Stony Brook iGEM is not only the best iGEM team to have ever existed, but also the best team to have ever existed. We've got people. We've got teamwork. We've got trips to adventureland. We've got trips to Taco Bell. We've got trips to Taco Bell. Take a few moments out of your day to bask in the glorious grid of colors and pictures that is our team. I can't come up with any more filler text. How's it going?
Brainstorming ideas for projects during our spring reading course. Our team split into five groups:
A meeting with Dr. Tae Jin Kim (Chemical Engineering, Stony Brook University) reveals the difficulty in converting lignocellulose to usable fuels, as well as the long chemical composition of diesel and gasoline and the chemical pathways involved in producing them.
“Biodiesel is hard”
We concluded that biodiesel from agricultural waste may be possible with genetic engineering, but “simpler” fuels like ethanol are a more realistic target for a summer project.
A meeting with Dr. Jackie Collier (SoMAS, Stony Brook University) provides valuable information on how to grow our strain of cyanobacteria. She aided us in our equipment setup, recommending that we bubble air through a flask of water to hydrate it. The hydrated air would then go to individual test tubes housing our cyanobacteria. The test tubes are placed in racks in a small aquarium with a heating element to maintain comfortable temperature. Dr. Collier informed us that the easiest way to keep stocks of our bacteria when we weren’t actively experimenting them was in agar plates.
Dr. Gabor Balazsi (Laufer Center, Stony Brook University) helped provide information during our project development stage in the spring reading course. In the beginning of our project development, we had two pathways in which the products would be a potential feedstock for biofuel. We were deciding between a pathway that produced chains of alkanes or a pathway that produced and excreted sucrose. Dr. Balazsi gave us advice regarding how we would design our project if we chose the sucrose pathway versus if we chose the alkanes pathway.
Neil Warner is the Director of Transportation and Parking at Stony Brook University. He looked very positively on our mission to use alternative fuels and carbon sequestration. He explained that when considering using alternative fuels in diesel engines designed for public highways, the effect these fuels have on the regenerative exhaust systems and the fuel pumps impacts whether people would adopt such a system. (We should actually reach out to a diesel company to see how ethanol could work in diesel engines). Thus, most institutions would reach out to the vehicle manufacturer first to see if a solution is viable. Also, he 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-base 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. (This led us down the path to economic math modeling).
Dr. Brian Baynes, former CEO of Joule Unlimited, discussed experimental strategies as well as the challenges of using cyanobacteria to produce alkanes. He recalled that Joule’s production of alkanes was successful at a small scale; large scale production required extraction and distillation to get desired purity. In addition, the photosynthetic efficiencies of their cyanobacteria reached a maximum of 10%, and the methods they utilized to enhance efficiency were very expensive. In essence, Dr. Baynes believed that such a venture would not currently be economically feasible. At the time we were deciding between an alkane-production project and our current project, so our conversation with Dr. Baynes greatly informed our ultimate project choice. 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 through sucrose production. One main paradigm of Joule’s experimental design was the separation of growth and stationary phases (where the cyanobacteria would start secreting product upon reaching stationary phase). 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.
Dr. John Aikens, former CEO of Proterro, emphasized the good our work could do for the environment Cyanobacteria are gentle; contamination is almost guaranteed once they start making sucrose (mostly by skin-related germs). Use bsl-2 level technique carbon flux: Desirable to have high CO2 input to reduce total amount of Cyanobacteria and water needed; however, not necessary Explained difficulty of working with Cyanobacteria polyploidy E. Coli conjugation is an effective but time consuming process Emphasized importance of a fast-growing strain Suggested investigating CRISPR as a solution to polyploidy
Hung Li is a part of the Department of Chemical Engineering of National Tsing Hua University and is a PhD student of Professor Hu who was the first to research on CRISPR-Cas9 and PCC 7942 in 2016. He explained that CRISPR-Cas9 would take the same amount of time as natural transformation, ______reread the email
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 used the same strain of cyanobacteria. 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 time-consuming process of transformation (without CO2) Can take 2 months Many parallel projects are necessary
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 Lux AB gene in pAM1414. She explained that BamHI and BsiWI should be suitable enzymes to insert our promoter sequences. (5/28) Dr. Golden also confirmed that DH5alpha would be a compatible growth strains for the cyanobacterial cloning vectors pAM1414, pAM2991, and pAM1579. She also answered our questions on which luminometers should be used for characterizing the promoters. Dr. Golden explained that adding decanal to the cells is fine if we don’t mind killing our sample. (5/31) She explained that for using liquid decanal “The dissolving in mineral or vegetable oil was just to make the vapor come off more gradually and protect the cells — not necessary if you don’t mind killing your sample.You can just add a tiny drop inside your closed measurement vessel. A little goes a long way. It stinks to high heaven - you’ll want to seal it up if you can. It dissolves adhesives, so be careful using it in any device that has a sticky seal (like the TopSeal on a microtiter plate). You can do it, but in a very short period of time the seal will dissolve and anything it is attached to, like s sheet of plastic, will float off into your machine if you aren’t careful.”
Dr. Devinder Mahajan is a professor and the graduate program director of our Chemical Engineering department. We met him at our bake sale at the library when he walked by and saw our poster. He questioned our team about what the bake sale was for and we introduced him to iGEM. So, we set up a meeting with him to talk about what our project is and what iGEM is. Dr. Mahajan works with research in low-carbon energy and in this meeting, we set up a meeting for a tour. We also discussed the implications our work could have for the biofuel industry and how carbon sinks and ethanol could work in the real world.
Professor of Michigan State University and he wrote a research paper in which he engineered cyanobacteria secrete sucrose. While speaking with him and discussing the creation of our constructs, he suggested using Gibson Assembly. During the video call, we were able to ask all questions we had in regards to cyanobacteria and the specifics of our project. We were considering using a quorum-sensing based promoter earlier on in our work, but during the meeting, made the final decision not to follow through with that due to the lack of research in the field.
Dr. Jarrod French, one of our team advisors and a professor in our Chemistry department, discussed plasmid construction and experimental design with us. He said 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 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. 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 urged us to consider the feasibility of large scale applications of our project. Theoretically, commercial companies would set up large vat photobioreactors. 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. After doing some research into this topic, we concluded that we would not introduce a peptide sequence for a tag since the 3D structure of cscB is not well characterized. Of course, 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.
Mirna Kheir is a researcher in Gabor Balazsi’s lab and she helped us with designing our constructs for HiFi Assembly. She also gave advice on the appropriate procedure to follow for using HiFi Assembly in the laboratory. Dr. Balazsi explained the Michaelis-Menten equation to us and showed how we could apply it to modeling extracellular sucrose production in our bacteria Modeling this system accurately could inform us on optimal growth conditions and if glycogen knockout is necessary
We met with Michael Axelrod who manages the Life Sciences Greenhouse at Stony Brook University. He answered a few of our questions on how to set up the incubators for our cyanobacteria. Suggestions on lighting (4100 kelvin fluorescent lamps) Using a quantum sensor to measure light intensity Offered to let us use a growth chamber
Dr. Devinder Mahajan brought us on a tour of his low carbon energy lab at the Advanced Energy Research and Technology Center at the Research and Development Park of Stony Brook. In this tour, we saw an eppendorf fermentation machine which we could potentially use to turn our sucrose into biofuel. 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.