Team:Virginia/GI
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Human Practices
“The potential for synthetic biology and biotechnology is vast; we all have an opportunity to create the future TOGETHER.” - Ryan Bethencourt
Human Practices
CHAPTER 4: GAIN INSIGHT
3A) Scientific Guidance
With our proposed solution at hand, Team Virginia contacted researchers within biotechnology, synthetic biology, chemical manufacturing, and pharmacology to gain insight into our proposed design. We wanted to engage in earnest conversations that provided constructive feedback to push Manifold towards a more sustainable and socially good design. Hence, in this phase, Manifold underwent several project-wide revisions as we integrated meaningful expert feedback into refining a modular and industrially-standardized version of Manifold, where our proof of concept was to produce a heart medication call “resveratrol”. Mainly, our team wanted these conversations to direct our team focuses and encourage greater perspective on the practical aspects of synthetic biology, like modeling and wetlab research. To begin this phase, our team identified 3 questions from our earlier conversations with scientists, stakeholders and end-users. These included “What bacterial microcompartment should we choose?,” “How do we make the DNA scaffold and enzyme binding domains more modular,” and “What would our bioreactor setup look like?” This led our team to Professor Martin Warren, whose recommendations on the bacterial microcompartment would later guide our conversations with experts involved with DNA scaffolds and experts involved with pathway enzymes. Ultimately, these conversations would allow us to conceptualize a working Manifold device, recognize future optimization through bioreactor design, and understand how Manifold could be implemented in the chemical manufacturing industry.
CHOOSING THE BACTERIAL MICROCOMPARTMENT
From the conceptualization of Manifold, Team Virginia recognized the important role of the protein shell. Not only does the it sequester our biosynthetic reactions from other cellular processes, but further bounds our pathway enzymes (attached to DNA scaffolds) to the interior shell. Thus, choosing the correct bacterial microcompartment was essential to solving both the toxic intermediates and pathway competition problems as explained by the scientific perspective. Although the 2020 Virginia iGEM Team initially designed Manifold using “propanediol utilization (PDU) bacterial microcompartments”, our vision for a modular and industrially-standardized version of Manifold required greater reflection on what protein shell to base Manifold around. As a result, Team Virginia carefully studied the literature, where we eventually narrowed our decision between PDU bacterial microcompartments and carboxysomes. Both microcompartments contain pores that permit diffusion of small polar molecules across the shell, while broadly excluding large or nonpolar molecules from entering or leaving. This regulation makes these microcompartments ideal, because industrial biosynthesis commonly involves polar starting materials that must enter the protein shell and non-polar intermediates that must remain in the protein for biosynthesis to occur. Furthermore, specific peptides have been identified in both carboxysomes and PDU bacterial microcompartments that allow researchers to attach molecules (like DNA scaffolds) to the interior shell. However, with the team split between using PDU bacterial microcompartments versus carboxysomes, Team Virginia needed expert feedback to determine which variant was best for our project?
Professor Martin Warren
HOW SHOULD OUR TEAM CHOOSE THE CORRECT PROTEIN SHELL FOR MANIFOLD?
Introduction: Dr. Warren is a professor of biochemistry at the University of Kent in Canterbury, England. Ever since completing his PhD studies at Southampton University, Dr. Warren has had the extensive opportunity of studying biosynthesis and observing its evolution throughout the chemical manufacturing industry. In 1989, he worked on engineering cells to produce vitamin B12. In 2007, he was awarded the prestigious BBSRC Professional fellowship for his work on bioengineering of complex metabolic pathways. Ever since, he has published numerous articles on tetrapyrrole biosynthesis, which eventually led to his current research focus in bacterial microcompartments. As a leading expert in bacterial microcompartment research, Team Virginia first contacted Dr. Warren to understand the key differences between PDU bacterial microcompartments and carboxysomes that current literature often overlooks.
HOW SHOULD OUR TEAM CHOOSE THE CORRECT PROTEIN SHELL FOR MANIFOLD?
Introduction: Dr. Warren is a professor of biochemistry at the University of Kent in Canterbury, England. Ever since completing his PhD studies at Southampton University, Dr. Warren has had the extensive opportunity of studying biosynthesis and observing its evolution throughout the chemical manufacturing industry. In 1989, he worked on engineering cells to produce vitamin B12. In 2007, he was awarded the prestigious BBSRC Professional fellowship for his work on bioengineering of complex metabolic pathways. Ever since, he has published numerous articles on tetrapyrrole biosynthesis, which eventually led to his current research focus in bacterial microcompartments. As a leading expert in bacterial microcompartment research, Team Virginia first contacted Dr. Warren to understand the key differences between PDU bacterial microcompartments and carboxysomes that current literature often overlooks.
Discussion: From our hours long conversation with Dr. Warren, we learned that although PDU bacterial microcompartments and carboxysomes were largely similar in function (encasing reactions that have toxic or volatile intermediates), carboxysomes differed from PDU bacterial microcompartments in two key respects. First, carboxysomes are composed of anywhere between 12-15 different polypeptides, whereas PDU bacterial microcompartments are composed of strictly 14 different polypeptides. Second, carboxysomes have a significantly smaller ratio of pentameric protein to hexamers than PDU bacterial microcompartments, meaning more simply, that transport across carboxysome pores is essentially insignificant compared to transport across pores in PDU bacterial microcompartments. As a result, this key difference eventually led our team to question, “PDU bacterial microcompartments may be better suited for our project, but with 14 different polypeptides, is engineering this protein shell even feasible?” Dr. Warren shared that although no research group has really engineered PDU bacterial microcompartments to the extent of our vision for Manifold, recent literature has successfully proved that expression of synthetic structures inside these bacterial microcompartments was possible through the manipulation of one polypeptide PduD. Here, our conversation with Dr. Warren strongly suggested that PDU bacterial microcompartments was the protein shell to base Manifold’s design around.
Reflection: From the discoveries made by Dr. Warren, Team Virginia immediately knew that Manifold could not be built around carboxysomes as transport of starting materials into the microcompartment was necessary to even initiating biosynthesis. As a result, this led us to question the feasibility of engineering PDU bacterial microcompartments, where we realized that this endeavor has never been done to the extent of our vision for Manifold. Although Dr. Warren reassured our team that scientists have successfully expressed artificial structures inside these bacterial microcompartments and even generously gifted Team Virginia a crude plasmid that expressed PDU bacterial microcompartments to encourage us further, an underlying hesitancy lingered across the team. Taking some time off to contemplate this decision, we repeatedly returned to the local clinic to serve our community further and encourage team bonding. Throughout the summer, the patients we interviewed earlier were excited to see how our project went. Although it was hard to see that many of their lives remained the same since we last talked, their joyful smiles from our visits reminded Team Virginia why this project needed to be made. Immediately, we returned to the bench to begin redesigning the Warren plasmid to express PDU bacterial microcompartments at consistent yields.
Reflection: From the discoveries made by Dr. Warren, Team Virginia immediately knew that Manifold could not be built around carboxysomes as transport of starting materials into the microcompartment was necessary to even initiating biosynthesis. As a result, this led us to question the feasibility of engineering PDU bacterial microcompartments, where we realized that this endeavor has never been done to the extent of our vision for Manifold. Although Dr. Warren reassured our team that scientists have successfully expressed artificial structures inside these bacterial microcompartments and even generously gifted Team Virginia a crude plasmid that expressed PDU bacterial microcompartments to encourage us further, an underlying hesitancy lingered across the team. Taking some time off to contemplate this decision, we repeatedly returned to the local clinic to serve our community further and encourage team bonding. Throughout the summer, the patients we interviewed earlier were excited to see how our project went. Although it was hard to see that many of their lives remained the same since we last talked, their joyful smiles from our visits reminded Team Virginia why this project needed to be made. Immediately, we returned to the bench to begin redesigning the Warren plasmid to express PDU bacterial microcompartments at consistent yields.
Associate Professor Keith Kozminski
WHAT DOES MAKING A MODULAR AND STANDARDIZED VERSION OF MANIFOLD MEAN?
WHAT DOES MAKING A MODULAR AND STANDARDIZED VERSION OF MANIFOLD MEAN?
Discussion After weeks of redesigning the Warren plasmid and introducing two other plasmids that expressed the DNA scaffold and resveratrol enzyme binding domains, our team met with Dr. Kozminski to review our plasmid design. What began as a simple request for approval quickly evolved into a philosophical debate, where we began questioning the importance of “modularity” and “standardization” across chemical manufacturing. Slowly, our conversation prompted our team to realize that our initial plasmid designs could be optimized even further. Firstly, as stated by Dr. Warren, PDU bacterial microcompartments are composed of 14 different polypeptides, each encoded by their respective gene. As a result, simply attaching 14 genes onto a plasmid backbone creates a recombinant plasmid with more than 10kbp of DNA. The overall effect is that our plasmid would compete with the native genome for cellular machinery, largely reducing both replication and expression efficiencies. This large plasmid construct further impacts the transformation efficiency, where our plasmid would have difficulties diffusing through the tiny cell membrane channels and overall preventing any expression from the beginning. Secondly, with vision of creating a modular Manifold device, chemical manufacturers need ways to modify the plasmid construct, such that they can swap any pathway enzyme to produce any chemical of interesting through biosynthesis. With the current design of using the same pJPO63 plasmid backbone for the other two plasmid constructs, we faced a serious problem of having little to no ways of modifying this construct in an industrial setting. Finally, because each of the three plasmids contained the same origin of replication (ORI), the possibility of plasmid incompatibility could occur. Essentially, our plasmid constructs would have to compete for the same machinery during DNA replication, creating an unstable environment where, for example, one plasmid gets inherited by daughter cells and the other two plasmids are lost.
