Point 1: Defining the Scope of Implementation for Our Project
This was our initial point of ideation . After several brainstorming sessions and team consensus, we thought that tackling innovation within the biofuels industry would be the most interesting and most realistic problem to solve given our constraint in time and resources. In order to learn more, we decided to reach out to Frank Fields, part of the California Algal Biotechnology Lab at UC San Diego. Established in part by a national initiative to further biofuel research across the United States, this particular lab's interests include expression of therapeutic proteins in algae and the structure of the chloroplast ribosome .
Frank helped us focus on some of the most relevant challenges in biofuel production, including:
- Producing on a scale large enough to actually contribute to energy needs
- Operational costs - this arose primarily from two issues:
- Lack of complete feedstock utilization made it an inefficient process
- These settings were often susceptible to bacterial contamination
- Achieving price parity with fossil fuels
Frank also cautioned us about this last concern in biofuel production, noting that the PI of the lab, Dr. Stephen Mayfield, had started several relevant companies in the biofuel industry, including Solazyme and Sapphire Energy. He mentioned that one of the biggest reasons that recent interest in biofuel had dipped was that the renewable energy has heavily struggled to achieve economic parity with oil prices . Because the government agencies refrained from increasing already-substantial subsidies for farmers, biofuel production eventually began to lag, leading to a complete stop in the industry. Thus, he thought that if our project was to be successful, it had to somehow lower production costs on a sizable industrial scale.
We were extremely thankful for Frank's comment that biofuel production was a very niche issue that would be difficult from both an industrial and entrepreneurial perspective. Thus, we decided to broaden the scope of our project to a bio-production platform to have a more tangible impact. This achieved two things: (1) It allowed for a general implementation of our project, offering versatility in its end products and introducing the quality of modularity, (2) It allowed us to incorporate our original goal of addressing some potential shortcomings in current biofuel production methods if the environment was optimal.
Point 2: Gaining Insight on Current Problems in Biologics Manufacturing and A Possible Solution
After our discussion with Frank, we also decided to get a more in-depth perspective of the challenges in metabolic engineering and associated fields. We reached out to Novogy, a Boston-based company that specializes in bio-based production of food, fuel, and pharmaceutical substances. This was particularly beneficial to us because talking to an industrial setting would allow us to see a cursory version of what we wanted to implement. Thus, we got in contact with Dr. Shaw, Senior Research Director .
Dr. Shaw helped us focus on the experimental approach that we planned on taking. He encouraged us to make sure that our approach was:
- Less susceptible to bacterial contamination
- Relatively easy to implement
To help, he also pointed us to a recent paper that he had published entitled Metabolic Engineering of Microbial Competitive Advantage for Industrial Fermentation Processes. In this technique, scientists engineered metabolic pathways into host organisms that allowed them to use unconventional compounds that contained nitrogen and phosphorus, allowing both bacteria and yeast to outcompete potential contaminants.
Dr. Shaw' s main concerns guided us throughout the project because it allowed us to have an end product that would actually have industrial relevance. We decided to tackle each point through further consultation.
Going back to Frank's point, we realized a key point of achieving economic parity with fossil fuels would be decreasing or eliminating the risk of bacterial contamination. We decided that implementing the ROBUST Method would provide a viable solution to this problem . In our research on industrial fermentation processes, we learned that the fact that glucose could be easily metabolized by many organisms, including potential pathogens, contributed to contamination.
We decided to implement the method in microbial organisms to synthesize valuable, unconventional carbon sources as feedstock that could similarly prevent contamination for our bio-production platform.
Point 3: Establishing Criteria for Carbon Source Selection
Up to this point, our team needed to decided what carbon source could serve as an alternative to current food-based crops that served as feedstock. We decided to talk to Dr. Dan Wichelenki at a startup in Virginia called Bonumose Biochem LLC which is currently involved in the synthesis of artificial sweeteners. We felt that this was most beneficial because it allowed us to understand the criteria for having an industrially viable manufacturing process, which was one of Dr. Shaw's key points.
