallery to enjoy its corresponding write-up.</p>
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<br><br>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Figure 2</b><i>P-LA!</i> and <i>DF-LA!</i> side by side</figcaption>
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</figure>
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<br>
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<hr>
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<br>
  
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<div id="carousel">
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<h3>Light Wait</h3>
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  <carousel-3d :autoplay="false" :controls-visible="true" :controls-prev-html="'&#10092;'" :controls-next-html="'&#10093;'" :controls-width="30" :controls-height="60" :width="720" :height="540" :display="3">
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<br>
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<h4>Function</h4>
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    <slide :index="0">
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<p><i>Light Wait</i> supports optogenetic research, especially in optogenetic biomanufacturing, by allowing researchers to scale up to a 500 ml working volume bioreactor.</p>
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      <button class="sponsor-boxes tablinks" onclick="openSponsor(event, 'IHP1')">
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<br>
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        <figure>
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<h4>Product Demonstration</h4>
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          <img src='https://static.igem.org/mediawiki/2018/0/0a/T--NUS_Singapore-A--IHP_FooJeeLoonSTD.jpg' style='max-height:100%;max-width:100%;'>
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<br>
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<video src="#" width:"300">Bioreactor Backup Video</video>
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<br>
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<figure><figcaption><b>Video 2</b>. <i>Light Wait</i> may be housed in a shaking incubator unit such as the one shown above.</figcaption></figure>
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<br>
  
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          <figcaption>"BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH"
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<h4>Validation</h4>
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          </figcaption>
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<br>
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<p><i>Light Wait</i> was validated through a series of experiments which first proved each component’s functionality, and then the functionality of the whole system when all the components were assembled. </p>
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<br>
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<h4>Experimental Plans</h4>
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<br>
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<h4>Experimental Results</h4>
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<br>
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<h4>How it Works</h4>
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<br>
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<p>This operation guide assumes that all components have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. For instructions on how to set up and operate each component of <i>Light Wait</i>, please refer to our dedicated component pages.</p>
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<br>
  
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        </figure>
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<h4>Operation</h4>
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      </button>
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<br>
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    </slide>
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<ol>
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  <li>Place <i>Light Wait</i> in a shaking incubator unit as shown in our Product Demonstration. Take care to ensure that all wires and tubing are slack and of sufficient length, else they may become disconnected during operation. </li>
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  <li>Fill and cover the fermentation chamber. </li>
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  <li>Connect the pump, 2-in-1 sensor, and the fermentation chamber with the silicone tubings in a loop as shown below (Figure _). The remaining 2 small tubes are for introducing more media, and an air pump. </li>
  
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    <slide :index="1">
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      <button class="sponsor-boxes tablinks" onclick="openSponsor(event, 'IHP2')">
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<figure class="figures">
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        <figure>
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  <img src="#" alt="Video">
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          <img src='https://static.igem.org/mediawiki/2018/1/14/T--NUS_Singapore-A--IHP_HolgerSchlaefke.jpg' style='max-height:100%;max-width:100%;'>
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  <figcaption><b>Figure __</b>. Illustration of how the pump, sensor, and fermentation chamber should be connected by silicone tubing.</figcaption>
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          <figcaption>"BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH"
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</figure>
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          </figcaption>
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<br>
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        </figure>
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      </button>
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    </slide>
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    <slide :index="2">
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<li>Turn on the AC adapters for the pump and the LEDs in the fermentation chamber.</li>
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      <button class="sponsor-boxes tablinks" onclick="openSponsor(event, 'IHP3')">
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<li>The pump should begin to rotate and the LEDs should light up. </li>
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        <figure>
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<li>Connect the Arduino controlling the 2-in-1 sensor to your PC. </li>
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          <img src='https://static.igem.org/mediawiki/2018/5/5e/T--NUS_Singapore-A--IHP_LeongMinyiSTD.jpg' style='max-height:100%;max-width:100%;'>
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<li>Load the code for the 2-in-1 sensor and open the Serial Monitor to check that the sensor is collecting data. </li>
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          <figcaption>"BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH"
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<li>After verifying that all the components are working to your satisfaction, close the shaking incubator door. </li>
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          </figcaption>
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<li>When the OD reaches your target levels, the LEDs in the fermentation chamber will turn off. The green LED in the 2-in-1 sensor will also turn off, and the red LED will turn on. The default OD in the code is 0.6.</li>
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        </figure>
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<li>When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are stressed, the LEDs in the fermentation chamber will turn on again.</li>
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      </button>
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<li>When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are NOT stressed, the LEDs in the fermentation chamber will turn off again.</li>
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    </slide>
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<li>Steps 7-8 will repeat indefinitely, unless you power the system off.</li>
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</ol>
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<br>
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<h4>Components</h4>
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<br>
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<p><i>Light Wait</i> comprises a peristaltic pump, a 2-in-1 OD and fluorescence sensor, and a fermentation chamber. Click on the picture of the component to be taken to its dedicated page!</p>
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<br>
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<figure class="figures">
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  <img src="#" alt="Video">
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  <figcaption><b>Video goes here</b> : labelled picture of pump</figcaption>
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</figure>
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<br>
  