Reflection: With several problems at hand, our team faced two big challenges. First, we needed to reduce the number of genes encoded by our proposed plasmid to less than 10kbp of DNA. This would require meticulous screening of our plasmids for unneeded segments (including entire genes) and planning out molecular biology procedures to surgically remove these segments. This is where our modeling team began creating simulations that showcased how the PDU bacterial microcompartment folded, which provided crucial information in determining which genes weren’t needed in the folding process. Second, we needed to transfer each of the plasmid construct into a more engineering-friendly plasmid vector. This was where we began looking to standardized plasmids that were commonly used across the molecular biology field for their polylinkers that contain as many as 10-20 cloning sites. Essentially, these polylinkers contain several restriction enzyme sites that allow both our team and future chemical manufacturers to swap, remove, and add any segment of DNA into our plasmid with ease. Third, to reduce the possibility of plasmid incompatibility, we further needed to identify a standard plasmid vector that was high-copy and contained different ORIs. This would prevent our plasmids from competing with one another, while ensuring their longevity across multiple generations of replications. With much work ahead, our team turned towards three goals:
1)Remove unnecessary DNA segments from our plasmid construct
2)Switch the plasmid vectors to standard plasmids that contain multiple cloning sites
3)Find plasmid vectors that are high copy and contain different ORIs respectively.
Reflection: With several problems at hand, our team faced two big challenges. First, we needed to reduce the number of genes encoded by our proposed plasmid to less than 10kbp of DNA. This would require meticulous screening of our plasmids for unneeded segments (including entire genes) and planning out molecular biology procedures to surgically remove these segments. This is where our modeling team began creating simulations that showcased how the PDU bacterial microcompartment folded, which provided crucial information in determining which genes weren’t needed in the folding process. Second, we needed to transfer each of the plasmid construct into a more engineering-friendly plasmid vector. This was where we began looking to standardized plasmids that were commonly used across the molecular biology field for their polylinkers that contain as many as 10-20 cloning sites. Essentially, these polylinkers contain several restriction enzyme sites that allow both our team and future chemical manufacturers to swap, remove, and add any segment of DNA into our plasmid with ease. Third, to reduce the possibility of plasmid incompatibility, we further needed to identify a standard plasmid vector that was high-copy and contained different ORIs. This would prevent our plasmids from competing with one another, while ensuring their longevity across multiple generations of replications. With much work ahead, our team turned towards three goals:
1)Remove unnecessary DNA segments from our plasmid construct
2)Switch the plasmid vectors to standard plasmids that contain multiple cloning sites
3)Find plasmid vectors that are high copy and contain different ORIs respectively.
What Did Our Conversations Discover?
After reviewing the literature in bacterial microcompartments, Dr. Warren was an invaluable resource to determining which bacterial microcompartment was best for Manifold. Ultimately, Team Virginia chose PDU bacterial microcompartments, because of its well characterization in literature, while allowing diffusion of starting materials and retention of intermediates during biosynthesis. However, upon presenting our plasmid design to our principle investigator Dr. Kozminski, our conversation led Team Virginia to reevaluate the importance of “modularity” and “standardization”. We learned that in order for Manifold to accomplish these two goals, we needed to optimize the plasmid for high expression efficiency, high transformation and ease of modification by chemical manufacturers. Thus, Team Virginia needed to condense our design to less than 10kbp of DNA and switch our vector for a standard, high copy plasmid containing multiple cloning sites and different ORIs. With these goals, our team was left questioning—What genes are necessary to express empty PDU bacterial microcompartments and what genes aren’t? This is where modeling became extremely helpful in ideating Manifold’s design.
Professor Jason Papin
HOW DO WE BEGIN OUR MODELING PROJECT?
Introduction: Dr. Jason Papin is a professor of biomedical engineering at the University of Virginia School of Engineering and Applied Science. Currently, his research involves developing computational models of cellular networks and performing high-throughput experiments to characterize biological systems relevant to human disease. After his training in Bioengineering at the University of California, San Diego, Jason Papin joined the faculty at the University of Virginia in 2005, where he founded the Papin Lab. His lab works on problems in systems biology, metabolic network analysis, infectious disease, toxicology, heart disease, and cancer, developing computational approaches for integrating high-throughput data into predictive computational models. With his extensive experience in modeling complex biological systems, we contacted Dr. Papin to understand how we could use modeling to answer our questions in engineering Manifold.
HOW DO WE BEGIN OUR MODELING PROJECT?
Introduction: Dr. Jason Papin is a professor of biomedical engineering at the University of Virginia School of Engineering and Applied Science. Currently, his research involves developing computational models of cellular networks and performing high-throughput experiments to characterize biological systems relevant to human disease. After his training in Bioengineering at the University of California, San Diego, Jason Papin joined the faculty at the University of Virginia in 2005, where he founded the Papin Lab. His lab works on problems in systems biology, metabolic network analysis, infectious disease, toxicology, heart disease, and cancer, developing computational approaches for integrating high-throughput data into predictive computational models. With his extensive experience in modeling complex biological systems, we contacted Dr. Papin to understand how we could use modeling to answer our questions in engineering Manifold.
Discussion: From our initial establishment of the modeling committee, our team needed direction as many of our team members had never modeled a biological system before. This is where Dr. Papin provided invaluable guidance in establishing our modeling committee. First, Dr, Papin gave our team a general workflow that allowed our modeling committee to streamline our research process. This included first questioning the problem, modeling the problem, interpreting the model for answers and finally, carrying out the experiment when possible. Second, Dr. Papin provided resources to modeling complex biological systems, proposing we look into developing a partial differential equation (PDE) model to understand how Manifold would function in a cell. This modeling assignment would later become one of the most important models for Manifold, which is described in greater detail under the Modeling tab. Finally, Dr. Papin directed our team to the modeling programs I-Tasser, stating these software will allow our team to start our modeling endeavor by answering our question “What genes are necessary to express empty PDU bacterial microcompartments and what genes aren’t”. With the important advice needed to establish our modeling committee, he directed us to Dr. Ruoshi Sun to learn more about how to use this software before pursuing our modeling questions.
Reflection: Dr. Papin was incredibly important in the founding of our modeling committee. As most of our members had never coded before, we needed direction of where to start. His advice allowed us to get hands-on experience with I-Tasser before meeting with Dr. Ruoshi Sun to understand how we could use I-Tasser to answer our modeling questions. But more importantly, Dr. Papin contextualized how our new modeling committee could impact Manifold’s design. Specifically, our modeling committee needed to focus on supporting our wet-lab and integrated human practices work. The proposal of the PDE model would later allow our team to effectively communicate Manifold to stakeholders through a visual and engaging manner. The introduction of I-Tasser would allow our wetlab team to understand which genes were not essential in forming an empty protein shell.
Reflection: Dr. Papin was incredibly important in the founding of our modeling committee. As most of our members had never coded before, we needed direction of where to start. His advice allowed us to get hands-on experience with I-Tasser before meeting with Dr. Ruoshi Sun to understand how we could use I-Tasser to answer our modeling questions. But more importantly, Dr. Papin contextualized how our new modeling committee could impact Manifold’s design. Specifically, our modeling committee needed to focus on supporting our wet-lab and integrated human practices work. The proposal of the PDE model would later allow our team to effectively communicate Manifold to stakeholders through a visual and engaging manner. The introduction of I-Tasser would allow our wetlab team to understand which genes were not essential in forming an empty protein shell.
Computational Scientist Ruoshi Sun
HOW CAN I-TASSER MODEL MANIFOLD’S BACTERIAL MICROCOMPARTMENT FOLDING?
Introduction: Dr. Sun is an experienced computational scientist that researches electronic structures that code for computational materials. He takes part in managing the Rivanna software stack, building Singularity and Docker containers, and providing user support and research consultation. Through our initial modeling attempts with I-Tasser, our modeling committee was able to perform crude models in our attempt to identify which genes that encoded empty bacterial microcompartments weren’t needed. However, with countless crashes and other processing problems, we decided to meet with Dr. Sun to figure out a solution on how to improve our modeling committee’s productivity.
HOW CAN I-TASSER MODEL MANIFOLD’S BACTERIAL MICROCOMPARTMENT FOLDING?
Introduction: Dr. Sun is an experienced computational scientist that researches electronic structures that code for computational materials. He takes part in managing the Rivanna software stack, building Singularity and Docker containers, and providing user support and research consultation. Through our initial modeling attempts with I-Tasser, our modeling committee was able to perform crude models in our attempt to identify which genes that encoded empty bacterial microcompartments weren’t needed. However, with countless crashes and other processing problems, we decided to meet with Dr. Sun to figure out a solution on how to improve our modeling committee’s productivity.
