Team:Virginia/future-directions
As aspects of our project fell short of some of our team’s expectations and goals, we recognize that there are many places for further development and improvement. Our team sees great promise in our device, which we hope to continue developing. Detailed below are a few areas our team hopes to continue integrating into our project.
Wetlab Future
Extracting PHB Sustainably
Given the environmental nature of this project, our team attempted to keep our protocols environmentally conscious. PHB extraction was particularly difficult to do this for as many extraction methods involve potent chemicals to lyse the cells and separate PHB from the cell debris. Towards the end of our project our team began working on our own PHB extraction method that couples sonication to lyse the cells, and density differential centrifugation, with a sucrose stock, to separate the cell debris. The most updated version of this protocol can be found under experiments in our protocol handbook.
This protocol however, is far from perfected. Sonication, for example, is very empirical in determining the most efficient procedure for lysing the cell, and would likely need to be performed many more times before our team is confident that we have found the most effective manner of sonicating. Similarly, the creation of a sucrose solution that effectively separated PHB from cell debris was not nearly as efficient as it had the potential to be. There were likely impurities of cell debris in the PHB pellet, as well as PHB in the cell pellet. Through more empirical trials this method could be improved to increase the precision of separation of the two pellets. Improvements made to sonication settings and the sucrose solution would also increase the purity and yield of PHB obtained from this method.
Quantifying sty Plasmid Functionality
Gas chromatography (GC) is a very precise and efficient method to determine the amount of styrene uptaken by bacteria by measuring the concentration of styrene contained within the biphasic mixture.1 We were unable to successfully perform GC because our solvent for styrene, dioctyl phthalate, interrupted the data with powerful peaks. Dioctyl phthalate is an efficient solvent for dissolving styrene and is harmless to the cells, however it was not compatible with GC. In the future, we have to select a solvent that fulfills these qualities in addition to working for gas chromatography.
Gas-Liquid Chromatography Machine depicting individual
parts necessary for its function.1
Detailed fractional distillation mechanism that highlights
the individual parts of the apparatus.2
Another possible method for determining styrene concentration is fractional distillation. The boiling points of styrene and dioctyl phthalate differ greatly, and thus fractional distillation is a simple technique to separate the styrene liquid from the dioctyl phthalate. Fractional distillation can be performed after sufficient culturing of bacteria to roughly determine how much styrene was used during the time of culturing.2
Testing Different Genes that Show Potential for the Biochemical Pathways
There are multiple bacteria with unique genes that are able to complete the biochemical pathways of styrene degradation and PHB production. The genes we selected for our pathway were from well characterized chassis organisms that showed promise to be efficient in these pathways. However, to further improve the efficiency of this process, our team would have liked to test other genes involved in this process to see if yield could be improved. sty genes have a few genetic variants, specifically in the styAB gene portion. For example, there exists a set of genes (styA1 and styA2B) which show promise to be an even more effective variant of the styAB gene set.3 Our team hoped to test styrene degradation with these genes as well, but did not due to time constraints.
Using an Optimized Chassis
Pyrolyzing polystyrene to produce styrene monomers result in toxic byproducts including aromatic compounds such as benzene and toluene.4 We did not consider these byproducts when choosing our chassis as E. coli K12 TG1 as pure styrene was fed to the bacteria during experimentation. However, future implementation must include mechanisms which manage these toxins. For example, Pseudomonas putida mt-2 and F1 strains have been shown to degrade toluene and ethylbenzene, the two major byproducts of pyrolysis.5 By re-engineering our device into these species, we can effectively control the entire flowthrough of pyrolysis.
