From 2012.igem.org

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

Background

The increasing problem of plastic waste.

Playing an integral role in our modern world, plastics account for over a third of products made today. They have a relatively low cost of production, serve to promote the development of industry, lower the cost of consumer goods, and are easy to produce on a large scale. Plastics, regardless of disposability, compose 10% of the waste generated in the world and an even higher percentage is carelessly thrown into our environment [1]. Plastic pollution discharges debris into the ocean that are ingested by wildlife, often resulting in injury or death [2].

The PUR Problem

A commonly used plastic, Polyurethane (PUR), is used to create a wide range of products including dense solids, rigid insulating foam, and thermoset elastics. Because of its extreme 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]. The leading method of recycling polyurethane is to mechanically grind the waste and rebind it into carpet cushioning, accounting 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

Current methods of disposal, like burning is harmful for our environment.

Non-biodegradable plastics are often removed from the chemosphere through waste incineration as it is the most efficient method of degradation. However, a major issue with this is that the products of plastic burning, which include polychlorinated di-benzo-p-dioxins/furans, are known to be carcinogenic [6]. Upon incineration, these products are released into the atmosphere and have the potential to cause future problems to human health. An additional concern, plastics constitute large volumes of space as they are habitually disposed of in landfills. It has been shown that plasticizers and plastic additives leak from the degrading plastics 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 IDT gBlocks 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 ([http://partsregistry.org/Part:BBa_K892012 BBa_K892012]). 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, [http://partsregistry.org/Part:BBa_K892008 BBa_K892008]), 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([http://partsregistry.org/Part:BBa_K892008 BBa_K892011]), 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

Schematic diagram of our turbidostat. It uses the optical density measurements to modulate how much media is pumped into the culture vessel every minute to maintain a constant cell density.

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 optical density (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.

By putting fucO and aldA onto a single plasmids with a single promote, we were able to create an operon that when transcribed and translated allows microbes to utilize ethylene glycol as a sole carbon source [10].

The next assay we ran was to transform MG1655 with the fucO-aldA operon ([http://partsregistry.org/Part:BBa_K892013 BBa_K892013]). This operon consisted of fucO and aldA being on a single plasmid that contained a single promoter. With this present, the evolved MG1655 strain would be theoretically able to digest the ethylene glycol in the media since fucO and aldA create an enzymatic pathway that converts ethylene glycol first into glycolaldehyde and then into glycolic acid, a central metabolite. By running the turbidostat in the same way as with the evolutionary experiment, the ability to see how efficiently the transformed cells could metabolize ethylene glycol could be gained from how much new media was needed to be input every cycle to maintain a constant OD.

Results Summary [Top]

PUR Esterase Assay Results

We ran the PUR esterase assay and we found that the foam we used did not degrade. We quantified this by measuring the weight of the foam pieces to the nearest milligram before and after we ran the assay. We actually ended up leaving the lysate with the foam for three days to improve degradation but this had no effect on the end result. The reason we think the foam was not degraded was because we did not know the full composition of the foam, it could of had other polymers mixed in with the polyurethane that prevented our enzyme from properly degrading it. Further testing will need to be done to properly assay functionality of our PUR esterase.

Exporter Protein OsmY Assay Results

Each histogram depicts the average normalized fluorescence values for cells plus supernatant, cells only, and supernatant only. The image in the center is a visual representation of the presented data.

When running the osmY assay described in the methods section, we saw that sfGFP fused to osmY resulted in a high overall amount of fluorescence in the supernatant versus when sfGFP was fused to mamI. Even though there the cells containing the sfGFP-osmY plasmid exhibited a quantifiable amount of fluorescence, the overall fluorescence of the liquid culture is equally distributed between the inside of the cells and the outside (supernatant) where as for the sfGFP-mamI construct almost all of the fluorescence exhibited in the liquid culture was due to sfGFP within the cells. It can be gathered from this data that osmY when fused to a protein will export it out into the supernatant.

Turbidostat Assay Results

Optical density measurements of E. coloi transformed with fucO pGA3K3 + aldA pGA1C3 within the turbidostat growing on M9 30 mM ethylene glycol media.

M9 30 mM ethylene glycol media injected into the culture vessel every minute to maintain the arbitrary optical density of 0.3.

Following the turbidostat assay protocol, we used transformed MG1655 with fucO ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K892009 BBa_K892009]) on a Kanamycin resistant backbone and aldA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K892010 BBa_K892010]) on a Chloramphenicol resistant backbone and cultured it up in the turbidostat. The turbidostat was operated overnight and the above data illustrates that the transformed MG1655 grew using ethylene glycol as its sole carbon source. This is the case because the optical density (OD)/cell density rose from an initial arbitrary OD of 0.18 (time of inoculation) to an OD of 0.3, which was maintained throughout the time of the experiment (15 hours). The turbidostat maintained this OD through constantly imputing amounts of new media into the culture vessel based on a software implemented P.I.D. controller that modulated in real time how much media needed to be input every cycle to maintain an OD of 0.3. From this data, it can be seen that when fucO and aldA genes are over expressed in E. coli, E. coli gains the ability to utilize ethylene glycol as its sole media source.

Future Directions [Top]

Putting it All together

The parts as they are right now have not been put into one cell strain and tested as a system. The obvious next step would be to test the efficacy of the entire plastic degrading construct against polyurethane. Using the turbidostat, we have the luxury of having an optimized and continual growth environment for our system to mutate towards improved functionality. Better cell strains would be periodically saved and tested in harsher selective conditions until eventually the strain is able to survive and reproduce off of polyurethane as its sole carbon source. We recognize that this may take many iterations to complete but the end product could potentially be a cell that is able to consume plastic much faster and safer than traditional recycling methods.

Trash to Treasure

After stable growth off of plastic is achieved the next step would be to clone in a third plasmid that would produce some valuable commodity. Washington 2011 demonstrated that diesel fuel can be synthesized from central metabolites using their Petrobrick platform. The addition of the Petrobrick to the system would allow plastic waste to be processed by e. coli which would then turn it into biofuels.

Parts Submitted [Top]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K892009 Glycolaldehyde Reductase: fucO]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K892010 Glycolaldehyde Dehydrogenase: aldA]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K892011 Super Folder GFP Linked to Osmotically Inducible Protein Y: sfGFP-osmY]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K892012 Polyurethane Esterase: PUR Esterase]

[http://partsregistry.org/wiki/index.php?title=Part:BBa_K892013 fucO-aldA Operon]

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.