Discussion: From our initial hands-on-experience with I-Tasser, the modeling committee faced a serious problem of routine crashes as a result of not having enough graphic processing unit to support the graphic intensive process of modeling. Although we came to Dr. Sun to learn more about how to apply I-Tasser to support wet-lab research, Dr. Sun introduced us to a program called Rivanna that expedited our ability to model complex biological systems. With Rivanna, a high-performance computing system offered by the University of Virginia, this system allowed our modeling team to improve and accelerate all of our computing processes. Dr. Sun helped our team run I-Tasser on Rivanna and later, the better modeling software Alpha Fold. But, besides these processing problems, we further encountered a problem with I-Tasser, where proteins containing more lengthy primary structures weren’t being outputted. This resulted in our modeling committee unable to identify which genes were necessary to form empty bacterial microcompartments. To troubleshoot this problem Dr. Sun guided our modeling committee through standard troubleshooting tests and eventually resolved the problem by adding a more powerful graphics processing unit to Rivanna just for our modeling committee. Ultimately, these technical supports progress our modeling committee into modeling the empty bacterial microcompartment.
Reflection: With the technical assistance of Dr. Sun, our modeling endeavors progressed significantly as we had the processing capabilities to fully model an empty bacterial microcompartment. As a result, we began modeling the empty PDU bacterial microcompartment using I-Tasser to understand which genes encoded by the Warren Plasmid were necessary to form our empty PDU bacterial microcompartment and which one weren’t. Over the course of a month, this led our modeling committee to narrowing our results from the initial 14 polypeptides to pduABJKNUT. But with the introduction of a more accurate modeling software called AlphaFold, our modeling committee agreed that we need to switch to this software in order to better model empty PDU bacterial microcompartments. This led our team to research countless guides and even contacting other faculty members like Dr. Karsten Siller, Dr. Charles M. Grisham and Dr. Michael Wormington to understand more about how to use AlphaFold. Nevertheless, once our team became proficient with AlphaFold, we developed our first model in mid August. However, with our model variably folding with and without the polypeptide pduK, our modeling committee sought expert insight to understand why exactly this result occurred.
Reflection: With the technical assistance of Dr. Sun, our modeling endeavors progressed significantly as we had the processing capabilities to fully model an empty bacterial microcompartment. As a result, we began modeling the empty PDU bacterial microcompartment using I-Tasser to understand which genes encoded by the Warren Plasmid were necessary to form our empty PDU bacterial microcompartment and which one weren’t. Over the course of a month, this led our modeling committee to narrowing our results from the initial 14 polypeptides to pduABJKNUT. But with the introduction of a more accurate modeling software called AlphaFold, our modeling committee agreed that we need to switch to this software in order to better model empty PDU bacterial microcompartments. This led our team to research countless guides and even contacting other faculty members like Dr. Karsten Siller, Dr. Charles M. Grisham and Dr. Michael Wormington to understand more about how to use AlphaFold. Nevertheless, once our team became proficient with AlphaFold, we developed our first model in mid August. However, with our model variably folding with and without the polypeptide pduK, our modeling committee sought expert insight to understand why exactly this result occurred.
Professor Todd O. Yeates
HOW DO WE INTERPRET OUR MODELING RESULTS?
Introduction: Professor Todd Yeates earned his Ph.D. at University of California in Los Angeles, developing X-ray crystallographic methods while working on early structures of membrane proteins, followed by postdoctoral research at the Scripps Research Institute elucidating the structures of viral capsids. Yeates joined the faculty at UCLA, where his research combines interests in molecular biology, biophysics and computational methods, applied to problems from molecular structure to genomic sequence analysis. With Dr. Yeates extensive knowledge in understanding the role of polypeptides in protein shells, we contacted him to help elaborate on our interpretation of the modeling results.
HOW DO WE INTERPRET OUR MODELING RESULTS?
Introduction: Professor Todd Yeates earned his Ph.D. at University of California in Los Angeles, developing X-ray crystallographic methods while working on early structures of membrane proteins, followed by postdoctoral research at the Scripps Research Institute elucidating the structures of viral capsids. Yeates joined the faculty at UCLA, where his research combines interests in molecular biology, biophysics and computational methods, applied to problems from molecular structure to genomic sequence analysis. With Dr. Yeates extensive knowledge in understanding the role of polypeptides in protein shells, we contacted him to help elaborate on our interpretation of the modeling results.
Discussion: From our conversation with Dr. Yeates, he immediately recognized that the problem with pduK involved an issue with the crystallographic data uploaded to the Protein Data Bank, which our AlphaFold model was based on. “With the Protein Data Bank, the primary researcher will try and characterize all the parts to the crystallography. But sometimes, they get it wrong which explains why pduK doesn’t fold in your model,�Dr. Yeates explained. “Instead, it could be that pduK is just a fragment and isn’t necessary for folding. Or, the primary researcher may not have included another pdu protein that pduK must interact with to fold correctly.” With this interpretation in mind, Dr. Yeates helped our team make an important decision that determined whether or not pduK would be included in our plasmid construct that encoded PDU bacterial microcompartments.
Reflection: From Dr. Yeates critical interpretation, our team decided to retest the model with and without pduK. As a result, we ended with the same conclusion that pduK sometimes was needed to facilitate folding of the PDU bacterial microcompartment and other times it wasn’t. This led the modeling committee to present their findings in our weekly meeting, where our team unanimously agreed that we needed to keep pduK in our plasmid construct, such that we ensure the protein shell always folds even at the expense of precious base pairs in our plasmid construct. But besides this key determination, our modeling committee further identified an important pdu protein that was needed to diffuse resveratrol out of the bacterial microcompartment—pduA. This key polypeptide contains a pore that allows Manifold to continuously synthesize resveratrol by permitting the diffusion of product to the cytoplasm and preventing buildup in the protein shell. But upon applying this to our PDE model, we encountered a problem that required expert insight.
Reflection: From Dr. Yeates critical interpretation, our team decided to retest the model with and without pduK. As a result, we ended with the same conclusion that pduK sometimes was needed to facilitate folding of the PDU bacterial microcompartment and other times it wasn’t. This led the modeling committee to present their findings in our weekly meeting, where our team unanimously agreed that we needed to keep pduK in our plasmid construct, such that we ensure the protein shell always folds even at the expense of precious base pairs in our plasmid construct. But besides this key determination, our modeling committee further identified an important pdu protein that was needed to diffuse resveratrol out of the bacterial microcompartment—pduA. This key polypeptide contains a pore that allows Manifold to continuously synthesize resveratrol by permitting the diffusion of product to the cytoplasm and preventing buildup in the protein shell. But upon applying this to our PDE model, we encountered a problem that required expert insight.
Associate Professor Peter Kasson
HOW DO WE HANDLE CONFLICTING MODELING RESULTS WITH LITERATURE?
Introduction: Dr. Peter Kasson is an associate professor at the University of Virginia, teaching molecular physiology and biomedical engineering in the engineering department. As the founder of the Kasson Lab, Dr. Kasson addresses fundamental questions about infectious disease by studying the membrane biology of virus-host cell interactions using both computational models and experimental approaches. Currently, Dr. Kasson is interested in applying the biophysics to viral infection, particularly influenza and Zika virus. This has allowed Dr. Kasson to become a leading research in developing computation models and new physical tools to gain greater insight into viral infection, extreme drug resistance in bacteria and engineering diagnosis/therapy for drug-resistant infections.
HOW DO WE HANDLE CONFLICTING MODELING RESULTS WITH LITERATURE?
Introduction: Dr. Peter Kasson is an associate professor at the University of Virginia, teaching molecular physiology and biomedical engineering in the engineering department. As the founder of the Kasson Lab, Dr. Kasson addresses fundamental questions about infectious disease by studying the membrane biology of virus-host cell interactions using both computational models and experimental approaches. Currently, Dr. Kasson is interested in applying the biophysics to viral infection, particularly influenza and Zika virus. This has allowed Dr. Kasson to become a leading research in developing computation models and new physical tools to gain greater insight into viral infection, extreme drug resistance in bacteria and engineering diagnosis/therapy for drug-resistant infections.
Discussion: With the creation of our PDE model, we wanted to develop a model that was entirely different from typical ordinary differential equations (ODE) models used in iGEM, encompassing the entire cellular dynamics of Manifold in E. coli. This included understanding the division of bacteria and the movement of substrates, ligands and resveratrol into and out of the protein shell. With such a complex model, Dr. Kasson was the perfect expert to contact when we approached the problem of resveratrol unable to diffuse out of our protein shell in our PDE model. Although our AlphaFold model and published literature indicated that pduA permitted diffusion of products, we wanted to understand precisely why our PDE model didn’t showcase this. Talking to Dr. Kasson, he explained that the problem involves our lack of information provided to our PDE model. We needed to include molecular dynamics simulation to inform our PDE model on how it should how predict resveratrol interacting with the pduA pore. In lay man’s terms, this molecular dynamics simulation add-on predicts the permeability values of resveratrol through the pduA pore and the mass transfer coefficient. These information not only tell if resveratrol can diffuse through the protein shell, but can additionally provides a rough estimate to the rate at which resveratrol flows through. With this key advice from Dr. Kasson, we included this molecular dynamics simulation to our model.
Reflection: From the advice of Dr. Kasson, we began exploring how to implement molecular dynamic simulations into our PDE model. As a result, this led us to meet with Dr. Kasson throughout our iGEM experience, where we eventually agreed that we should follow an “umbrella sampling” to find the permeability of pduA and then use that data in our PDE model to predict how resveratrol should interact with pduA. Overall, this led us to create a model that we would later be used to communicate our project to stakeholders.