Modeling Future
Modeling the Fed-Batch Bioprocess
Our model determined that PHB production is unable to be growth-coupled in our bacterial device. Due to inevitable evolutionary strain on the metabolic network, the bacteria will have to grow for a shorter period of time through a fed-batch process rather than in a continuous bioreactor. To further reduce evolutionary and metabolic strain, the promoter of the PHB production genes should be induced at a specific time point on the bacterial growth curve while the promoter for styrene processing genes should be constitutively expressed. PHB production will be induced using a chemical inducer, such as IPTG, or a growth-associated inducer based on quorum-sensing (Virginia iGEM 2018). While the theoretical time point is established as the time the bacterial culture reaches carrying capacity, a bacterial kinetics model, combined with experimental data, will be used to determine the experimental time point for maximum PHB production in an unsteady state system.
The bacterial kinetics for the fed-batch process will be represented using the Verhulst logistic growth model (Equation 1) combined with the Luedeking-Piret equation, which separates growth associated and non-growth associated PHB production (Equation 2).6
The model accounts for the inducement of PHB production as production is 0 until the time point of induction t0. Because production was shown to not be growth-associated from growth-coupling analysis, ⍺ is assumed to be 0 and an equation for β is derived. (Equation 3).6
Equation 4 shows styrene consumption based on bacterial growth, bacterial maintenance, and PHB production.6
The parameters for the ordinary differential equations will then be determined through experimentation (Table 1).
Table 1: description of parameters in bacterial growth model
Conducting a Financial Analysis
To understand how industrial implementation of our device would disrupt the market, we would need to conduct a thorough financial analysis. Points of interest include: shipment and processing of waste polystyrene to styrene, the maintenance cost of the most effective upscaled method of production, and the processing and sale figures of PHBs produced. While we did learn that PHAs can replace petroleum-based plastics for the same uses, after our conversation with Danimer Scientific, a more comprehensive comparison between PHA and PET/PP properties would more strongly support the idea that our device could reduce the demand of petroleum-based plastics like PET and PP.
Human Practices & Outreach Future
Using Wikipedia
An easy way to continue to positively influence public knowledge is to continue to monitor and create Wikipedia pages describing subjects we have amassed knowledge in. As it pertains to Transfoam, we will focus on pages related to plastics and synthetic biology in the environment, though this is not exclusive in the future. A page we are currently planning would consist of several lists of “best plastics” based on various properties, including production emissions, recyclability and associated costs/price. We are always open to suggestions and encourage other iGEM and research teams take similar steps to promote their work and make an impact!
Putting ASTM Revision on Agenda
We were fortunate to hear back from the American Society for Testing and Materials regarding a revision to the standard definition of “biodegradable plastic.” Our short-term goal is to participate in task groups to promote our revision proposal to the November agenda, and our long-term goal is to ultimately have the revision passed.
Pushing Bill Proposal Forward
Even though our project is coming to a close, we will without a doubt continue our efforts to pass legislation that will enable plastic bans to be passed reasonably in Virginia. We plan to speak with several candidates running this November in an attempt to encourage our directives into their political agendas as they enter election season. Specifically, we plan to meet with Delegate Plum, Delegate Krizek, and Delegate Guzman to discuss the best course of action.
References
- Kanazawa, J., Kubo, H. & Sato, R. Gas-Liquid Chromatography of Organophosphorus Pesticides. Agricultural and Biological Chemistry 29, 56–60 (1965).
- Fractional Distillation. http://www.pitt.edu/~ceder/lab7/fractionaldistillation.html.
- Karaduman, A. Pyrolysis of Polystyrene Plastic Wastes with Some Organic Compounds for Enhancing Styrene Yield. Energy Sources 24, 667–674 (2002).
- Nogales, J., García, J. L. & Díaz, E. Degradation of Aromatic Compounds in Pseudomonas: A Systems Biology View. in Aerobic Utilization of Hydrocarbons, Oils and Lipids (ed. Rojo, F.) 1–49 (Springer International Publishing, 2017). doi:10.1007/978-3-319-39782-5_32-1.
- Tischler, D. et al. StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System. J. Bacteriol. 192, 5220 (2010).
- Divyashree, M. S., Rastogi, N. K. & Shamala, T. R. A simple kinetic model for growth and biosynthesis of polyhydroxyalkanoate in Bacillus flexus. New Biotechnology 26, 92–98 (2009).