We discussed several different topics in a tele-conference call with Dr. Wichelenki. First, he expressed great interest in our project, noting that his company manufactures two sugars that serve as artificial sweeteners: allose and tagatose . When we explained to him that we were trying to use a different feedstock, he suggested that we do in depth analysis of the carbon source because that was going to be a central aspect of our project. He urged us to look at factors including (1) cost of manufacturing for each, (2) any documented effects of that sugar, and (3) chemical properties that could rely on less expensive methods of purification. He said that most carbon sources would be more expensive than the typical glucose, but its long-term benefits in preventing contamination outweighed any short-term savings.
After a team meeting following the Skype with Dr. Wichelenki, we agreed that he had given some invaluable advice regarding the assessment criteria for choosing these carbon sources. However, we felt that we had to add another criteria to that list, namely the idea that the carbon source had to have at least some detailed research on its potential applications. The reason we wanted to make this distinction was because we believed that in order to minimize our environmental impact, we needed to use a rare carbon source that had already been well characterized by others, especially use on an in industrial scale.
After in-depth analysis of 4 sugars (xylose, mannose, lactose, and tagatose) using Dr. Wichelenki's criteria, we managed to narrow it down to xylose and tagatose. After another round of consideration, we ultimately decided on tagatose because of its recent traction on the artificial sweetener market.
Another additional benefit was the length of the biochemical pathway
It confirmed tagatose was good because it was a shorter biochemical production pathway that required fewer enzymes and produced fewer intermediate metabolites
Point 4: Closer Look at Tagatose
At this point, we had identified tagatose as the primary source. Although it would not really impact our carbon source selection, we decided to get a deeper understanding of the appeal for tagatose. It would be prudent for when scientists and other individuals questioned us about the rationale for our overall design. Therefore, we turned to Richard Alejandro, director of the Bay Area chapter of the American Diabetes Association .
Although Mr. Alejandro was impressed with our idea in the broadest terms, he admitted that his position gave him more credibility to discuss the tagatose and its overall appeal. He also suggested that despite recent traction of tagatose in the artificial sweetener market and its Generally Regarded as Safe status, public perception should become an important factor in our decision. He also gave us helpful advice in constructing a public questionnaire and administering it in an unbiased way as to avoid introducing bias and error into our assessment .
We decided that it was important to carry out a public survey for the sake of thoroughness; thus, the first step was to determine the questions that we were going to ask and word them in an unbiased manner. This was extremely important because individuals were already likely to have a negative bias against genetically modified organisms and any approach with GMOs. In addition, we had to decide the appropriate medium for the survey to be effective. Thus, we decided to administer it at the Pleasanton Public Library in August, and used the results to assess the efficacy of our approach. Public perception suggested that 37% of individuals were comfortable with our xenobiotics approach and tagatose biosynthesis mechanism; however, only 30% of the respondents were familiar with tagatose and wanted more information before backing our proposal.
Point 5: Discussion for Organism Choice
At this point, we had selected the carbon source for the implementation of the ROBUST method. However, we were struggling to decide as a team on which microbe to use for exporting the tagatose metabolizing pathway. Therefore, we turned to Pengfei Gu, part of the pHD program at Washington University in St. Louis. His research interests include metabolic biochemistry and DIY Biology processes. We thought that his familiarity with aligning research fields would definitely help narrow down our decision.
Pengfei was impressed with the scope of our project and thought the use of xenobiotic compounds to prevent bacterial contamination was an idea that needed to be explored further. He believed that exporting the tagatose metabolism pathway to prevent contamination was a smart idea but we also had to choose the right organism to be able to have the most effective solution. He pointed out that many organisms possess biochemical pathways that utilize biomass and conversion into products that resemble biofuels . However, these native micro-organisms are often ineffective in industrial processes. Furthermore, these isolated strains typically suffer from a lack of genetic and molecular biology tools. These traits are often limited and regulated by complex cascades of cellular control, and some additional functions need to be added to produce an integrated, multifunctional biocatalyst.
