Difference between revisions of "Team:RHIT/Description"

Description

Design

Experiments

Notebook

Results

Achievements

InterLab

Attributions

The Plastic Problem

We live in a plastic world. From our grocery bags and disposable bottles to straws and coffee cups, plastic has become an integrated part of our everyday lives. Since plastic production started in the 1950s, more than 6.9 billion tons of plastic have become waste. According to a study in 2017, only 9% of this waste has been recycled. [1] If these products never reached the recycling plant, where did 6.3 billion tons of plastic end up? Unfortunately, the plastics that are never recycled end up in landfills, on the sides of roads, and in our lakes, rivers, and oceans.

Because of its unique structure, plastic does not degrade. Instead, the plastic just breaks up into smaller and smaller pieces, eventually becoming microplastics. Smaller pieces of plastic and microplastics are more dangerous because they are easier for sealife to ingest. Because of the currents in the oceans, the plastic dumped in the ocean can travel around the world, leaving no parts unaffected. As much as 15% of sand on certain beaches in Hawaii is made of microplastics. [1]

Previous Work

In March of 2016, an article was released about the discovery of a bacterium that could degrade PET, polyethylene terephthalate. Ideonella sakaiensis 201-F6 was found to contain a PET hydrolase and a MHET hydrolase, named PETase and MHETase respectively. PETase introduced on a PET film degraded PET into MHET, a monomer of the PET chain, along with minimal amounts of terephthalic acid. In combination with MHETase, the PET film was degraded into the final products of terephthalic acid and ethylene glycol. After 6 weeks, the PET film was almost completely degraded. [2]

Two years later, in April of 2018, an article was released with extensive research on wild-type PETase and select PETase mutations. The W159H/S238F double mutant of PETase showed significant improvement in crystallinity reduction and product release over the wild-type in just 96 hours. The percent crystallinity change is a result of the pitting on the film caused by the PET degradation. [3]

Our Project and Inspiration

For our project, we have designed a plasmid that secretes MHETase and the double mutant PETase to increase the rate at which PET is degraded compared to the previous PETase sequence. We inserted the plasmid into an E. coli MG1655 strain. Because of the toxicity of ethylene glycol, a second plasmid was designed to allow the bacteria to break down the ethylene glycol and utilize its products as a carbon source. These enzymes include glycolaldehyde reductase, glycolaldehyde dehydrogenase, glycolate oxidase, and malate synthase. This series of enzymes will turn the ethylene glycol, released from the breakdown of PET, into malate which can be used by the cell as a carbon source via the citric acid cycle.

Much of the inspiration for our project came from the many changes that have taken place on Rose-Hulman’s campus. Within the last few years, recycling and decreasing plastic waste have become important aspects of campus. Many of these changes are due to the Six Sigma class. Six Sigma does projects where the students collect data before and after an improvement phase. Some of their past projects have included collecting data on the amount of recyclables in the trash and the use of plastic straws on campus. Recycling areas were set up throughout the academic buildings, and the campus community was educated about what and how to recycle. The plastic straw project initiated a decline in plastic straw use around campus by offering a biodegradable alternative to plastic straws at the eateries on campus and selling reusable straws to students. Figure 2 and Figure 3 show data collected by the Six Sigma class during their recycling project. Figure 2 is before the improvement phase and Figure 3 is after. This data shows a noticeable change in the campus community. [4]

References

• [1] Parker, L. (2018). We Depend On Plastic. Now, We’re Drowning in It.. [online] Nationalgeographic.com.
• [2] Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., Toyohara, K., Miyamoto, K., Kimura, Y. and Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate). Science, 351(6278), pp.1196-1199.
• [3] Austin, H., Allen, M., Donohoe, B., Rorrer, N., Kearns, F., Silveira, R., Pollard, B., Dominick, G., Duman, R., El Omari, K., Mykhaylyk, V., Wagner, A., Michener, W., Amore, A., Skaf, M., Crowley, M., Thorne, A., Johnson, C., Woodcock, H., McGeehan, J. and Beckham, G. (2018). Characterization and engineering of a plastic-degrading aromatic polyesterase. Proceedings of the National Academy of Sciences, 115(19), pp.E4350-E4357.
• [4] D. Evans and P. Olejnik, “Tightening Rose-Hulman's Wasteline: Using a Standard Operating Procedure to Reduce Trashed Recyclables on a College Campus,” rep.