  
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    <slide :index="3">
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  <button class="accordion"> TEST </button>
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      <button class="sponsor-boxes tablinks" onclick="openSponsor(event, 'IHP4')">
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    <div class="panel" style="line-height: 17em;">
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          <img src='https://static.igem.org/mediawiki/2018/f/f8/T--NUS_Singapore-A--IHP_Gerard_Talhoff.jpg' style='max-height:100%;max-width:100%;'>
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           <figcaption>"BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH"
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           <table>
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                <h3><i>Abs<sub>600</sub></i></h3>
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              </td>
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              <td>
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                <ul style="list-style: none; margin: 0; padding: 1em; text-align:left; border-left: .5px solid black">
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                  <li> Wavelength: 600nm </li>
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                  <li> Read Speed: Normal </li>
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                  <li> Delay: 100 msec </li>
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                <h3><i>Fluorescence</i></h3>
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                  <li> Excitation: 485 </li>
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                  <li>Emission: 525</li>
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                  <li>Optics: Top</li>
 +
                  <li>Gain: 50</li>
 +
                  <li>Light Source: Xenon Flash</li>
 +
                  <li>Lamp Energy: High</li>
 +
                  <li>Read Speed: Normal</li>
 +
                  <li>Delay: 100 msec</li>
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                  <li>Read Height: 7 mm</li>
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                <h3><i>Abs<sub>600</sub></i></h3>
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          <img src='https://static.igem.org/mediawiki/2018/9/97/T--NUS_Singapore-A--IHP_VinodAgnihotri.jpg' style='max-height:100%;max-width:100%;'>
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                  <li> Wavelength: 600nm </li>
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                  <li> Read Speed: Normal </li>
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                  <li> Delay: 100 msec </li>
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                <h3><i>Fluorescence</i></h3>
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                <ul style="list-style: none; margin: 0; padding: 1em; text-align:left; border-left: .5px solid black">
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                  <li> Excitation: 485 </li>
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                  <li>Emission: 525</li>
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                  <li>Optics: Top</li>
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                  <li>Gain: 50</li>
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                  <li>Light Source: Xenon Flash</li>
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                  <li>Delay: 100 msec</li>
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                  <li>Read Height: 7 mm</li>
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<h2> CONFIGURATIONS </h2>
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<p> KITTY IPSUM dolor sit amet discovered siamesecalico peaceful her Gizmo peaceful boy rutrum caturday enim lived quis Mauris sit malesuada gf's saved fringilla enim turpis, at mi kitties ham. Venenatis belly cat et boy bat dang saved nulla other porta ipsum mi chilling cat spoon tellus.</p>
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<p> KITTY IPSUM dolor sit amet discovered siamesecalico peaceful her Gizmo peaceful boy rutrum caturday enim lived quis Mauris sit malesuada gf's saved fringilla enim turpis, at mi kitties ham. Venenatis belly cat et boy bat dang saved nulla other porta ipsum mi chilling cat spoon tellus.</p>
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<h2>Bio-production</h2>
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<p>It’s important to have automation in bioproduction especially in industrial level. We designed a small bioreactor system which incorporated optical density (OD) and fluorescence sensors to control the metabolic behaviours in E. coli. </p><br>
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          <figcaption>"BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH BLAH"
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<h4>Automated Control through feedbacks</h4>
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<p>IN progress. </p>
  