Therefore, he proposed the following criteria for the ideal bioprocessing microbe:
- Needs to be easily manipulated
- Proven successful in an industrial setting and scale
- Superior conversion yields
Pengfei suggested that using synthetic biology to discover interesting bio-mass degrading and biofuel-producing genes can be transferred into a genetically tractable organism. Keeping in mind that we planned to do a proof of concept (for either this year's project or future implementation), we wanted organisms that have had preliminary work done regarding tagatose metabolism. After some literature research, it seemed that E. coli would be the most ideal choice: key parts of its genome are well characterized and it has widespread tools for genetic manipulation. In addition, it is one of a few model species that can be used as a launching point for other scientists in the future.
In addition, the Joris Lab in Belgium had performed gene knockouts in E. coli and enabled them to be able to metabolize tagatose. As a result, the convenience of tools at our disposal convinced us that changing the feedstock to tagatose and exporting fermentation biochemical pathways in E. coli would be the primary aim of our project.
Point 6: Discussion with Ideker Lab and Consultation for S. elongatus
At this point, our team had identified several aims of the research project, with the key challenge being the elimination or at least the containment of bio-contamination in industrial fermentation processes. At this stage, we wanted to get input from industry sponsors and turned to Dr. Kreisberg and the San Diego Center for Systems Biology. This was because his group has assisted past iGEM teams and includes several graduate students with competition experience, making it a valuable asset for us to discuss how to analyze our progress. We also wanted some secondary confirmation regarding our research proposal and thought that Dr. Kreisberg and his group could help us out.
Dr. Kreisberg and his group were very impressed with what we were trying to accomplish; however, they also helped us rein in our expectations. They suggested that when we constructed the work flow for the project, it include a short-term vision and a long-term rollout for implementation.
They also noted that our solution was lacking because it was not very cost-effective. Dr. Kreisberg suggested that we do a quick economic analysis and calculation to determine which factors in the bio-production process could be handled more efficiently. He also encouraged us to keep the system easy to use because that would be a key selling point if we decided to commercialize the technology.
After discussion with Dr. Kreisberg and his team, we decided that one way to drive down the cost of our system was to synthesize the tagatose within our proposed system. Instead of having an external source produce tagatose and then introduce it to the environment for E. coli to metabolize, we decided that having a multi-organism approach would be more cost-effective This would also help reduce the costs to a point where our solution would become economically relevant in industry applications.
Our Applied Design section includes some economic calculations based on our implementation of a co-culture system with a ROBUST approach, leading to a nearly 50 fold reduction in costs.
Another key decision that our team had to make was the choice of organism to produce the tagatose. After several hours of discussion, our team settled on using S. elongatus PCC 7492 for several reasons: cyanobacteria naturally grow on light and carbon dioxide, bypassing the need for fermentable plant biomass and arable land Because lowering costs was a key objective for our solution, cyanobacteria would play a key role in achieving that objective.
Point 7: Discussion about the Ethics of a Xenobiotics Approach for Our Project
Although we were curious to see how the implementation of the modified ROBUST method would affect the overall goals of our project, we also realized that there were ethical ramifications to our approach. Therefore, we decided to discuss potential drawbacks and ways to address them. We also wanted a non-research but still scientific perspective that would allow us to acknowledge some of the larger challenges in synthetic biology. Thus, we met with Dr. Reuther, an associate lecturer at UCSD who actually teaches a class based on synbio principles.
In our discussion with Dr. Reuther, he also expressed wonder at the goals of our project. However, he cautioned us about some of the drawbacks of using a xenobiotics approach:
- Toxicity to host organism
- Concern about environmental release
- Lack of predictive modeling regarding intermediate metabolites
In addition, he cited that one of the biggest challenges in synthetic biology was the lack of standardization and well-characterized parts. Although the Registration Center of Standard Biological Components contains more than 5000 parts, most of them have only preliminary levels of characterization, thus raising the possibility of an inaccurate description.
From the perspective of an educator, he also suggested that we could use our project as an opportunity to get students more excited about synthetic biology. He noted that although there are a number of biology textbooks, they all follow the same pattern of information, leading to redundancy and lack of creativity.
