Project Description

Existing Solutions

There have been many different proposals that have been made addressing this problem. Here are some of the most prominent:

Project Description

For our project, we have decided to improve our project from last year. We are working on breaking down PET plastics by using PETase, and then MHETase to further degrade the by-products produced from the initial reaction. According to the Maritime Aquarium, last year, Americans used around 500 billion plastic water bottles, with less than one-third to one-fourth of these water bottles being recycled. Many of these un-recycled water bottles end up in the ocean, causing aquatic ecosystems to be disrupted with the high levels of plastic, especially microplastics. Here in San Diego, we have seen firsthand the amount of plastic waste at our beaches, which has caused us to continue to research solutions to this problem. We have decided to continue our research on degrading the microplastic polyethylene terephthalate (PET). This process revolves around using PETase (isolated from Ideonella Sakaiensis) to split the PET microplastic chunks into monomers of mono (2-hydroxyethyl) terephthalate (MHET), and then MHETase to break the product into terephthalic acid and ethylene glycol. To create these enzymes in a natural environment, we plan to insert PETase and MHETase plasmids into the bacteria Alteromonas macleodii, which will then release the enzyme into the environment. We decided on this bacteria because it is commonly found in seawater habitats, making it an ideal candidate when seeking to break down ocean microplastics. This year, we are researching a new version of PETase, called Fast PETase, which was created by researchers using machine learning algorithms. It has a variety of amino acid mutations that were found to increase the enzymatic rate significantly in a lab environment. However, the improvement in enzymatic rate has not been tested in the pH and temperature fluctuation observed in a marine environment, and thus, we plan to use research methods such as FTIR Spectroscopy and GCMS to observe the presence and efficiency of degradation of FastPETase as well as wild-type PETase. We hope to analyze the statistical difference between each transformed organism, ideally in presence of several common marine conditions (with variations in different temperature, salinity, pH). As a whole, we want to analyze how to best optimize PET plastic degradation in Alteromonas macleodii to provide a solution to this serious problem.

Fast PETase vs PETase

An enzyme, PETase, has previously been isolated from a bacterium, Ideonella Sakaiensis. It was found to degrade Polyethylene Terephthalate (PET) into BHET and MHET monomers, which can then be degraded by MHETase, another enzyme similarly found in Ideonella Sakaiensis. Recently, researchers have utilized chemical engineering and machine learning to add mutations to FastPETase, making it more thermostable and active. If FastPETase is efficient enough, implementation through genetic engineering may be a possible solution to decrease and control PET microplastic pollution into the future. One of the goals for our project is to determine the efficiency of FastPETase and PETase in an in-vivo environment to determine the feasibility of this possibility.

Our process

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There are several steps that must be taken before the Fast PETase gene can be engineered into Alteromonas. With IDT’s codon optimization tool, we were able to create a g-block for Fast PETase, which we ordered (along with sequencing primers) from IDT. Then, through a series of steps in-lab, we have used JCVI’s promoter and terminator parts to create our L0 and L1 plasmids. In the next few steps, we will use our MHETase g-block that we have saved from last year to create our L2 plasmids. This work is all done in E. Coli cells. Our final step in transforming our Alteromonas cells with the L2 plasmid created in E. Coli. After this step is complete, we will move on to the results and analysis stage.

Our New and Improved Parts

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As our project this year is very similar to last year’s project, where we also created bacteria with PETase and MHETase, this year’s project is a little different. This year, we are using Fast PETase, which is a more thermostable and active enzyme than traditional PETase. Last year, due to problems with our parts, we were unable to complete the results and analysis phase of our project before the Giant Jamboree. This year, we hope to re-test our traditional PETase cells, which have been saved by JCVI, and compare the results with our new Fast PETase cells. However, we have run into issues with our parts, similar to last year. The MHETase gene, and the production of the enzyme, has stressed the cells out, seeming to cause them to drop the gene, resulting in negative gel electrophoresis. Nonetheless, we have begun to test our last year’s cells containing the PETase gene, and this year’s cells containing the Fast PETase gene to see if there is a different in results, and if the more thermostable and active Fast PETase enzyme has an impact on the rate of PET plastic degradation.

image sources: https://inhabitat.com/new-report-says-plastic-trash-to-exceed-fish-in-the-sea-by-2050/, https://happyeconews.com/indonesian-program-pays-fishers-to-collect-plastic-trash-at-sea/, https://theoceancleanup.com/ocean-plastic/