Team:Stony Brook/Human Practices

Integrated Human Practices

A brief timeline

February 20: Meeting with Dr. Tae Jin Kim (Chemical Engineering Department, Stony Brook University)

The group researched potential renewable biofuel products and decided to meet with Dr. Tae Jin Kim, an expert at Stony Brook University’s Chemical Engineering department early on in their project development. They learned about the current state of biofuels and synthetic biology. They met with Dr. Kim, who explained the difficulty in converting lignocellulose, a form of plant-derived sugar, to usable fuels. From the meeting, we learned that it is difficult because of the chemical composition of diesel and gasoline and the long chemical pathways involved in producing them. According to Dr. Kim, decades of research and funding from the Department of Energy has already gone into biofuel production. However, Dr. Kim saw some potential in engineered enzymes, which he thought may have potential to overcome some issues, especially with algae as possible next-generation feedstock. Though originally we planned on focusing on the fermentation process and form lipid biodiesel, this discussion led us to pursue algae and other photosynthetic chassis. We also decided to focus on producing products that have far less complex synthesis pathways, thereby allowing us to focus on the production of feedstock. We also learned that some companies, such as Joule Unlimited, Algenol, HelioBioSys, and Proterro, attempted to work with algae-based biofuel but had become bankrupt. From what we learned in scientific literature on algae-based biofuels, we started to reach out to and investigate these companies, so that we could identify any faults in the system and learn what issues we could attempt to solve. 

March 28: Meeting with Dr. Jackie Collier (School of Marine and Atmospheric Sciences, Stony Brook University)  

We wanted to meet with an expert who works with cyanobacteria because we lacked knowledge of how to grow our strain of cyanobacteria. We chose to meet in person and tour her lab because were interested in learning how to properly culture cyanobacteria. Touring her lab allowed us to see how microbiologists working with cyanobacteria perform research. She also aided us greatly in our equipment setup and culture conditions. For one, she recommended that we bubble air through a flask of water to hydrate it. Through this method, we learned that the hydrated air would then go to individual test tubes housing our cyanobacteria. The test tubes would be placed in racks in a small aquarium tank with a heating element to maintain comfortable temperature. Thus, initially, we aimed to replicate Dr. Colliers setup with the aquarium tank and the flasks that allowed for air to be bubbled into them. However, we later obtained a CO₂ incubator from Dr. Malbon’s lab in the Department of Pharmacology. Cyanobacteria growth in a CO₂ appeared to be a better setup because it required less materials and maintenance. In addition, Dr. Collier agreed that incubating cyanobacteria in CO₂ as opposed to bubbling gas into a flask of cyanobacteria would allow them to grow faster. Furthermore, we did not use test tubes to grow cyanobacteria as Dr. Collier initially suggested but rather we used flasks because they provided a larger surface area. After being informed of this change, Dr. Collier further advised us to use stoppers made of cotton to stopper the flasks. Thus, through our correspondence with Dr. Collier, we considered different experimental setups and based on knowledge from Dr. Collier about optimal cyanobacteria growth conditions and we designed and improved our setup accordingly.

April 13: Meeting with Dr. Gabor Balazsi (Department of Biomedical Engineering, Stony Brook University)

At this stage in the project development, two potential routes we pursued for our project were the alkane and ethanol pathways in cyanobacteria. We sought to determine a method to measure alkane and sucrose (for ethanol production) output. To measure sucrose output, there were commercial kits available to allow us to measure sucrose content in a liquid solution. However, measuring alkane production was more complicated. We decided to reach out to Dr. Gabor Balazsi, an expert in biosensors at Stony Brook University to determine if we could make an alkane biosensor, similar to that in existing literature. Dr. Balazsi strongly suggested using a negative feedback loop, allowing for a linearized signal from the sensor, such that a measurement device would form. A measurement device, as opposed to a typical biosensor, he explained, does not have a threshold, but rather is able to indicate different concentrations of a molecule. This measurement device for alkane output sounded almost as feasible as the method to measure sucrose. Ultimately, Dr. Balazsi’s advice would come to greatly influence our decision between our choice for alkanes or sucrose.   