Reflection: From the advice of Dr. Kasson, we began exploring how to implement molecular dynamic simulations into our PDE model. As a result, this led us to meet with Dr. Kasson throughout our iGEM experience, where we eventually agreed that we should follow an “umbrella sampling” to find the permeability of pduA and then use that data in our PDE model to predict how resveratrol should interact with pduA. Overall, this led us to create a model that we would later be used to communicate our project to stakeholders.
Where Did We Go From Here?
After our exploration in modeling, we revisited Dr. Warren, where he assisted our team in finalizing which genes were needed to express our protein shell. Coupling this meeting with the folding simulation of the PDU bacterial microcompartment made by our modeling committee, we reduced our plasmid construct to only seven genes—pduABJKNUT. With the Warren plasmid kindly gifted to Team Virginia earlier, we immediately began our wet-lab experience, while finalizing the design of our plasmid constructs. Following the advice of Alex Zorychta, Team Virginia created a flow chart to outline each step of our engineering design process and support research transparency. First, Team Virginia isolated the pduABJKNUT genes from the plasmid backbone pJP063 provided by Dr. Warren. Second, Team Virginia inserted these pduABJKNUT genes into a high copy biobrick assembly plasmid called pSB1C3, containing several restriction sites that allow for future engineering by chemical manufacturers. Finally, Team Virginia removed any illegal sites found in our plasmid construct. These steps would later allow Team Virginia to create a modular and industrially-standardized plasmid that expressed the first part to Manifold—empty bacterial microcompartments. Now, Team Virginia moved on to the next step, “How do we attach DNA scaffolds inside PDU bacterial microcompartments, while insuring our design remains both modular and standardized.”
STANDARDIZING AND MODULATING THE DNA SCAFFOLD
From our choice of PDU bacterial microcompartments and reemphasis on “modularity” and “standardization”, Team Virginia refocused our attention towards a structural peptide labeled pduD. Naturally occurring in PDU bacterial microcompartments, this peptide spontaneously fuses with either pyruvate decarboxylase or alcohol dehydrogenase to bound these enzymes to the protein shell interior. However, if Team Virginia could replace these enzymes with our DNA scaffolds attached to any enzymatic binding domains, then Manifold could position multiple enzymes in series, target them inside the protein shell, and synthesize any biosynthetic product. With this concept in mind, Manifold would solve the remaining problems to large-scale biosynthesis as described by the scientific perspective: intermediate loss and flux imbalance. By positioning pathway enzymes in series and in close proximity, substrates are immediately catalyzed by the next enzyme along the DNA scaffold, preventing a buildup or loss of those intermediates. Hence, we immediately began designing an approach that expressed DNA scaffolds within the protein shell interior, while modulating and standardizing our scaffold design.
Our initial concept for our DNA scaffolds involved creating a monomeric complementary structure composed of a short single-stranded DNA sequence hybridized to its reverse complement. As such, Team Virginia could modify the length of our DNA scaffolds, simply by adding or removing monomeric sequences that code for the polymeric DNA scaffold. Then, by using zinc-finger fusion proteins, we can bind pathway enzymes to our DNA scaffold in whatever position we’d like. This meant in practice, chemical manufacturers could produce any chemical of interest through Manifold, simply by varying the length of our DNA scaffolds to accommodate more or less enzymatic binding domains and using zinc-finger fusion proteins to position enzymes anywhere along the scaffold. However, the major problem we faced was that large amount of literature showing unsuccessful results at expressing these scaffolds. We questioned, “How do we ensure our DNA scaffolds are built at consistent qualities and yields, while keeping the overall design simple?” If this problem was not correctly accounted for, then Manifold would express too few DNA scaffolds, lowering the amount of enzymes participating in biosynthesis and reducing product yield. With this problem at hand, Team Virginia returned to Dr. Kozminski, because of his expertise in scaffold design.
Associate Professor Keith Kozminski
HOW BEST TO DESIGN A MODULAR SCAFFOLD?
HOW BEST TO DESIGN A MODULAR SCAFFOLD?
Discussion From the initial introduction of our scaffold design, Dr. Kozminski commended our team for returning to our vision of creating a modular and industrially-standardized version of Manifold. But with the problem of expressing DNA scaffolds at consistent qualities and amounts, Dr. Kozminski couldn’t provide our team with a definitive solution. Turning information from genes into RNA and back into DNA introduces many error-prone steps that could result in inconsistent expression and quality of DNA scaffolds, especially when these DNA scaffolds are composed of polymerized DNA repeats. As a result, these errors in expressing the DNA scaffold translate to our Manifold device containing less enzymes and overall producing less product. With this problem at hand, Dr. Kozminski instead pointed our team towards two textbooks “Biochemistry” by Garrett and Grisham and “Molecular Biology of the Cell” by W.W. Norton. “If we want to know how to do something, look to literature. If we want to know why it works, look to textbooks,” suggested Dr. Kozminski.
Reflection: From these wise words, our team realized that we were approaching the problem entirely wrong. Before engineering a solution to our problem, we needed to understand precisely why expressing DNA scaffolds was a problem at all. From our days of reading these textbooks and learning new concepts that were never taught in biology class, our pursuit for the question “Why?” led us to entirely new areas of research. We learned that chromosomes take on a genetic scaffold structure, which allows for the removal of DNA and histone proteins in preparation for replication. We learned that reverse transcriptases are usually expressed in viruses like HIV that oddly store their genetic information as RNA and need reverse transcriptases to integrate their RNA into the host’s DNA genome. But most importantly, we learned that the reason why so many papers yield unsuccessful assembly of DNA scaffolds is because of recognition. Reverse transcriptases requires a primer to bind to RNA. But, due to the complicated secondary structure of our DNA scaffold, simply expressing a DNA primer that binds to our scaffold creates even more difficulties in plasmid design. As a result, even though the DNA scaffold is properly designed, the reverse transcriptase will still have trouble binding, causing the scaffold’s RNA form to be degraded. From this new understanding of the problem, our team now needed to design a solution.
Reflection: From these wise words, our team realized that we were approaching the problem entirely wrong. Before engineering a solution to our problem, we needed to understand precisely why expressing DNA scaffolds was a problem at all. From our days of reading these textbooks and learning new concepts that were never taught in biology class, our pursuit for the question “Why?” led us to entirely new areas of research. We learned that chromosomes take on a genetic scaffold structure, which allows for the removal of DNA and histone proteins in preparation for replication. We learned that reverse transcriptases are usually expressed in viruses like HIV that oddly store their genetic information as RNA and need reverse transcriptases to integrate their RNA into the host’s DNA genome. But most importantly, we learned that the reason why so many papers yield unsuccessful assembly of DNA scaffolds is because of recognition. Reverse transcriptases requires a primer to bind to RNA. But, due to the complicated secondary structure of our DNA scaffold, simply expressing a DNA primer that binds to our scaffold creates even more difficulties in plasmid design. As a result, even though the DNA scaffold is properly designed, the reverse transcriptase will still have trouble binding, causing the scaffold’s RNA form to be degraded. From this new understanding of the problem, our team now needed to design a solution.
What Did We Discover?
From our conversation with Dr. Kozminski, our team realized that we were approaching the problem entirely wrong. Instead of immediately trying to figure out a solution to our problem, we should have been looking towards the underlying principle. As a result, the members of the Virginia iGEM Team began refreshing our knowledge through textbooks to understand why exactly countless of papers have documented immense difficulty in expressing DNA scaffolds. As a result, this led our team to adopt a growing mindset, where we began exploring new concepts and areas of research that would allow us to improve Manifold further. Unbeknownst to us, our readings in reverse transcriptases would later inspire one of our teammates to pursue undergraduate research at the Thaler Center for HIV and Retrovirus Research at the University of Virginia’ School of Medicine. Nevertheless, we discovered that the reason DNA scaffolds are difficult to express involves the complicated secondary structure of our DNA scaffold during its RNA form. With our newfound perspective on DNA scaffold expression and reverse transcriptases, we began designing a solution.
Where Did We Go From Here?
Combining our newly obtained knowledge in DNA scaffold expression and reverse transcriptases with our vision of creating a modular and industrially-standardized version of Manifold, we returned to the drawing board to engineer a solution. Of our countless attempts at ideating a solution, we realized that if the problem was centered around the reverse transcriptase not properly binding to the complicated secondary structures found in the RNA form of the DNA scaffold, then we needed some way to facilitate this process. Through our countless meetings to design a solution, we took inspiration from the former Virginia iGEM Team’s design and ideated one solution termed the “R-olio Flipper”. This part modifies the recognition site of reverse transcriptase by introducing an HIV terminator binding site “HTBS”. As a result, when DNA polymerase transcribes the gene coding for our DNA scaffold, HTBS is also expressed alongside the RNA form of the DNA scaffold. So, instead of having to express an in-vitro DNA primer, our HTBS RNA sequence induces a natural affinity towards the reverse transcriptase, allowing reverse transcriptase to properly bind and reverse transcribe the RNA form of the DNA scaffold. More information about this part can be found under Parts, Additionally, from our review of the literature, we learned that by further expressing two types of reverse transcriptase's: the Human Immunodeficiency Viruses Reverse Transcriptase (HIV-RT) and Murine Leukemia Virus Reverse Transcriptase (ML-RT), this would ensure that our scaffold would assemble correctly. These solutions allowed Team Virginia to focus on our wet-lab and modeling endeavors as we attempted to express DNA scaffolds attached to pathways enzymes using zinc-finger fusion proteins. This would make up the bulk of our iGEM experience, before realizing that there were even more ways to optimize Manifold.
