From 2012.igem.org

Background

“Plastics: made to last forever, designed to throw away”--5gyres.org

Plastics play an integral role in our modern world. Due to their relatively low cost of production, they serve to promote the development of industry and lower the cost of consumer goods but perhaps the largest advantage to using plastics is their ease of production on a large scale. Because of this ease of production, products using plastics account for over a third of the products manufactured today. However, because this plastic is marketed as disposable, much of it ends up in landfills or in the ocean. Although 10% of the waste generated in the world is plastic, plastic makes up a much greater percent of the waste that is strewn about in the environment [1]. Plastic pollution is more than a problem of aesthetics. One of the many environmental problems associated with plastic pollution is that debris in the ocean are ingested by wildlife and often result in injury or death [2].

The PUR Problem

One commonly used plastic, Polyurethane (PUR), is extremely versatile and is thus used to create a wide range of products including dense solids, rigid insulating foam, and thermoset elastics. Because of this versatility, the world consumption of PUR is continually increasing. According to current market analysis, the global production of PUR is estimated at 27 billion pounds per year and is expected to increase to 36 billion pounds by the year 2016 [3]. This increase of demand can largely be attributed to the enormous population of Asia-Pacific countries and this region’s recent move toward modernization. Over half of the produced polyurethane is used in the construction and furniture industries, both of which are expected to experience widespread growth in the Asia-Pacific and South America [3]. All polyurethanes can be recycled. The leading method of recycling polyurethane is to mechanically grind the waste and rebond it into carpet cushioning. This accounts for about 4% of waste polyurethane [4]. However, due to the high costs of transportation and chemical degradation, most PURs are incinerated to recoup some of the energy used to make them [5].

Current Means of Disposal are Undesirable

Nonbiodegradable plastics are often removed from the chemosphere through waste incineration because it is the most efficient way to degrade these plastics. However, a major issue with this is that the products of waste incineration, which include polychlorinated di-benzo-p-dioxins/furans, are known to be carcinogenic [6]. When the plastics are incinerated, these products are released into the atmosphere and have the potential to cause problems in the future. Plastics are also often disposed of in landfills where they occupy a large volume and create future problems as the plastics degrade. It has been shown that plasticizers and plastic additives leak from degrading plastics in the landfills and contaminate aquatic environments [7].

Engineering Microbes to Degrade PUR

The complete system pathway takes PUR, an undesirable waste product and converts it into food for the bacterium.

To solve the problem of PUR recycling, we propose a bacteria that is able to both degrade PUR and subsist off of the products of degradation as its sole carbon source. To this end, we propose a two plasmid system. The first plasmid would have two genes. The first gene, polyeurethane esterase [8], will encode an enzyme that is able to break down the PUR polymer structure into two molecules, one of which, ethylene glycol, can diffuse across the membrane of the bacterium. The second gene, osmotic inducible protein Y (osmY) [9], would encode a protein that is fused to PUR esterase and exports the enzyme through the cell membrane and into the supernatent. The second plasmid would have an operon composed of, glycolaldehyde dehydrogenase (fucO) and glycoaldehyde reductase (aldA), that allow the bacterium to use ethylene glycol as its central metabolite [10]. This system would allow the bacteria to turn the plastics plaguing our landfills into bacterial biomass which would in turn degrade more PUR.

Methods [Top]

PUR Esterase Assay

To assemble the polyurethane esterase, we used 4 gene blocks and Gibsoned them together onto a pGA backbone.

Polyurethane esterase degrades polyurethane by cleaving the ester bonds to produce one of a variety of monomers with isocyanate groups on either end and (poly)ethylene glycol. For our circuit, we have chosen to use the esterase estCS2. Originally isolated and characterized by Kang et al (Microbial Cell Factories 2011), the sequence of estCS2 is available at GenBank (GU256649). Since their DNA constructs were unavailable and the authors did not respond to requests, we synthetically constructed the entire 1.7kb estCS2 gene from four 500-base gBlocks provided by IDT using Gibson cloning.

To assemble for activity of polyurethane esterase, cells transformed with the PUR esterase plasmid were grown to saturation, sonicated, then a piece of pre-weighed polyurethane foam was inserted into the culture tube.

To assay esterase activity we made one 50mL TB + kan overnight culture each for cells containing the esterase plasmid and cells without the plasmid. After leaving the cultures overnight, we spun them down to pellet the cells. After pouring out the supernatant, we resuspended the cells in enough water for them to be equalized at an arbitrary OD of 1.4 and aliquoted out each of the two cell types 1mL at a time into 3 different eppendorf tubes. We then sonicated the cells at an amplitude of 20 with 1 second pulses on and off for 30 seconds. We then applied the entire 1mL of lysate to preweighed samples of foam with 5 mL of LB. We made 3 replicates of each cell type and left the tubes in room temperature overnight.