April 16: Correspondence with Neil Warner (Director of Transportation and Parking, Stony Brook University)

In addition to reaching out to experts in synthetic biology, we wanted to understand how administrators in transportation would view our project. We reached out to Neil Warner, the director of transportation and parking at Stony Brook University, in order to get his input. He responded quickly to us and admired our research in producing alternative fuels. He explained the effect these alternative fuels have on the regenerative exhaust systems, and the fuel pumps that impact whether people would actually adopt such a system. He also explained that NY State Agencies are exempt from the state sales tax on highway fuel. Therefore, the cost of fuel for such agencies is equal to the price of diesel at the pumps. For ethanol-based biofuel to be adopted, it must be able to compete with that price and have a decent fuel economy. Fuel economy is directly related to the energy that fuels produce from combustion, and so our ethanol-based biofuel needs to have a fair cetane rating. Furthermore, Mr. Warner suggested that we reach out to Caterpillar and other diesel engine companies to learn more about the impacts of ethanol on exhaust systems and fuel pumps. Unfortunately, we did not receive a response from them.  

May 3: Meeting with Dr. Brian Baynes (Former CEO, Joule Unlimited)

Dr. Brian Baynes discussed with our team the experimental strategies as well as the challenges of using cyanobacteria to produce alkanes. He recalled that Joule’s production of alkanes was successful but only at a small scale; large scale production required extraction and distillation to get a desired purity. In addition, the photosynthetic efficiencies of their cyanobacteria reached a maximum of only 10%, and the methods they utilized to enhance efficiency were very expensive. In essence, Dr. Baynes believed that such a venture would not be economically feasible. Our conversation with Dr. Baynes encouraged us to pursue the sucrose-based project. Considering the amount of time and resources we had, the team felt it would be more feasible to do a project centered around carbon sequestration. 

May 11: Meeting with Dr. John Aikens (Former CEO, Proterro)

Dr. John Aikens, former CEO of Proterro, talked about how one of the main paradigms of Proterro’s experimental design was the separation of growth and stationary phases, where cyanobacteria would start secreting product upon reaching stationary phase. This was due to rerouting the carbon flux early on, endangering the cyanobacteria themselves. We used this model as an inspiration for our experimental design in Part III, where sucrose production can be easily induced at stationary phase in response to certain environmental stimuli. However, at the same time, he stressed how cyanobacteria are extremely under-studied and that most promoters need further characterization. As a result, we wanted to characterize many promoters to help further the cyanobacterial synthetic biology community, and made that a main goal of our project. He informed us that it was desirable to have high CO₂ input to reduce total amount of cyanobacteria and water needed, although this was not necessary. He also suggested using carbon dioxide controlled conditions, which prompted us to secure a cell culture incubator. Moreover, he informed us that contamination is almost guaranteed once cyanobacteria start making sucrose, mostly by skin-related germs. This comment encouraged us to use cell culture level precaution and techniques. Lastly, Dr. Aikens addressed some potential issues of working with cyanobacteria, one of which was polyploidy. One method he recommended was E. coli conjugation, but explained that despite its effectiveness, that it was a time consuming process. To counteract this issue, our team suggested using the CRISPR-Cas9 system, which he was very enthusiastic about. He also emphasized the importance of experimenting with a fast-growing strain, which we sought out. 

May 13: Correspondence with Hung Li (Martin) (Department of Chemical Engineering, National Tsing Hua University)

Based on the knowledge and advice of people we met with up until this point, we have some concerns regarding the length of time cyanobacterial transformation takes. Thus, we considered using CRISPR-Cas9 gene editing technology to insert our genes and promoters of interest into cyanobacteria. We reached out to Hung Li who is a part of the Department of Chemical Engineering of National Tsing Hua University and is a PhD student of Professor Hu, the first to research on CRISPR-Cas9 with PCC 7942 in 2016. Mr. Li explained that CRISPR-Cas9 would take the same amount of time as natural transformation under normal conditions, so we decided that taking traditional transformation processes would ultimately be more feasible for our team.  