We confirmed that an accidental release of tagatose-producing cyanobacteria would not pose a sizable environmental risk. In fact, we also modified the genetic constructs to address Dr. Reuther's first concern regarding a mechanism control toxicity from potential. We included a potential riboswitch in the BioBrick construct because it allowed the cyanobacteria to stop producing tagatose when concentrations of substrates and intermediate metabolites became unmanageable.
Point 8: Consultation with the Vlachos Group at the University of Delaware
After constructing the metabolic pathway to theoretically enable cyanobacteria to synthesize tagatose starting with naturally present sucrose as a beginning point, we knew that within the time frame, the furthest we would probably get is confirm that the cyanobacteria had produced tagatose as a proof of concept. Therefore, we wanted to get insight from the Vlachos Group about ideal detection methods.
In our discussion with one of the postdocs, we realized that our initially proposed method of engineering biocatalysts to bind to tagatose and emit a fluorescent dye was unnecessarily complex and expensive. In trying to keep it simple and easy to detect, we opted to use HPLC (high performance liquid chromatography) . However, the postdoc also expressed doubt that the current metabolic pathway could produce enough tagatose to be chemically distinguished from the other sugars naturally present within the cyanobacteria.
She suggested that we do the following two things to ensure a higher chance of success for this part of the project:
- Find the specific HPLC column that has been documented for tagatose detection, because most columns can detect galactose but not really rare carbon sources such as tagatose.
- Find a method to increase either the conversion rate for tagatose production or increase the overall amount of tagatose that was being produced. She suggested that modeling specific parameters would be useful in assessing our next step
From this discussion, we changed our detection method to HPLC because it was simpler and more convenient. We also changed the construct of the BioBrick by introducing a flag tag . In the event that tagatose was not successfully synthesized, we would be able to identify at which point the pathway had failed.
Another very important change that we made was a modification to the metabolic pathway. Because we wanted to increase overall concentration of tagatose present within the cell, we did some flux balance analysis modeling and confirmed that a shift to a two loop system instead of a one loop system did not pose any singificant increases. Although there was no way of knowing for certain, our team felt that using this strategy would get tagatose concentration to at least the minimally detectable level based on the enzymatic pathway that we had constructed.
Point 9: Learning about Bioprocessing Design
Going back to one of our initial discussions, we felt that we had not done our due diligence in presenting an industrially viable solution for bacterial contamination. While our solution is economically viable, it was ultimately about being able to visualize some sort of prototype for a bioreactor that contains our ROBUST technology. Therefore, we thought that it would be important to understand the basic parameters of bioreactor design and general bioprocessing techniques. To get more insight, we discussed potential solutions with the Golden Lab about how to put all the components of our project over together.
We learned that in order to have an industrially acceptable project, we needed to differentiate between the different stages of bioprocessing, specifically upstream processing and downstream processing.
The upstream process is the primarily lab-based component. This involves the step in which microbes are grown and engineered to produce the substances of interest. Factors contributing to upstream processing include growth rate, typical duration of fermentation, production rate, medium cost, and strain development costs.
Factors contributing to downstream processing are harvesting and extraction of a purified product. Typical difficulties include treatment with chemical solvents and expensive techniques to harvest the substances . The lab suggested that we look into ways to minimizing costs of these features because these can account for up to 75% of overall costs in a system.
By doing so, the lab also suggested that we could think about the different kinds of bioreactors and think about how we were going to house two different types of organisms, one photosynthetic and one non-photosynthetic. They also suggested that we do research into existing solutions, which we have documented on the Applied Design section of our website.
We decided against using a single space to enclose both organisms because there would be no way to really control and optimize growth control for both of these organisms if they were in a close proximity to one another. Instead, we decided that a separate two-compartment design would be more ideal and help us maximize the potential of our system.
