Introduction

Plastic is a ubiquitous material in modern consumer goods. One of the most common plastics used today is polyethylene terephthalate (PET). Chemically, it is a polymer consisting of ethylene glycol and terephthalic acid subunits. With the increasingly short life cycles of consumer products, PET waste is accumulating around the globe at an uncontrollable rate. Moreover, PET is a plastic that does not biodegrade well, and degradation often has to use industrial processes. Given the rapid accumulation of PET and the difficulties associated with biodegradation by ecosystems, it is paramount to find a more efficient method to degrade PET.
Our iGEM project was inspired by the results of a collaborative study between Keio University and Kyoto Institute of Technology, two prestigious Japanese universities. Their research investigated the ability of PETase, an enzyme, to degrade PET plastic using a bacterium known as Ideonella Sakainesis. The success of PETase in the degradation of PET plastic into Polyethylene Terephthalate and Ethylene Glycol sparked our interest in improving the Japanese ecological environment. Thus, the goal of our iGEM project is to create a biobrick incorporating an ideal Anderson promoter to optimize the use of PETase in an E. coli bacteria system. Past iGEM projects centered on plastic degradation, such as those of Turkey 2014, University of Washington 2012 and Darmstadt 2012, also provided us with inspiration on the potential applications for our project, as well as lab protocols.
Our team focused on making a biobrick featuring the PETase enzyme to contribute to the IGEM registry. In addition, we hoped to optimize the initial step of PET breakdown, or depolymerization, so as to expedite the overall process of degradation. To accomplish these goals, we selected several Anderson promoters of different strengths (weak, moderately-strong, and strong) to test for optimizing PETase production. The faster the rate of transcription for the PETase gene, the more PETase enzyme is produced. The greater the volume of enzyme, the faster the rate of depolymerization, since there is more enzyme available to ‘work’ on a plastic sample.

Project Design

Each of our constructs featured an Anderson promoter of a given strength, followed by an oSMY gene, and then PETase. This is demonstrated in the construct diagram below:


Figure 1: Diagram of ASIJ Tokyo PETase Construct

Why Anderson Promoters?

Our team chose to use Anderson promoters in our construct for their versatility and the variety of strengths they came in. It was crucial for our experiment to be able to test a range of Promoter strengths to determine which was optimal for promoting transcription of the PETase gene. Further, Anderson promoters work for most general protein expression in E. Coli systems, so we thought that it would be compatible with the PETase gene for our project.

Why osmY?

The role of osmY in our construct was to aid in secretion of PETase. osmY is a hyperosmotically induceable protein located in the periplasm, which acts as a transporter across the cell membrane. We needed the osmY within our construct to make the rate of secretion of PETase optimal, so as to increase the amount of the enzyme in solution with each PET sample. As stated prior, the method by which to expedite PET degradation is through maximizing the volume of PETase each sample is exposed to. This means increasing the rates of both the synthesis and secretion of PETase.

Abstract

In recent years, the production of Polyethylene Terephthalate (PET) has increased rapidly, as a result of low production costs and consumer demands. PET is one of the most common plastic polymers, comprised of repeating monomeric subunits of Terephthalic Acid and Ethylene Glycol. It is frequently used in the manufacture of plastic bottles and clothing fibres, especially in Japan. As avid users of PET-based products, our team decided to research how to optimise the degradation of PET, which takes an average of 450 years to degrade naturally. We were further inspired to pursue this goal with Keio University’s recent discovery of Ideonella sakainesis — a unique bacteria capable of PET degradation. Thus, we have focused our project on the synthesis of an optimal PETase biobrick, which would be included in the iGEM database. Ultimately, our goal is to find an ideal promoter to expedite the production and secretion of PETase.

References

Keio University and Kyoto Institute of Technology. (2016, March 30). Discovery of a Bacterium that Degrades and Assimilates Poly(ethylene terephthalate) could Serve as a Degradation and/or Fermentation Platform for Biological Recycling of PET Waste Products [Press release]. Keio University. Retrieved June 30, 2016, from

https://www.keio.ac.jp/en/press_releases/2016/cb96u90000005501-att/160330_2.pdf
P. (2015). FAQs - Frequently Asked Questions. Retrieved June 30, 2016, from http://www.petresin.org/faq.asp
Hampson, M. (2016, March 09). Science: Newly Identified Bacteria Break Down Tough Plastic. Retrieved June 30, 2016, from http://www.aaas.org/news/science-newly-identified-bacteria-break-down-tough-plastic
How Long does it take to Decompose - Facts Analysis. (2012, January 24). Retrieved June 30, 2016, from http://www.hoaxorfact.com/Science/how-long-does-it-take-to-decompose.html

T. (n.d.). Labjournal Metabolism. Retrieved June 30, 2016, from https://2012.igem.org/Team:TU_Darmstadt/Labjournal/Metabolism
M. (n.d.). Team:METU Turkey project. Retrieved June 30, 2016, from https://2014.igem.org/Team:METU_Turkey_project
What is PET? (2015). Retrieved June 30, 2016, from http://www.napcor.com/PET/whatispet.html

Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016). A bacterium that degrades and assimilates poly(ethylene terephthalate) (Doctoral dissertation, Kyoto Institute of Technology, Keio University) [Abstract]. U.S. National Institutes of Health's National Library of Medicine. Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/26965627.