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<h4>OD/F sensor</h4>
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<p>IN progress.</p>
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<h4>Pump</h4>
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<p>IN progress.</p>
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    <img src="https://static.igem.org/mediawiki/2018/a/a6/T--NUS_Singapore-A--IHP_FooJeeLoon.jpg" class="sponsor-pic" alt="Dr Foo Jee Loon"></a>
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    <span class="sponsor-description"> 25 May 2018 - Dr. Foo Jee Loon
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<br> <br>
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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.
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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. 
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<br><br>
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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.
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    <span class="sponsor-description"> 14 June 2018 - Mr. Holger Schlaefke
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<br> <br>
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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.
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<br> <br>
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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.
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<br> <br>
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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.
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<br> <br>
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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.
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<br> <br>
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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!
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    <img src="https://static.igem.org/mediawiki/2018/b/b6/T--NUS_Singapore-A--IHP_LeongMinyiSolo.jpg" class="sponsor-pic" alt="Miss Leong Minyi"></a>
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    <span class="sponsor-description"> 18 June 2018 - Miss Leong Minyi
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<br> <br>
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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.
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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.
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<br> <br>
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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. 
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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.
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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.
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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.
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<br> <br>
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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.
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    <span class="sponsor-description"> 28 June 2018 - Mr. Gerard Talhoff
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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. 
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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.
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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. 
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<br> <br>
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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.
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<br> <br>
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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.
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<br> <br>
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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.
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    <span class="sponsor-description"> 11 July 2018 - Mr. Vinod Agnihotri
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<br> <br>
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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.
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<br> <br>
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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.
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<br> <br>
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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. 
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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.
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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.
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Latest revision as of 16:03, 15 October 2018

CONNECT WITH US

Introduction


Our hardware team developed two sets of hardware to address two problems in synthetic biology, and complement the work of the wet lab team to complete our optogenetic biomanufacturing platform.


Problem #1


The first problem is that while there is a rapidly-growing interest in using optogenetics for biomanufacturing, development of custom tools to support the research of optogenetic circuits cannot match this pace, and is insufficient to meet user needs[2]. An example of the most current hardware tools available is a modified Tecan microplate reader, which provides controlled illumination on top of its usual measurement capabilities[3]. Such an approach is costly and requires specialized knowledge of the microplate reader model. Another example would be the open-source light exposure tool constructed for a 24-well plate[4]. To our team, it seemed that scaling-up in optogenetic research (Figure 1) was not well-supported by current hardware solutions, which only cater to microwell plates.


Video
Figure 1. Scaling-up in optogenetics research - from the microplate to small-scale bioreactor

Yet, the biomanufacturing industry is expected to deliver products to the market, in high volumes, at high quality, and at competitive prices[5]. If we are ever to bring our optogenetic biomanufacturing platform to an industrial scale, it is necessary to bridge the gap between the microplate and the industrial bioreactor, and adapt our cells for actual large-scale bioreactor conditions. We thus designed a suite of three devices, called PDF-LA!, which enables the characterization of optogenetic circuits at different scales - 12-well Plate, petri Dish, and conical Flask. We also created a bench-top optogenetic bioreactor, Light Wait. It is our vision that these devices will empower optogenetic researchers to make great leaps forward in their research, although we acknowledge that there is a still-greater leap between our humble bioreactor and an industrial bioreactor (Figure 2). For now, it is enough for us to have taken the first few steps.