May 24: Correspondence with McKenna Hicks (Former iGEM Member, 2017 UCSC Team)

McKenna Hicks was a member of the 2017 UCSC iGEM team. We emailed her to ask her questions about her team’s project related to cyanobacteria, especially since that team worked with the same strain of cyanobacteria that we were planning to work with. In particular, we asked for advice on constructs and culturing techniques. McKenna advised us to add terminators to our constructs because the plasmid backbones we were using did not have correctly oriented ones, as her team discovered during their work. She also supplied information and specifications about their culturing setup. Emphasized that cyanobacteria transformation may be a time-consuming process of transformation without carbon dioxide so we should consider running our experiments in parallel projects if necessary.

May 25 to May 31: Correspondence with Dr. Susan Golden (Director of Center for Circadian Biology, University of California - San Diego)

We reached out to Dr. Susan Golden upon review of the literature, we discovered that she wrote the protocol for S. elongatus transformation and created the promoterless luxAB NS1 vector, pAM1414 in which we would eventually use in our final project. We wanted her advice on performing her protocols in optimal conditions. More specifically, we emailed Dr. Susan Golden to ask about her vectors on Addgene. We asked her questions regarding the restriction enzyme sites for our promoter sequences which we would put upstream of the LuxAB gene in pAM1414. She explained that BamHI and BsiWI should be suitable enzymes to insert our promoter sequences. Dr. Golden also confirmed that DH5alpha would be a compatible growth strain for the cyanobacterial cloning vectors pAM1414, pAM2991, and pAM1579. She also answered our questions regarding which luminometers should be used for characterizing the promoters. Regarding promoter characterization, we were unsure of whether to use a luciferase- related plasmid such as luxCDE or use decanal and Dr. Golden explained that adding decanal to the cells would be adequate. Additionally, when talking with Dr. Golden, we learned about neutral sites in her recommended cyanobacteria “suicide vectors.” These types of vectors integrate into the genome through double homologous recombination and have no origins of replication. The neutral sites match sequences in the genome where the recombination occurs. As a result, E. coli conjugation was no longer necessary, resolving issues that Dr. Aikens brought up. Furthermore, through correspondence with Dr. Golden, she also confirmed that we should characterize the light and iron induced promoters during experimentation. Overall, Dr. Golden provided advice that validated our decisions regarding which promoters to characterize and helped us to confirm our use of decanal in testing these promoters.  

May 30: Meeting with Dr. Daniel Ducat (Department of Biochemistry & Molecular Biology, Michigan State University)

Dr. Daniel Ducat is a Professor of Michigan State University, and he wrote a research paper in which he engineered cyanobacteria to secrete sucrose. We reached out to him because he attempted the kind of research we were about to perform. During the video call, we were able to ask all questions we had in regards to cyanobacteria and the specifics of our project which was very beneficial in our experimental design. While discussing the creation of constructs, he suggested using Gibson Assembly. Additionally, Dr. Ducat provided us with research materials involving quorum sensing as a method by which to regulate gene expression, and later on sucrose production,using cell- density population. A review of the literature showed that there is a lack of research in the field of quorum sensing, however, this research gave us the idea to use nutrient-repressible promoters which may act as viable density-dependent regulators.   

June 1: Meeting with Dr. Jarrod French (Department of Biochemistry and Cell Biology, Stony Brook University)