To make our system commercially viable, we realized that another component to this project (not the focus for this year) would be to engineer a transport mechanism for tagatose out of the cyanobacteria. At the current stage, lysing the cell and checking with HPLC would be sufficient, but we thought that a transport mechanism would make the bio-production platform more cost-effective. Thus, the entire scope of our project now included a 3 step cost-reduction strategy:
- Reducing long term costs and increasing sustainability via the ROBUST method
- Using a co-culture design to start the first metabolic production pathway
- Genetic engineering to help transport the tagatose directly into the growth medium for the E. coli and make a more sustainable platform
Point 10: Factors to Consider for the Upscaling of Cyanobacteria
Although E. coli expression platforms have been popularized in biotechnology over the last several decades, even a cyanobacteria model organism such as S. elongatus has only recently become noticeable for purposes of genetic manipulation. We realized that the scale of the work we were doing in the lab was not even close to the industrial scale we were envisioning, so we thought that it would be important to discuss the factors and considerations when trying to increase the production from cyanobacteria. Thus, we decided to get in contact with a local San Diego company, Triton Algae, to discuss the challenges of working with S. elongatus.
Our discussion with research scientists at Triton Algae centered around strategies to decrease costs of producing the tagatose even further. At this point, by switching to a genetic engineering - based method rather than chemical-based, we had already significantly decreased the cost of synthesizing tagatose. The implementation of the four-step metabolic pathway was an important step, but he thought that we could also consider the environmental impact of our project. We learned that the current methods for algae cultivation include open and closed systems, including raceway ponds and photo-bioreactors (PBR) . He invited us to consider the lifecycle of the platform and choose to optimize our design accordingly.
In addition, one of the research scientists also pointed us to one of his publications and emphasized that the success of this project from an engineering perspective also required some research about materials science and how it pertains to bioreactors. He also suggested that we try to perform a
Now that we knew about the importance of differentiating between upstream and downstream bioprocessing, we also wanted to do our best so that our product could become even more cost-effective. Since we were now using a co-culture design, it was important to consider the upstream processing for the cyanobacteria component of our platform.
Point 11: Entrepreneurship and concept validation
Because we wanted to ultimately make a product that could be utilized by industry, we decided to get advice about how to proceed with commercialization of our wet lab work, including concept validation and tapping into the Bay Area ecosystem of thriving startups. We decided to go to the Livermore iGATE Innovation Hub because it represents a unique combination of technology based startups, science innovation through partnerships with Sandia National Laboratories, and a growing network of life sciences investors. Specifically, we were looking for the key criteria that would be hit or miss for most investors.
We first met with Mr. Brandon Cardwell, the director of this setup. He explained the original thoughts behind iGATE and how it would serve as a
Our discussion with Mr. Cardwell gave us some great insights about what needed to occur from both a scientific and entrepreneurial perspective. He agreed that it was definitely an interesting project, and offered us resources and other contacts who could help us with this specifics of entering the biotechnology industry.
- Have a proof of concept
- Protect our research through the proper intellectual property channels
- Be aware of the regulatory aspects and potential obstacles for the commercialization of the ROBUST technology
- Keep a reasonable expectation about the timeframe for the implementation of the bio-production pathway, and try to keep it as simple as we could
We laid out a pathway to commercialization in a reasonable timeframe, hoping that our proposed bio-production platform can help solve issues such as bacterial contamination while also paving the way for the next generation of biologics manufacturing.
Point 12: Visual Storytelling consultant, Jen Boyce
As we began to put together our work so that people could view it on the 2017 UCSD iGEM wiki, we realized that there were a number of components that we had overlooked, namely the method of presenting information throughout the website. After going through nearly 200 websites from last year's competition, we set out to redefine the style of conveying information, relying on more graphic design and clearer organization of information. To get some creative ideas, we consulted Ms. Jennifer Boyce, a visual storytelling expert from Oregon.
Because we understand that the iGEM project should be a reflection of our ideas, not someone else's, we had already laid out some ideas to discuss with Ms. Boyce. Her key advice was that at every stage during the website, we had to keep in mind our audience. Because we are presenting highly technical information, it would be important to find the balance between simplification and visual appeal. She also suggested that because most people are visual learners, we should try to find opportunities to convey information through infographics and structuring the information, both literally and figuratively.
From this point, we created a design philosophy for our website. We wanted to modify the existing paradigms of information giving and allow the user to control the overall experience. We also realized that we needed unique ways of conveying the different points, so we held multiple brainstorming sessions. The result is that every page on this website has a very specific purpose with clear organization of information, wrapped in a sleek design that enhances the information provided.