Difference between revisions of "Team:ASIJ Tokyo"

Project Overview

What is PET?

PET is a combination of two monomers known as ethylene glycol and purified terephthalic acid that when combined form a polymer of polyethylene terephthalate. Considered as the most common type of polyester, PET is a plastic resin often used for packaging consumer products. The problem with PET is that, though it is a convenient material for humans to use, it is also very hard to biodegrade. As PET accumulates in the ecosystems around the world, it poses detrimental effects on habitats worldwide.

http://www.petresin.org/faq.asp

FAQs - Frequently Asked Questions. FAQs. PET Resin Association, n.d. Web. 07 June 2016.

http://www.napcor.com/PET/whatispet.html

"What Is PET?" NAPCOR. NAPCOR, n.d. Web. 07 June 2016.

http://www.aaas.org/news/science-newly-identified-bacteria-break-down-tough-plastic

“Newly Identified Bacteria Break Down Tough Plastic” AAAS.AAAS, n.d. Web. 25 June 2016

Our project focuses on using the enzyme PETase for breaking down PET hoping to increase the efficiency with which the breakdown occurs. PETase uses the process of hydrolysis to breakdown PET, enabling PET as an energy source for the PETase producing strain 201-F6, and its ability to exist in natural surroundings.

PET (Polyethylene Terephthalate) is a polymeric plastic resin comprised of monomers of ethylene glycol and terephthalic acid.

Inspirations

Using Experimental aids and inspiration from the joint collaboration research project from Keio University and the Kyoto Institute of Technology, and their research on PET degradation to Terephthalic acid and Ethylene glycol using the bacterium Ideonella Saikaiensis. Experimental description of the experiment:

http://www.keio.ac.jp/en/press_releases/2016/cb96u90000005501-att/160330_2.pdf

Our Plan and Goal

Combining research methods from previously done experiments with what we learn from our own experiments (though we haven’t done many yet!). Right now we want to isolate the first step of our procedure and increase its efficiency, before delving in any deeper. We do have a goal in mind: to add in a new biobrick using PETase to increase efficiency and usability in the plastic degradation process.

However, we’d like to also think about the practicality of our experiment in the real world. Questions we may ask ourselves during our experimental procedures will help us seek solutions useful if applied in the real world. Such questions include: “how long will the degradation take?” and “will the byproducts of the degradation be harmful in any way to the environment or to humans?”. In regards to the first question, to our current knowledge, 6 weeks is the record for the biodegradation of PET. In our eyes, this is much too inefficient. As a result, our experiment will focus on increasing efficiency of the degradation of plastic through the manipulation of PETase. These are just some examples of the types of questions that we must first consider. Once our confidence in research progresses, we can then focus on individual procedural steps, such as western blot tests, of our project.

The End Products

We’d like to synthesize a plasmid coded to efficiently secrete PETase. The results we hope to see include a larger amount of PET degraded in a shorter amount of time. Hopefully, we can break the record of 6 weeks. This is our main objective in terms of the lab. Theoretically, our project extends to the production of a feedback loop in which we take advantage of tryptophan to create a trp operon system through a series of combined intermediate enzymes. If ever this theory is substantiated, we hope to see bacteria use PET as a food source in which PETase will biodegrade PET when it needs to. In a real world situation, if PET is accumulated in one area where our genetically engineered bacteria have habituated, then we predict that we can leave the PET there to be degraded by the bacteria and basically forget about it. It will disappear eventually. This would requires minimal human and industrial intervention of the disposal of PET. We hope our project will be able to reduce the impact of PET as a pollutant on the environment.

Project Description

We are surrounded by plastic everyday, specifically PET. Considered as the most common type of polyester, PET is a plastic resin often used for packaging consumer products. We can also find it in our water bottles, plastic toys, and even in the fiber of our clothing. PET is a combination of two monomers known as ethylene glycol and purified terephthalic acid that, when combined, form a polymer of polyethylene terephthalate or PET. The problem with PET is that, though it is a convenient material for humans to use, it is also very hard to biodegrade. As PET accumulates in the ecosystems around the world, it poses detrimental effects on habitats worldwide. That is why it is crucial that we find an efficient way to degrade PET.

Upon conducting our research on plastic degradation, shortly after deciding to focus on this topic, our team came across a joint collaboration research project from Keio University and the Kyoto Institute of Technology, two popular universities in Japan. Their paper explained their recent research on PET degradation into terephthalic acid and ethylene glycol using a new bacterium originating from Japan called Ideonella Saikaienis. We were inspired by Ideonella Saikaienis because it was a wonderful PET degrader (containing PETase) from Japan, our current home. Given this, when we realized that the production of PETase was from a uniquely Japanese context, we saw it only apt to center our project around this as it showcases a special (and scientifically recent) feature of the Japanese environment. Consequently, our aim and experimental idea were born: to optimize the use of PETase and hopefully add a new biobrick to the iGEM catalog. Turkey’s 2014 project on PET degradation as well as University of Washington's 2012 project and Darmstadt Project 2012 also provided us with inspiration on the potential applications for our project in addition to lab protocol ideas.

We chose to optimize PETase as our goal to make a new type of biobrick available to future iGEM teams. We hope that this will make experiments in the following years easier to conduct. However in order to even get to that stage, we recognized that we needed a well thought out procedure. Our team is currently working on this step, but thus far we have determined we will be using a western blot test to help us deal with the signal peptides. Currently we also have three promoters (one on the slightly weaker side and two on the stronger side) taken from the list of Anderson promoters to test in order to determine how to make PETase most efficient. Before we do this though, we need to isolate the initial aspect of our procedure and evaluate its effectiveness. This information is essential before we delve deeper into our next steps and think about real world applications. Questions we may ask ourselves following this first step include: “How long will the degradation take?” and “Will the byproducts of the degradation be harmful in any way to the environment or to humans?” Our aim, as stated above, is to find a faster, more efficient process to degrade plastic. Answers to such questions will be essential in seeking solutions to global environmental problems. In regards to the first question, to our current knowledge, 6 weeks is the record for the biodegradation of PET. From our perspective, this is much too inefficient when you consider how much PET we use daily. As a result, our experiment will focus on increasing efficiency of the degradation of plastic through the manipulation of PETase. These are just some examples of the types of questions that we must first consider. Once our confidence in research progresses, we can then fully focus on individual procedural steps of our project such as the Western Blot.

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., Maehada, Y., . . . Oda, K. (2016, March 11). Supplementary Materials for a Bacterium that Degrades and Assimilates Poly(ethylene terephthalate). Science, 351, 1196th ser. Retrieved June 30, 2016, from

file:///Users/lisawatanuki/Downloads/PETase supplemental (2).pdf

Yoshida, S., Hiraga, K., Takehana, T., Taniguchi, I., Yamaji, H., Maeda, Y., . . . Oda, K. (2016, March 11). A Bacterium that Degrades and Assimilates Poly(ethylene terephthalate). Science, 351(6278), 1196-1199. doi:10.1126/science.aad6359