We met with Dr. Jarrod French to discuss plasmid construction and experimental design. We wanted to meet with him because his research on characterizing metabolic pathways in cells gave us insight to our cyanobacteria’s sucrose pathway and how we can manipulate it in our experiments.We learned that the rate of sucrose production won’t be drastically higher in our sps- overexpressing cyanobacteria because another step in the sucrose production pathway will inevitably become the rate limiting step.
For our experiments, Dr. French also recommended having a positive and negative control wherever possible. For example, we decided that in part II, the trp-lac promoter would serve as negative control and the rbc promoter would serve as positive control. Unfortunately, in later stages, the trp-lac promoter was unable to be synthesized and our rbc stock died. In part I, Dr. French and the team concluded that an empty vector would serve as a suitable control, and that we should also use WT untransformed bacteria, which should die and bear no results.
Dr. French also urged us to consider the feasibility of large scale applications of our project which is a consideration we made when talking to Dr. Mahajan. Theoretically, commercial companies could set up large vat photobioreactors. However, energy expenditure to maintain all of the cyanobacteria would be high, and the sheer population of the cyanobacteria would cause all sorts of light scattering issues.
He also recommended that we consider tagging our cscB protein to ensure proper expression, transport, and embedding in the cell membrane. Thus, we decided to perform more research on the types of tags that we could potentially use, such as a poly(His) tag and FLAGtag. We attempted to FLAtag the sps gene, however, sps was too expensive to purchase with the incorporation of a tag. We also considered tagging the cscB gene but we concluded that we would not introduce because the 3D structure of cscB is not well characterized; should either the C or N terminus (or both) be tucked on the inside of cscB, producing a tag at the affected terminus may have drastic consequences on the function of cscB. Lastly, we researched corresponding measurement techniques such as dot blot that could be used with protein tagging. Thus, meeting with Dr. French helped to fine tune some aspects of our project and provided us with more to consider regarding our experimental setups and development of our gene circuits.  

June 1: Meeting with Mirna Kheir (Laufer Center, Stony Brook University)

Mirna Kheir is a researcher in Gabor Balazsi’s lab and she helped us with designing our constructs for Gibson assembly. More specifically, she showed us how to design overlapping ends between adjacent sequences and recommended an overlap length of at least 30 base pairs. We then applied these principles to all of our genetic constructs. She also gave advice on what procedures to follow with Gibson assembly in the laboratory and recommended what products to order for Gibson assembly.

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June 1: Meeting with Dr. Gabor Balazsi (Department of Biomedical Engineering, Stony Brook University)

Dr. Balazsi explained steady-state approximations and Michaelis-Menten kinetics to us. Within this context, he showed us various possible parameters that we could apply to our specific system. Specifically, he told us to focus on carbon dioxide intake, light intensity, redirection of carbon flux to natural pathways, the production of intracellular sucrose, and the rate of secretion of this sucrose. He showed us how to set up various generalized differential equations that we could then use in our system given rate constants that we would have to find in literature.
Modeling this system accurately could inform us on optimal light and atmospheric conditions for maximum sucrose production. It could also inform us on the value of knocking out other pathways for carbon flux, such as glycogen anabolism, for the purpose of retaining more carbon for sucrose. 

June 8: Tour of the Research and Development Park Center with Dr. Devinder Mahajan (Department of Chemical and Molecular Engineering, Stony Brook University)

Dr. Devinder Mahajan is a professor and graduate program director for Chemical and Molecular Engineering at Stony Brook University who researches about low carbon energy technology development and implementation. We used this opportunity to grasp the potential our project has after our laboratory studies. Once we introduced him to iGEM, he offered to meet with us to talk more about our project in the scope of biofuels and energy, as he researches low-carbon energy. We wanted to make sure that our project would aid in the development of biofuels and would be relevant to current advancements in carbon sequestration and renewable energy technology. During the meeting, we learned about the research he was conducting on carbon sequestration and renewable energy technology. He recounted the emission numbers of local Long Island landfills and powerplants and gave us the idea to use landfills as a site of carbon sinking for biofuel production. We toured his laboratory at Research and Development Park in Stony Brook where he showed us his eppendor fermentor machine, a device that is capable of turning the sucrose potentially produced by cyanobacteria into ethanol for biofuel. After the tour, he invited us to visit the facility again and to have bi-weekly meetings and correspondence to address our progress on the project and any questions we may have.