Video
Figure 2. The components in Figure 1 (bottom right-hand corner) are still dwarfed by an industrial bioreactor.

Problem #2

The second problem is that although a proof-of-concept already exists for optogenetic biomanufacturing, the process can be further optimized to bring the vision of an industrial-scale optogenetic bioreactor closer to reality.


For some background, Zhao et al. have increased yield of isobutanol from yeast by using a blue light repressible system in a simple bioreactor, showing the potential of optogenetics in biomanufacturing[6]. However, they did not optimize the duration or intensity of blue light, instead shining blue light periodically. We discovered that dynamic regulation is a good method for optimizing biomanufacturing, because prioritization of growth and production can be achieved simultaneously. We distilled this observation from both literature[7] and our Human Practice activities. Dynamic regulation can be achieved through computer-assisted feedback control, and we found that Argeitis et. al developed automated optogenetic feedback control for precise and robust regulation of gene expression and cell growth[8]. So far this is the most recent and sophisticated feedback system for optogenetics. However after examining his method, we found that while his feedback control system was closed-loop, his physical system was open. Measurement samples were discarded as waste. This is not advantageous to biomanufacturing, as this will lead to much product being wasted, lowering effective yield.


To solve this, we combined the insights and design features from these two systems (Zhao and Argeitis) to create an automated, closed-loop feedback control system for Light Wait.

PDF-LA!


Function


Plate-Dish-Flask Light Apparatus (PDF-LA!) supports optogenetic research by allowing researchers to investigate cells cultured in 12-well plates, petri dishes, and Erlenmeyer flasks.


Product Demonstration


Video
Figure 2. gif in progress
Video
Figure 1. Showcase of PDF-LA!

Video 1. With PDF-LA!, you’ll be light-years ahead of the competition! At the very least, you can program your own snazzy light show and be the envy of other optogenetics researchers.

The utility and functionality of PDF-LA! was validated by user feedback. We also used it when characterizing the behaviour of EL222 in repressible and inducible systems, thus producing what you see on our Results page.


How it Works


Operation


This operation guide assumes that all electronics have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. Instructions may be found on our dedicated page for PDF-LA!.


  1. Place your container into the required holder. If using an Erlenmeyer flask, first rest the flask on D-LA!, then place the flask adapter over the flask to form F-LA!. Keeping a firm grip on F-LA!, pull the flask upwards sharply to ensure a tight fit.

    2. Video
    Figure 2GIF of plate, dish, flask going into each container

  2. Connect the AC adapter to the Arduino and wall socket.

    3. Video
    Figure 2arduino, AC adapter picture, wall socket picture, arrows to indicate

  3. Turn on the wall switch controlling the AC adapter.


The devices should light up as shown in the product demonstration video above.


Possible Configurations


DF-LA! was designed with modularity and flexibility as fundamental guiding principles. Many configurations are possible, enabling researchers to customize their experimental setups to a greater degree. While P-LA! was designed separately and thus does not have this functionality, a final solution, PDF-LA! 2.0, to provide a truly integrated solution was designed and can be found on our dedicated page for PDF-LA!. Unfortunately, while we could not actualize this solution due to time constraints and limits on our 3D printing equipment, it is our hope that future iGEM teams may be able to experience, test, and improve PDF-LA! 2.0’s utility and functionality.


Examples of the possible configurations can be found below.


  • 1 x D-LA!, bottom illu, bottom and top illu
  • 1 x F-LA!, bottom illu, bottom and top illu
  • N x DF-LA!, bottom-bottom and bottom and top illu

Components


P-LA! comprises a tech holder and a lighting plate


Video
Figure 2picture of tech holder, picture of lighting plate, GIF of collapsed assembled P-LA!

Collectively, a single unit of DF-LA! comprises a tech holder, a petri dish illumination column, and a flask adapter.


Video
Figure 2picture of tech holder, a petri dish illumination column, and a flask adapter, GIF of collapsed assembled DF-LA!

Presenting, PDF-LA! Click here for its dedicated page.