BIOREACTOR DESIGN
While continuing our wet-lab, modeling and integrated human practices work in parallel, our early conversation with Affinity Chemical and Barrier Plastics showed that there were greater ways to improve Manifold through bioreactors. Like the chemical plant designed by Affinity Chemical, we were surprised to learn how some manufacturers have turned a pollution-intensive process like chemical synthesis into a more environmentally friendly process through bioreactors. With this impressive feat, our team pondered, “If we were to combine an already environmentally friendly and sustainable process like biosynthesis with a well-designed bioreactor, could we make Manifold a 100% pollution free solution?” This question lingered behind our team’s priorities as we dedicated much of our time to wet-lab and modeling. But, upon meeting with Affinity Chemical again to update them on the new direction for Manifold after that conversation, we were reminded yet again about the immense potential bioreactors could improve Manifold and further support the UN’s Sustainable Development Goals. We envisioned a 100% environmentally friendly bioreactor that continuously transferred excess nutrients and substrates back into Manifold. Additionally, this bioreactor would recycle cells for further iterations of product synthesis and convert cellular waste into the bioreactor’s power source. As such, we began our exploration into bioreactors by attending several seminars hosted by the University of Virginia School of Engineering and Applied Science, the nanoSTAR Institute, and the American Chemical Society in Green Chemistry . Although most of our team members had never heard of a bioreactor before, we became excited by the possibility of optimizing Manifold through a bioreactor set up. This led us back to Dr. Berger, a professor and experienced engineering in bioreactor design, as we attempted to figure out how our group of scientists and engineers should even begin.
Associate Professor Bryan Berger
HOW DO WE EVEN START DESIGNING A BIOREACTOR FOR MANIFOLD?
Discussion: After attending Dr. Berger’s yearly presentation on his startup company Lytos Technologies, our team was interested to learn more about how their bioreactor design has further enhanced their production of enzyme-based fungicides. We learned that without a proper bioreactor setup, a platform technology like Lytos Tech or Manifold could never be scaled onto the industrial level. “You need liters of cells making your product non-stop, just to have a product at all”, stated Dr. Berger. “Otherwise, your project is just a concept and it’s not helping society if its just a concept.” With such surprising truth to his advice, our team began investing more and more time to the implementation approach and the societal impact of Manifold as our project neared the Giant Jamboree. While many of us continued to work on our wet-lab and modeling research even throughout the start of our college semester, we were simultaneously trying to envision Manifold in an industrial setting. Attending Dr. Berger’s office hours for over a month, he directed our team towards important resources, while guiding us through the endless types of bioreactors. Firstly, from our conversations with Dr. Berger, he advised our team to learn the basic principles behind bioreactors as most of our team of scientists and engineers had never even seen a bioreactor before. As such, he directed us to the textbook Wei-Shou Hu’s Engineering Principles in Biotechnology, where our team began hosting journal meetings to discuss chapters involving bioreactors. From these simple readings, our team was surprised to learn how a proper bioreactor design equally contributed to the success of the biosynthesis process itself. This exploration into the mechanisms behind bioreactors explained why some chemical manufacturing companies have successfully scaled-up green chemical synthesis simply by recycling the chemical waste back into the process. Secondly, Dr. Berger shared that if we were to make Manifold on the industrial scale, he recommended we explore the continuous stirred-tank reactor (CSTR) as this type of bioreactor is easy functioning and efficient. This led our team to appreciate the high level of sophistication that goes into designing a bioreactor. Engineers must select the correct height to diameter ratio of the tank. They must choose the correct rotor to maximize mixing. They must verify that proper amount of oxygen enters the tank and gets dissolved by the liquid culture. If a recycling mechanism is introduced, they must figure out a way to separate the effluent stream and reintroduce certain isolated chemicals back into the reactor. Besides these process elements, engineers must further account for the pressure of the tank, the rate at which substrates enter the reactor, the amount of energy consumed, and many other variables to ensure a steady production of the desired chemical. As our team began realizing the immense complexity in bioreactor design, we wanted to understand how these decisions translated in the manufacturing process. Dr. Berger suggested we contact the University of Virginia School of Engineering and Applied Sciences to learn about industrial bioreactor setups and answer our important question.
Reflection: Dr. Berger was an incredibly useful resource to introducing our team to the novel field of bioreactor design. Coincidentally, these conversations with Dr. Berger paralleled our earlier conversations with Dr. Kozminski as it reminded our team that synthetic biologists should always be looking to expand our knowledge and improve our project design towards a more sustainable solution. This realization led our team to treat bioreactor design seriously, eventually inspiring two members of our team to enroll in Introduction to Biotechnology by Dr. Berger to expand our understanding of bioreactors even further. Over the course of the early fall semester, we began designing a concept to our bioreactor setup, while simultaneously finishing up our wet-lab and modeling research. This eventually led us to take up Dr. Berger’s suggestion of meeting with the University of Virginia School of Engineering and Applied Sciences to understand how a proper bioreactor setup can improve industrial biosynthesis.
Reflection: Dr. Berger was an incredibly useful resource to introducing our team to the novel field of bioreactor design. Coincidentally, these conversations with Dr. Berger paralleled our earlier conversations with Dr. Kozminski as it reminded our team that synthetic biologists should always be looking to expand our knowledge and improve our project design towards a more sustainable solution. This realization led our team to treat bioreactor design seriously, eventually inspiring two members of our team to enroll in Introduction to Biotechnology by Dr. Berger to expand our understanding of bioreactors even further. Over the course of the early fall semester, we began designing a concept to our bioreactor setup, while simultaneously finishing up our wet-lab and modeling research. This eventually led us to take up Dr. Berger’s suggestion of meeting with the University of Virginia School of Engineering and Applied Sciences to understand how a proper bioreactor setup can improve industrial biosynthesis.
University of Virginia School of Engineering and Applied Sciences
WHAT DOES A BIOREACTOR SETUP LOOK LIKE IN PRACTICE?
Introduction: University of Virginia School of Engineering and Applied Sciences is one of the colleges established by the University of Virginia. Out of the many departments under this college, their biomedical engineering, chemical engineering and materials science department partnered together to invest in large-scale reactors for academic purposes and collaborative projects with outside institutes. To understand more about bioreactor design, our team met with bioreactor technicians to understand the process of how we could introduce proper bioreactor design to our project Manifold.
WHAT DOES A BIOREACTOR SETUP LOOK LIKE IN PRACTICE?
Introduction: University of Virginia School of Engineering and Applied Sciences is one of the colleges established by the University of Virginia. Out of the many departments under this college, their biomedical engineering, chemical engineering and materials science department partnered together to invest in large-scale reactors for academic purposes and collaborative projects with outside institutes. To understand more about bioreactor design, our team met with bioreactor technicians to understand the process of how we could introduce proper bioreactor design to our project Manifold.
Discussion: From our meeting with bioreactor technicians at the University of Virginia School of Engineering and Applied Sciences , Team Virginia had the amazing opportunity to visit their facility and physically observe industrial biosynthesis in action. With so many bioreactor setups to study, we quickly became interested in one setup that could continuously culture and recycle used cells back into the biosynthesis process. From this visit, we learned that in a typical, continuous, stirred-tank bioreactor, the stream of liquid that exits your bioreactor (containing all your products, cellular waste and used cells), is generally just flown into a container where chemical manufacturers isolate the product through complicated chemical procedures. But in a bioreactor setup that recycles cells, the cell mass is also separated and fed back into the reactor to assist with further iterations of biosynthesis. With such a novel process, we consequentially asked these bioreactor technicians how our team could begin to design bioreactors and eventually integrate these bioreactors into our project Manifold. From their final comments, they recommended our team seriously understand the process behind designing bioreactors, before integrating this into our project.
Reflection: From our earnest conversations with bioreactor technicians at the University of Virginia School of Engineering and Applied Sciences , we recognized designing bioreactors was no easy feat and required almost an entirely new project on its own. Yet, from our realization that this bioreactor designs like the continuous stirred-tank reactors with cell recycling capabilities existed, we became highly intrigued by the possibility of combining this similar concept with a future Manifold design. We asked, “Instead of only separating the cellular content, could we design a bioreactor that could recycle our cellular waste back into our reactor? Could we use that cellular waste to help with cell growth or could we use it to reduce the energy consumption of our bioreactor? Could we recycle our cells (the major waste produced of Manifold) to assist with further iterations of product synthesis?” Our visit left us even more intrigued about bioreactor design.
Reflection: From our earnest conversations with bioreactor technicians at the University of Virginia School of Engineering and Applied Sciences , we recognized designing bioreactors was no easy feat and required almost an entirely new project on its own. Yet, from our realization that this bioreactor designs like the continuous stirred-tank reactors with cell recycling capabilities existed, we became highly intrigued by the possibility of combining this similar concept with a future Manifold design. We asked, “Instead of only separating the cellular content, could we design a bioreactor that could recycle our cellular waste back into our reactor? Could we use that cellular waste to help with cell growth or could we use it to reduce the energy consumption of our bioreactor? Could we recycle our cells (the major waste produced of Manifold) to assist with further iterations of product synthesis?” Our visit left us even more intrigued about bioreactor design.