June 5th: Meeting with Michael Axelrod (Instructional Support Specialist/Greenhouse Curator, Stony Brook University)

We met with Michael Axelrod, who manages the Life Sciences Greenhouse at Stony Brook University. We met with Mr. Axelrod to discuss the optimal conditions to grow cyanobacteria. We also discussed the practicality of growing cyanobacteria for biofuels on a commercial scale to meet the demand of consumers. He answered a few of our questions regarding how to set up the incubators for our cyanobacteria. Mr. Axelrod also suggested we used 4100 kelvin fluorescent lamps as a light source in the incubators. He taught us about the limitations of growing plants (difficulty of controlling CO2, humidity, etc.) and lead us to pursue making our own growth chamber. In addition, he let us borrow a quantum sensor to measure light intensity as well as offered to let us use a growth chamber. Thus, we learned much about growing cyanobacteria under optimal growth conditions and considered the feasibility of our project and in turn Mr. Axelrod, who was enthusiastic in helping us troubleshoot aspects of our experimental design, and we were also able to engage him in a discussion of plants as well as cyanobacteria as a source of biofuel.   

June 20th: Meeting with Zahid Syed (Commissioner of the Nassau County Commission on Human Rights, Nassau County)

Zahid Syed, a politician from East Meadow, New York, has had an active role in his community. Having been involved in civic, politics, labor, and human rights issues throughout Long Island, we thought he would be an informative person to speak with in order to gain some insight to our project. After exchanging emails for a week, Mr. Syed and our team thought it would be much more beneficial to meet in person. After listening to our project and our outlook, he provided us with advice and possible logistics we may face. Understanding that our project may have the potential to end world hunger, Mr. Syed was fascinated, and he invited us back to many political events, allowing us to present our project and meet with more members of the local government.

July 5th: Meeting and Presentation with Advisors (Stony Brook University)

At this meeting with the advisors Dr. Balaszi and Dr. French we presented on the progress of our research as well as issues we were currently troubleshooting in the lab. Regarding modeling, Dr. French recommended that we consider flux balance analysis, a mathematical model for simulating metabolism if given specific input and constraints of a certain metabolite, over the incorporation of the Michaelis- Menten equation in our model. He explained that use of the Michaelis- Menten equations in models is only useful in “test- tubes” situations where all of the the constants and parameters are known and fixed. However, this this is not true for a cell in which concentrations of substances and the rate of protein synthesis are constantly changing. He went on to explain that while the Michaelis- Menten equation uses steady state approximation to find all rates of enzymes involved, you cannot do that in a cell whose rates of enzymes are changing in a realistic situation. In other words, you cannot find steady state approximation of cell. Furthermore, the Michaelis-Menten equation only concerns one substrate molecule, however our research concerns bimolecular substrate reactions. Thus, upon advice from Dr. French regarding the direction of our modeling, we decided to conduct more research on flux balance analysis. We found that flux balance analysis may be a more conducive model for our work with cell biology because flux balance analysis assumes that all chemical reactions within a cell have reached equilibrium almost as if the cell were in a steady state. Furthermore, using flux balance analysis, a model can be built from a matrix of stoichiometric reactions taking place within the cell. Thus, we made the decision to shift our model focus from the Michaelis- Menten equation to flux balance analysis.  

October 5th: Meeting with Laura Curran and Staff Members (County Executive, Nassau County)  

Members of our iGEM team met with County Executive Laura Curran and two of her staff members, Sean Sallie and David Viana, to discuss the science behind our project, the implications of biofuel use on the economy, and the process of making biofuel. More specifically, one topic we discussed was how the revenue from producing biofuels could trickle back into funding the research behind these improving these processes. To this extent, her staff was very interested in increasing the sustainability of our project, and so we agreed to send our project poster and full wiki information after the Jamboree, such that they could help reach out to other leaders to help making carbon sinking more of a reality. We reached out to Laura Curran because she is a powerful and respected leader within Nassau County’s local government and we not only wanted her perspective on the importance of funding biofuel research but also we aimed to share our knowledge with an individual who had the ability to make impactful change in the community. The success of this meeting with a local member of the government informed us of society’s growing acceptance towards using biofuel in reducing carbon emissions as well as the receptiveness of local politicians towards the concerns of local citizens. While most of our outreach events involve young children, this meeting provided us a chance to engage with a political leader on the topic of synthetic biology and create a dialogue centered around synthetic biology.