Exporter Protein OsmY Assay

Since polyurethane is a large polymer, larger than the cell itself, it cannot be imported for degradation and therefore our esterase must be exported. It has been shown in Bolkinsky, Gregory et al [9] that a protein, osmotically inducible protein Y (osmY), is naturally exported by e. coli. Furthermore this paper shows that osmY can be used as an export tag in e. coli when it is translationally fused to a protein.

Our exporter protein (osmY) is linked to sfGFP through a serine glycine linker.

To test for exportation of osmY into the supernatant, we used our osmY fused to sfGFP and as a control sfGFP fused to mamI in the same conformation. These cells were grown up and then they were spun down and the fluorescence of the supernatant was measured.

To assay the exportation system we created a fusion protein. The fusion protein was sfGFP fused to osmY, which we can use to visually assay for exportation. If cells expressing sfGFP-osmY are spun down in a centrifuge and the supernatant is still fluorescent then the fusion protein must have been exported by the cells. If the supernatant of the osmY-sfGFP is not significantly more fluorescent than a control, the construct must still be contained in the cell and therefore not have been exported.

Turbidostat Assay

To optimize growth in ethylene glycol, we evolved a population of MG1655 e. coil to be more resistant to ethylene glycol in a turbidostat using homemade software. A turbidostat is a closed-loop continuous culture device that maintains a constant cell density and chemical environment. Without a constant environment, the selective pressures (in this case ethylene glycol) would change with time and our evolutionary trajectory would drift. A constant environment will maintain constant selective pressures and speed up the rate of evolution. Also, in a turbidostat, cells are always in exponential growth so you get more generations per day and fitness enhancing mutations rapidly propagate throughout the culture population.

The assay we performed was running the turbidostat using pure M9 with 30 mM ethylene glycol media as our sole media source. The culture chamber was blanked on pure M9 ethylene glycol media and MG1655 cells that were in log phase were washed with PBS and then added to the culture vessel to an OD of 0.2. This allowed for the cells still in log phase to divide without exceeding the OD threshold of 0.3. The turbidostat was then left running overnight and culture density and amount of fresh M9 ethylene glycol media added was checked in the morning.

Results Summary [Top]

Future Directions [Top]

Parts Submitted [Top]

Sources [Top]

1. Barnes, D. K. A., F. Galgani, R. C. Thompson, and M. Barlaz. "Accumulation and Fragmentation of Plastic Debris in Global Environments." Philosophical Transactions of the Royal Society B: Biological Sciences 364.1526 (2009): 1985-998. Print.
2. Gregory, M. R. "Environmental Implications of Plastic Debris in Marine Settings--entanglement, Ingestion, Smothering, Hangers-on, Hitch-hiking and Alien Invasions." Philosophical Transactions of the Royal Society B: Biological Sciences 364.1526 (2009): 2013-025. Print.
3. "Global Polyurethane Market to Reach 9.6 Mln Tons by 2015." Plastemart.com. N.p., 30 Aug. 2011. Web.
4. "Polyurethane Recycling." Polyurethanes. American Chemistry Council, n.d. Web. .
5. "Frequently Asked Questions on Polyurethanes." Polyurethanes.org. European Diisocyanate and Polyol Producers Association, n.d. Web. .
6. Takasuga, T., N. Umetsu, T. Makino, K. Tsubota, KS Sajwan, and KS Kumar. "Role of Temperature and Hydrochloric Acid on the Formation of Chlorinated Hydrocarbons and Polycyclic Aromatic Hydrocarbons during Combustion of Paraffin Powder, Polymers, and Newspaper." Archives of Environmental Contamination and Toxicology (2007): 8-21. Print.
7. Teuten, E. L., J. M. Saquing, D. R. U. Knappe, M. A. Barlaz, S. Jonsson, A. Bjorn, S. J. Rowland, R. C. Thompson, T. S. Galloway, R. Yamashita, D. Ochi, Y. Watanuki, C. Moore, P. H. Viet, T. S. Tana, M. Prudente, R. Boonyatumanond, M. P. Zakaria, K. Akkhavong, Y. Ogata, H. Hirai, S. Iwasa, K. Mizukawa, Y. Hagino, A. Imamura, M. Saha, and H. Takada. "Transport and Release of Chemicals from Plastics to the Environment and to Wildlife." Philosophical Transactions of the Royal Society B: Biological Sciences 364.1526 (2009): 2027-045. Print.
8. Kang, Chul-Hyung. "A Novel Family VII Esterase with Industrial Potential from Compost Metagenomic Library." Microbial Cell Factories 10.41 (2011): n. pag. Print.
9. Bokinsky, Gregory, Et. Al. "Synthesis of Three Advanced Biofuels from Ionic Liquid-penetreated Switchgrass Using Engineered Escherichia Coli." Proceedings of the National Academy of Sciences of the United States of America 108.50 (2011): 19949-9954. Print.
10. Boronat, Albert, Estrella Caballero, and Juan Aguilar. "Experimental Evolution of a Metabolic Pathway for Ethylene Glycol Utilization by Escherichia Coli." Journal of Bacteriology Jan. (1983): 134-39. Web.