A Future Extension of Manifold through Bioreactor Design
From our conversations with Dr. Berger, stakeholders, and bioreactor technicians, we realized that a proper bioreactor set up had the potential to make Manifold into a more environmentally friendly and sustainable solution. Instead of lowering the pollution generated from inefficient chemical synthesis through Manifold, we could couple Manifold’s large-scale biosynthesis with a bioreactor that entirely removes pollution from the environment. Cellular waste could be converted into biofuel or other basic chemicals like ethanol. CO2 produced by E. coli could be converted into reusable energy through carbon dioxide conversion technology. These processes create renewable energy from Manifold’s waste products, sustaining these bioreactors with the energy to carryout large-scale biosynthesis, support large-batch culturing or even synthesize new biosynthetic chemical that only form at extreme temperatures. On the other hand, the cells could be separated from the cellular waste too. This allows our cells to be reused in further iterations of product synthesis. With these possibilities, a proper bioreactor set up could truly make Manifold a 100% self-sustainable and environmentally friendly replacement to chemical synthesis. But with all our project goals, our team unfortunately agreed that our priorities needed to be elsewhere.
Where Did Our Decision Lead Us?
From the start of our iGEM experience, our primary goal was to finish the work of the 2020 Virginia iGEM Team by engineering a working prototype of Manifold. As such, we dedicated much of our iGEM experience to wet lab and modeling research. But, as our team embarked on our journey through integrated human practices, we needed to change Manifold’s design to address the problems facing the chemical manufacturing industry, including high manufacturing costs, drug shortages, and global pollution. Although many of our conversations sparked a modification to Manifold’s design, we unfortunately couldn’t invest enough time to add bioreactor design to our list of priorities. Our conversation with Dr. Berger and meeting with bioreactor technicians revealed we needed to learn much more if we were to make a proper bioreactor set up for Manifold. This led to our team agreeing that we couldn’t pursue this project addition. Instead, as our wet-lab and modeling research continued to engineer a working Manifold device, our journey through integrated human practices shifted away from the scientific aspects of Manifold and pivoted towards understanding the societal impact of Manifold. Our team had the device. Now, we needed to figure out how we would implement Manifold in the real-world.
3B) Societal Impact
From our Team Values document, we wanted to build a synthetic biology project that was sustainable, environmentally friendly and socially good. Although we met with scientists, stakeholders, and end-users to ideate a responsible solution, we needed to understand how our solution impacted the social landscape. Did the creation of Manifold serve as a net positive for society? Are there any ethical concerns involved with Manifold? If adopted by the chemical manufacturing industry, would we see an improvement in global pollution? By answering these questions and understanding Manifold’s impact on society, we could ideate an implementation approach that upheld our central purpose of improving the lives of people. Opening meaningful conversations with sociologists, ethicists, environmental scientists, and economists, we began our exploration into Manifold’s societal impact with Dr. Carlson, a renowned historian and sociologist. His words would shape our team’s perspective of technology in society, guiding our conversations with other experts in the humanities and ultimately our decision of how to implement Manifold in the real-world.
Professor W. Bernard Carlson
HOW DO WE UNDERSTAND THE ROLE AND ETHICS OF TECHNOLOGY IN SOCIETY?
Introduction: Dr. Bernie Carlson is a historian of technology who studies the societal impact of technology throughout history. He earned his Ph.D. in the history and sociology of science, and pursued a postdoc in business history at the Harvard Business School. During his early career as a researcher, Dr. Carlson published his world-renowned biography on Nikola Tesla, sparking his career as both a professor at the University of Virginia and co-editor of a book series with MIT Press, "Inside Technology". Dr. Carlson has been featured in countless documentaries on the History Channel, including “The Machines That Built America” which launched on July 18th, 2021. Furthermore, Dr. Carlson has presented his work internationally at “Talks at Google” and “Microsoft Research”. Because of his immense knowledge in technology and their societal impact, Dr. Carlson was the first expert we met to discuss Manifold’s role and impact in society.
HOW DO WE UNDERSTAND THE ROLE AND ETHICS OF TECHNOLOGY IN SOCIETY?
Introduction: Dr. Bernie Carlson is a historian of technology who studies the societal impact of technology throughout history. He earned his Ph.D. in the history and sociology of science, and pursued a postdoc in business history at the Harvard Business School. During his early career as a researcher, Dr. Carlson published his world-renowned biography on Nikola Tesla, sparking his career as both a professor at the University of Virginia and co-editor of a book series with MIT Press, "Inside Technology". Dr. Carlson has been featured in countless documentaries on the History Channel, including “The Machines That Built America” which launched on July 18th, 2021. Furthermore, Dr. Carlson has presented his work internationally at “Talks at Google” and “Microsoft Research”. Because of his immense knowledge in technology and their societal impact, Dr. Carlson was the first expert we met to discuss Manifold’s role and impact in society.
Discussion: Our conversation with Dr. Carlson was incredibly helpful in framing our perspective of technology in society. He recommended we approach technology with an optimistic but careful outlook, stating “All technology is good, but how we implement and use that technology is up to our ethical code.” In adopting this outlook, Dr. Carlson agreed that creating Manifold was a socially good decision. We wanted to reduce the chemical waste produced in chemical synthesis, increase chemical manufacturing yields and lower carbon emissions by making biosynthesis industrially viable. Even though these intentions were good, Dr. Carlson advised our team to be extremely cautious on how we implemented Manifold in society. Using the case study of McDonalds, Dr. Carlson explained, “McDonalds provides affordable, delicious food to consumers. But in the process of being a business, they’ve contributed significantly to the rise in diabetes and even adopted predatory strategies to increase sales in poorer areas.” From Dr. Carlson’s earnest advice, our journey to understand Manifold’s societal impact revolved around the implementation approach and not the project itself. Dr. Carlson recommended we learn about the ethical aspects of Manifold and consider the consequences of implementing Manifold in society.
Reflection: After meeting with Dr. Carlson, our team needed to identify the ethical concerns related to Manifold before crafting our implementation strategy. At this point, it became a great priority for our team to learn more about ethical perspectives and how to apply their reasonings to Manifold’s implementation. After learning that the 2017 Technion-Israel Team developed an introductory guide to ethics, we carefully reviewed their work to understand synthetic biology through an ethical framework. The 2017 Technion-Israel Team’s Ethics Handbook provided excellent explanations to all moral reasoning approaches, answering questions like “Why do different ethical approaches lead to different classifications of an ethical action?”, “With so many schools of ethical thought, which moral reasoning approach justifies our project decisions best?” and “How should ethics correctly guide iGEM teams towards a moral implantation approach?” From these questions, our team eventually chose consequentialism as the best moral reasoning approach for Manifold. Our conversation with Dr. Carlson led our team to this decision, as we weren’t concerned about if creating Manifold was an immoral action, rather if its consequences on society were ethical. By choosing this ethical approach, we needed to meet with an ethicist to understand specifically how we could apply consequentialism to Manifold. But before contacting an ethicist, our team began reflecting on the direct and indirect consequences of implementing Manifold, recognizing two major ethical dilemmas existed in implementing Manifold in the pharmaceutical industry. These ethical dilemmas were prompted by our conversation with patients at the free clinic and Adial Pharmaceuticals.
Reflection: After meeting with Dr. Carlson, our team needed to identify the ethical concerns related to Manifold before crafting our implementation strategy. At this point, it became a great priority for our team to learn more about ethical perspectives and how to apply their reasonings to Manifold’s implementation. After learning that the 2017 Technion-Israel Team developed an introductory guide to ethics, we carefully reviewed their work to understand synthetic biology through an ethical framework. The 2017 Technion-Israel Team’s Ethics Handbook provided excellent explanations to all moral reasoning approaches, answering questions like “Why do different ethical approaches lead to different classifications of an ethical action?”, “With so many schools of ethical thought, which moral reasoning approach justifies our project decisions best?” and “How should ethics correctly guide iGEM teams towards a moral implantation approach?” From these questions, our team eventually chose consequentialism as the best moral reasoning approach for Manifold. Our conversation with Dr. Carlson led our team to this decision, as we weren’t concerned about if creating Manifold was an immoral action, rather if its consequences on society were ethical. By choosing this ethical approach, we needed to meet with an ethicist to understand specifically how we could apply consequentialism to Manifold. But before contacting an ethicist, our team began reflecting on the direct and indirect consequences of implementing Manifold, recognizing two major ethical dilemmas existed in implementing Manifold in the pharmaceutical industry. These ethical dilemmas were prompted by our conversation with patients at the free clinic and Adial Pharmaceuticals.
The Ethical Dilemmas Facing Manifold
Ethical Dilemma 1:
Manifold reduces the cost that chemical manufacturing companies pay to make by optimizing and streamlining the chemical manufacturing process. Ideally, this allows pharmaceutical companies to supply more medications throughout the medical field, resulting in patients paying lower drug prices. However, because the pharmaceutical industry is currently an oligopoly, these companies have the opportunity to fix drug prices at unfairly high prices. As a result, the savings that pharmaceutical companies get from having a more efficient manufacturing process won’t go to patients, but are rather kept internally as profit. How should Team Virginia approach this dilemma of whether or not sharing Manifold to the pharmaceutical industry is even ethical?