Video
Figure 2P-LA! and DF-LA! side by side


Light Wait


Function

Light Wait supports optogenetic research, especially in optogenetic biomanufacturing, by allowing researchers to scale up to a 500 ml working volume bioreactor.


Product Demonstration



Video 2. Light Wait may be housed in a shaking incubator unit such as the one shown above.

Validation


Light Wait was validated through a series of experiments which first proved each component’s functionality, and then the functionality of the whole system when all the components were assembled.


Experimental Plans


Experimental Results


How it Works


This operation guide assumes that all components have been assembled and programmed. Ensure that this has been completed before operation, else results may vary. For instructions on how to set up and operate each component of Light Wait, please refer to our dedicated component pages.


Operation


  1. Place Light Wait in a shaking incubator unit as shown in our Product Demonstration. Take care to ensure that all wires and tubing are slack and of sufficient length, else they may become disconnected during operation.
  2. Fill and cover the fermentation chamber.
  3. Connect the pump, 2-in-1 sensor, and the fermentation chamber with the silicone tubings in a loop as shown below (Figure _). The remaining 2 small tubes are for introducing more media, and an air pump.

    4. Video
    Figure __. Illustration of how the pump, sensor, and fermentation chamber should be connected by silicone tubing.

  4. Turn on the AC adapters for the pump and the LEDs in the fermentation chamber.
  5. The pump should begin to rotate and the LEDs should light up.
  6. Connect the Arduino controlling the 2-in-1 sensor to your PC.
  7. Load the code for the 2-in-1 sensor and open the Serial Monitor to check that the sensor is collecting data.
  8. After verifying that all the components are working to your satisfaction, close the shaking incubator door.
  9. When the OD reaches your target levels, the LEDs in the fermentation chamber will turn off. The green LED in the 2-in-1 sensor will also turn off, and the red LED will turn on. The default OD in the code is 0.6.
  10. When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are stressed, the LEDs in the fermentation chamber will turn on again.
  11. When the fluorescence from the stress reporter reaches your predetermined value indicating that the cells are NOT stressed, the LEDs in the fermentation chamber will turn off again.
  12. Steps 7-8 will repeat indefinitely, unless you power the system off.

Components


Light Wait comprises a peristaltic pump, a 2-in-1 OD and fluorescence sensor, and a fermentation chamber. Click on the picture of the component to be taken to its dedicated page!


Video
Video goes here : labelled picture of pump

Abs600
  • Wavelength: 600nm
  • Read Speed: Normal
  • Delay: 100 msec

Fluorescence
  • Excitation: 485
  • Emission: 525
  • Optics: Top
  • Gain: 50
  • Light Source: Xenon Flash
  • Lamp Energy: High
  • Read Speed: Normal
  • Delay: 100 msec
  • Read Height: 7 mm

Abs600
  • Wavelength: 600nm
  • Read Speed: Normal
  • Delay: 100 msec

Fluorescence
  • Excitation: 485
  • Emission: 525
  • Optics: Top
  • Gain: 50
  • Light Source: Xenon Flash
  • Lamp Energy: High
  • Read Speed: Normal
  • Delay: 100 msec
  • Read Height: 7 mm


CONFIGURATIONS


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Video
Video goes here : blah blah 3


Video
Video goes here : blah blah 3


Video
Video goes here : blah blah 3

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Bio-production

It’s important to have automation in bioproduction especially in industrial level. We designed a small bioreactor system which incorporated optical density (OD) and fluorescence sensors to control the metabolic behaviours in E. coli.


Automated Control through feedbacks

IN progress.

OD/F sensor

IN progress.

Pump

IN progress.



HEAR OUR STORY

We’re using synthetic biology to change the way the world thinks and functions.

We’re a little disruptive, dangerous and maybe a tad too bold. But the world needs some shaking up every now and then, and we think we’re just the right people for that.

Together we’re going to engineer biology and create something awesome.

CONTACT US

nusigem@gmail.com

21 Lower Kent Ridge Rd, Singapore 119077