Ethical Dilemma 2: Let’s assume that Team Virginia makes Manifold with the condition that pharmaceutical companies use this technology to reduce the cost that patients pay for drugs. If we accept this assumption, lowering drug prices allows more patients to afford life-saving pharmaceuticals and effectively save more patient lives. However, by lowering drug prices, less money goes to pharmaceutical companies which prevents researchers from having enough capital to discover new drugs. Because pharmaceutical companies cannot manufacture new drugs, more patients will die in the long run. Thus, we questioned, "Does lowering drug prices actually benefit patients?"
Ethical Dilemma 2: Let’s assume that Team Virginia makes Manifold with the condition that pharmaceutical companies use this technology to reduce the cost that patients pay for drugs. If we accept this assumption, lowering drug prices allows more patients to afford life-saving pharmaceuticals and effectively save more patient lives. However, by lowering drug prices, less money goes to pharmaceutical companies which prevents researchers from having enough capital to discover new drugs. Because pharmaceutical companies cannot manufacture new drugs, more patients will die in the long run. Thus, we questioned, "Does lowering drug prices actually benefit patients?"
Before Our Meeting With an Ethicist
Our team explored the philosophy of consequentialism before meeting with ethicist Dr. Shepherd to apply its logic in determining the ethics of implementing Manifold. As a result, we returned to Dr. Carlson for further preparation, where he recommended we review two books, including Peter Singer’s Practical Ethics and Avram Hiller’s Consequentialism and Environmental Ethics. Throughout our weekly journal meeting, these books brought insight into the perspective of an ethicist. Contrary to a scientist’s focus on observations or an engineer’s dependence on data, we learned that ethicists rely heavily on moral intuitions—these instinctive, raw feelings towards a right and wrong decision. But, as we learned more and more about consequentialism, our book discussions began fostering a deep appreciation for ethics among the team. We realized without ethics, science would become dangerous and harmful to society as there wouldn’t be a framework to conduct honest, safe and respectful research. This led our team to seriously prepare for the Dr. Shepherd interview, such that we could accurately represent our two dilemmas through the lens of an ethicists.
Professor Lois Shepherd
HOW DO WE UNDERSTAND THE ROLE AND ETHICS OF TECHNOLOGY IN SOCIETY?
Introduction: Lois Shepherd is an expert in the fields of health law and bioethics. Her primary appointment is in the University of Virginia’s School of Medicine’s Center for Biomedical Ethics and Humanities where she directs the center’s programs in medicine and law. She teaches courses in health care law and ethics at both the law school and the medical school. After receiving her law degree from Yale University, where she served as a senior editor of the Yale Law Journal, Dr. Shepherd practiced corporate law for six years with the Charlotte, N.C., firm of Robinson, Bradshaw & Hinson, P.A. She began her academic career in 1993 at the Florida State University College of Law. Prior to joining the UVA faculty, Dr. Shepherd was the Florida Bar Health Law Section Professor and D’Alemberte Professor of Law at Florida State. Within the field of bioethics and law, Currently, Dr. Shepherd’s scholarly and teaching interests are focused on legal and ethical issues at the end of life and in human subject research. With Dr. Shepherd’s broad experience in ethics and its application in the sciences, we contacted her to assist our team in applying consequentialist moral reasoning to Manifold’s implementation.
HOW DO WE UNDERSTAND THE ROLE AND ETHICS OF TECHNOLOGY IN SOCIETY?
Introduction: Lois Shepherd is an expert in the fields of health law and bioethics. Her primary appointment is in the University of Virginia’s School of Medicine’s Center for Biomedical Ethics and Humanities where she directs the center’s programs in medicine and law. She teaches courses in health care law and ethics at both the law school and the medical school. After receiving her law degree from Yale University, where she served as a senior editor of the Yale Law Journal, Dr. Shepherd practiced corporate law for six years with the Charlotte, N.C., firm of Robinson, Bradshaw & Hinson, P.A. She began her academic career in 1993 at the Florida State University College of Law. Prior to joining the UVA faculty, Dr. Shepherd was the Florida Bar Health Law Section Professor and D’Alemberte Professor of Law at Florida State. Within the field of bioethics and law, Currently, Dr. Shepherd’s scholarly and teaching interests are focused on legal and ethical issues at the end of life and in human subject research. With Dr. Shepherd’s broad experience in ethics and its application in the sciences, we contacted her to assist our team in applying consequentialist moral reasoning to Manifold’s implementation.
Discussion: Our team presented two ethical dilemmas to Dr. Shepherd to investigate how we could apply consequentialism in ideating an ethical implementation approach for Manifold. But before examining these dilemmas, Dr. Shepherd wanted to understand our central purpose. “How do you envision Manifold in the real world?” she questioned. We explained to her our Team Values document, affirming our goal of creating Manifold to help people by increasing chemical production, reducing global pollution, and ensuring all people always have access to life-saving medications. Recognizing this vision, Dr. Shepherd approached our dilemmas meticulously. She interrogated each dilemma through the lens of consequentialism, then recommended solutions through the lens of our team values. By following this approach, Dr. Shepherd provided invaluable insight that would later help our team ideate an implementation approach that was ethical, socially good and improved end-user lives. Dr. Shepherd empathized with our team’s fear of implementing Manifold in the pharmaceutical industry. We explained that that without some way to hold pharmaceutical companies accountable, our technology could be exploited for profit at the expense of end-user lives. Conversely, if we drastically reduced drug prices, we could unintentionally harm the pharmaceutical industry by drawing away profit invested into the drug discovery process (a high-risk and resource-intense process, where most resources go as stated by Adial Pharmaceuticals). This prevents life-saving medications from begin discovered, ultimately harming patients. But through our conversation with Dr. Shepherd, we realized a similarity with our conversation with Dr. Carlson. Dr. Shepherd explained “These ethical dilemmas aren’t a question of whether implementing Manifold is unethical. It’s a commentary on the state of the pharmaceutical industry.” Surprised by her earnest statement, our team seriously questioned her statement, explaining the immense problems that sharing our technology could create in the pharmaceutical industry. Yet, Dr. Shepherd explained, “When applying consequentialism, we look at the direct consequences of an action (in this case sharing Manifold to the pharmaceutical industry) … If the direct consequence of implementing Manifold is reducing pollution and increasing chemical production, then the ethical dilemma of what pharmaceutical companies do afterwards isn’t ours to bear.” Immediately after this statement, Dr. Shepherd quickly shared, “However, this doesn’t mean it’s not in your control to do something.” She recommended learning more about the patent process and talk with the University of Virginia Licensing and Ventures Group. There we might have our solution.
Reflection: After talking with Dr. Shepherd, our team was overjoyed to learn that implementing Manifold was an ethical decision as long as it was directed at being socially good and improving end-user lives. But before meeting with the University of Virginia Licensing and Ventures Group to ideate an implementation strategy that helped both patients and pharmaceutical companies, we still wanted to meet with other experts to discuss the other facets of Manifold’s social impact. This included learning more about the environmental impact and economic impact. At this point of the project, our integrated human practices journey identified a problem, modified and improved our a solution, showed that Manifold was a responsible, socially good, and ethical project, but we still needed to understand to what extent our project could help society.
Reflection: After talking with Dr. Shepherd, our team was overjoyed to learn that implementing Manifold was an ethical decision as long as it was directed at being socially good and improving end-user lives. But before meeting with the University of Virginia Licensing and Ventures Group to ideate an implementation strategy that helped both patients and pharmaceutical companies, we still wanted to meet with other experts to discuss the other facets of Manifold’s social impact. This included learning more about the environmental impact and economic impact. At this point of the project, our integrated human practices journey identified a problem, modified and improved our a solution, showed that Manifold was a responsible, socially good, and ethical project, but we still needed to understand to what extent our project could help society.
Professor Scott Doney
TO WHAT EXTENT CAN MANIFOLD REDUCE GLOBAL POLLUTION?
Introduction: Dr. Scott Doney is a professor in environmental change. His research at the Doney lab spans multiple disciplines within environmental change, including oceanography, climate, biogeochemistry, biotechnology and industry. As the prestigious Joe D. and Helen J. Kington Professor in Environmental Change at the University of Virginia, Dr. Doney has become an expert in understanding how the global carbon cycle and ocean ecology respond to natural and human-driven climate change signals such as ocean warming, sea-ice loss, and ocean acidification due to the invasion of carbon dioxide from fossil fuel burning. These qualifications made Dr. Doney an excellent contact, allowing us to learn more about how Manifold could positively impact the climate.
TO WHAT EXTENT CAN MANIFOLD REDUCE GLOBAL POLLUTION?
Introduction: Dr. Scott Doney is a professor in environmental change. His research at the Doney lab spans multiple disciplines within environmental change, including oceanography, climate, biogeochemistry, biotechnology and industry. As the prestigious Joe D. and Helen J. Kington Professor in Environmental Change at the University of Virginia, Dr. Doney has become an expert in understanding how the global carbon cycle and ocean ecology respond to natural and human-driven climate change signals such as ocean warming, sea-ice loss, and ocean acidification due to the invasion of carbon dioxide from fossil fuel burning. These qualifications made Dr. Doney an excellent contact, allowing us to learn more about how Manifold could positively impact the climate.
Discussion: From the start of our conversation, Dr. Doney immediately commended our team for attempting to make biosynthesis an industrially viable replacement to traditional manufacturing solutions. In fact, Dr. Doney shared that in a presentation to the National Science Foundation for Environmental Research and Education, he recommended that the best solution to combat climate change is to make a drastic shift towards adopting biotechnology across the chemical manufacturing industry. This recommendation was supported by his research at the Doney Lab. “Industrial biotechnology has already proven its worth in climate change mitigation.“ Dr. Doney stated. “If you look at herbicides and insecticides that have been made through biosynthesis, over 26 million tons of carbon dioxide has been removed in the past 16 years. This statistic means that by reducing greenhouse gases, we can limit the frequency and intensity of droughts, storms, heat waves, rising sea levels, melting glaciers, warming oceans, while preserving habitats, protecting animals and ensuring the livelihoods of people and their communities.” From our realization that adopting biosynthesis alone had the potential to significantly reduce climate change, our team became excited by the possibility of large-scale biosynthesis. We continued to ask questions about how adopting biosynthesis could positively impact society and make chemical manufacturing a green solution. But, Dr. Doney responded by saying it depends on how much the chemical manufacturing industry adopts biotechnology like Manifold. “Assuming that the entire chemical manufacturing industry adopts biotechnology like Manifold, I would say current models in environmental change research show anywhere between 50 to 90% of their current carbon emissions would disappear like that." But before parting ways, Dr. Doney challenged our team to come up with a model that shows the impact Manifold has on the environment. “If you could prove that your technology could significantly reduce carbon emissions, you could bring an end to human-caused climate change.”
Reflection: Our conversation with Dr. Doney reaffirmed our commitment towards bringing an end to environmental pollution caused by chemical synthesis. Although we knew in principle that industrial biosynthesis could reduce global carbon emissions, we never realized the larger contribution of biosynthesis. We weren’t just building Manifold to reduce carbon emissions or stop chemical waste from ending up in bodies of waters. We were building Manifold to entirely modernize the chemical manufacturing industry into a sustainable and environmentally solution that could bring an end to human-driven droughts, storms, heat waves, rising sea levels, environmental degradation, melting glaciers and so many other effects of climate change. With our conversation with Dr. Doney, we would later modify our implementation approach such that it emphasized our team’s mission towards environmental protection. But right now, our team wanted to meet with Dr. Kester again, the director of the nanoSTAR Institute, to ask about the economic impact of Manifold before truly calling our project a responsible, socially good and ethical solution.
Reflection: Our conversation with Dr. Doney reaffirmed our commitment towards bringing an end to environmental pollution caused by chemical synthesis. Although we knew in principle that industrial biosynthesis could reduce global carbon emissions, we never realized the larger contribution of biosynthesis. We weren’t just building Manifold to reduce carbon emissions or stop chemical waste from ending up in bodies of waters. We were building Manifold to entirely modernize the chemical manufacturing industry into a sustainable and environmentally solution that could bring an end to human-driven droughts, storms, heat waves, rising sea levels, environmental degradation, melting glaciers and so many other effects of climate change. With our conversation with Dr. Doney, we would later modify our implementation approach such that it emphasized our team’s mission towards environmental protection. But right now, our team wanted to meet with Dr. Kester again, the director of the nanoSTAR Institute, to ask about the economic impact of Manifold before truly calling our project a responsible, socially good and ethical solution.
Professor Mark Kester
TO WHAT EXTENT CAN MANIFOLD IMPACT THE ECONOMICS OF CHEMICAL MANUFACTURING?
TO WHAT EXTENT CAN MANIFOLD IMPACT THE ECONOMICS OF CHEMICAL MANUFACTURING?
Discussion: As the director of the nanoSTAR Institute, Dr. Kester was an invaluable resource in understanding the extent at which Manifold could impact the economics of chemical manufacturing. Not only does the nanoSTAR Institute itself manufacture industrial liposomes through biosynthesis, but more importantly, Dr. Kester possesses incredible knowledge in the economics of biosynthesis and current chemical manufacturing. From our initial conversation with Dr. Kester, we learned how current chemical manufacturing practices, including plant synthesis and chemical synthesis, do not meet the demands of modern society, often resulting in drug shortages and high prices for industrially-important chemicals. With this problem in mind, we returned to Dr. Kester to question, “If current manufacturing practices are unsustainable and costly, to what extent can biosynthesis technology like Manifold improve the economics of chemical manufacturing?” Although Dr. Kester couldn’t provide our team an estimate (because of the variability in biosynthesis across industries), he explained how adopting industrial biosynthesis removes many of the costs wasted in plant synthesis and chemical synthesis. In plant synthesis, companies must invest thousands-of-millions of dollars to purchase the land necessary to grow plants. Afterwards, they must continually invest in labor and supplies to grow these plants. Similarly in chemical synthesis, companies must also invest millions of dollars to purchase chemical plants that optimize chemical production, while further investing more money into chemical isolation and purification as similarly expressed in our earlier conversation with AMPAC Fine Chemicals. “This makes biosynthesis more attractive economically, because companies can purchase inexpensive bioreactors for a few thousand dollars,” stated Dr. Kester. “But besides that, it also doesn’t require the same amount of resources as plant synthesis and chemical synthesis, while producing chemicals at substantially higher yields and purity.” Dr. Kester then went on to explain how insulin extracted traditionally from pigs costed hundreds-of-dollars today, but through biosynthesis, a vial of insulin costs a few dollars to make. Instead, the problem with biosynthesis involves its variability and exploitation. Dr. Kester shared, “Because biosynthesis will differ depending on the chemical of interest (in terms of the organism, pathway and bioreactor setup used to synthesize the chemical), some biosynthetic chemicals like insulin can be produced cheaply. But as a result, pharmaceutical companies price these medications unnecessarily high.” Realizing this dilemma, we shared to Dr. Kester about our emphasis on engineering a standardized and modular version of Manifold. To his response, Dr. Kester stated, “If you can somehow make all biosynthesis processes inexpensive, then more and more companies will start making insulin for example, and we would actually see a reduction in insulin prices.”
Reflection: From our conversation with Dr. Kester, our team fully confirmed that by standardizing and modulating industrial biosynthesis through Manifold, we could significantly impact the economics of chemical manufacturing. Not only does industrial biosynthesis remove significant start-up costs associated with plant synthesis and chemical synthesis, but by adopting our standardized and modular version of Manifold, chemical manufacturing companies can save thousands-of-millions of dollars needed to design biosynthesis process and sustain them. But surprisingly, our team was again reminded about the potential for Manifold to be exploited by chemical manufacturing companies. “If pharmaceutical companies can inexpensively make insulin for a few dollars, what’s preventing them from withholding these savings to the consumer and continuing to charge hundreds-of-dollars for insulin?” our team questioned. Our journey through integrated human practices showed that Manifold was a responsible, socially good and ethical replacement to current chemical manufacturing practices. However, as we were inspired by the words of Dr. Shepherd and reminded by the possibility of Manifold being exploited for profit, our team agreed we needed to do something. This led our team to take up Dr. Shepherd’s offer and meet with the University of Virginia Licensing and Ventures Group to find a solution.
Reflection: From our conversation with Dr. Kester, our team fully confirmed that by standardizing and modulating industrial biosynthesis through Manifold, we could significantly impact the economics of chemical manufacturing. Not only does industrial biosynthesis remove significant start-up costs associated with plant synthesis and chemical synthesis, but by adopting our standardized and modular version of Manifold, chemical manufacturing companies can save thousands-of-millions of dollars needed to design biosynthesis process and sustain them. But surprisingly, our team was again reminded about the potential for Manifold to be exploited by chemical manufacturing companies. “If pharmaceutical companies can inexpensively make insulin for a few dollars, what’s preventing them from withholding these savings to the consumer and continuing to charge hundreds-of-dollars for insulin?” our team questioned. Our journey through integrated human practices showed that Manifold was a responsible, socially good and ethical replacement to current chemical manufacturing practices. However, as we were inspired by the words of Dr. Shepherd and reminded by the possibility of Manifold being exploited for profit, our team agreed we needed to do something. This led our team to take up Dr. Shepherd’s offer and meet with the University of Virginia Licensing and Ventures Group to find a solution.
A Sustainable, Socially Good, and Ethical Solution
From our journey through integrated human practices, our conversations with experts from diverse fields, ranging from wet-lab researchers to modeling simulators to bioethicists to environmental scientists, provided greater context to the impact Manifold served on society. From the very start of our iGEM experience, we envisioned Manifold as a project centered around helping people, whether this involved reducing global pollution, ensuring all patients have access to live-saving medications or assisting chemical manufacturers meet society’s demand for chemicals. In any case, this led us through a serious phase of contemplation, where we needed to understand two questions. First, was making Manifold good for society? And second, was implementing Manifold into the real-world good for society? Ultimately, our conversation affirmed our vision that making Manifold was a socially good decision. But, although Dr. Shepherd stated that risk of exploiting our project wasn’t our ethical dilemma to bear, a large part of our us felt obligated to address this problem of exploitation. We knew that Manifold directly reduced global pollution and chemical waste, lowered manufacturing costs, and optimized the entire chemical manufacturing process, we need to ideate a way to keep chemical manufacturers accountable that these benefits go directly to the end-user. With the guidance of Dr. Shepherd, we met with the University of Virginia Licensing and Ventures Group to look for a solution.
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