Plastics in Aquatic Environments and their Growing Numbers
The amount of plastics in oceans, water supplies, and general bodies of water is escalating at an alarming rate. Plastics have made themselves a home in crucial bodies of water, especially the ocean. It is estimated that plastics comprise 50%-80% of litter in the ocean (Cressey, 2016). Knowing that 5.25 trillion macro and microplastics are currently flowing in our ocean’s waters affecting millions of marine life and humans a year proves that the global plastic problem is in critical condition (Surfer's Against Sewage). The growing amounts of plastics in vital bodies of water is affecting a considerable amount of wildlife and human life combined.
Plastics are one of the most prevalent types of waste that are currently present in our oceans and exist in all kinds of shapes and sizes. Most plastics in the ocean break up into small pieces called microplastics (NOAA, 2023). Microplastics come from all sorts of waste such as microbeads that come from manufactured polyethylene plastic in beauty products, resin pellets, or larger debris such as plastic bags or water bottles that later break down into smaller particles. There are currently about 245 million tonnes of microplastics in marine environments, which severely damage the environment of sea life and the lives of people who depend on resources from these environments (Andrady, 2011). It is imperative that people take action to solve this issue.
Existing Solutions
There have been many different proposals that have been made addressing this problem. Here are some of the most prominent:
Our Solution
We took initiative and designed plasmids that had the ability to break down these plastics to combat the daunting statistics. Our research revealed that PET plastic could be broken down by the enzyme PETase and the byproducts could be further broken down by the enzyme MHETase. We bioengineered plasmids through IDT with these genetic sequences separately. We created 2 separate Level 1 plasmids with MHETase and PETase in different plasmids. We then transformed the bacteria to create Level 2 plasmids with both MHETase and PETase sequences in one plasmid. This Level 2 plasmid was then conjugated into Alteromonas which was tested in water to break down PET plastics. We are currently in the stages of testing the amount of plastic degradation by the plasmids conjugated into Alteromonas.
The goal of this was to grow colonies of these plasmids that could work in tandem to break down plastics. By adding these plasmids to bodies of water that house tonnes of plastic, the plasmids will be able to break down large amounts of plastic in aquatic environments. We expect a decrease in the mountains of plastic in aquatic environments, uplifting the pressure on those who use the environment as a resource.
What We Did
Our team recognized the cruciality of this issue in how quickly the amount of plastic was multiplying. We found two enzymes- MHETase and PETase, that could degrade plastic. We then discerned the genetic sequences of both and then incorporated the genetic sequences into a plasmid using SnapGene. Originally, the plasmids were transformed into E.Coli. However, E.Coli aren’t well suited for aquatic environments, therefore, our team searched for another answer. We found our answer in a bacteria that is found in sea water- Alteromonas. We then set a goal to conjugate the plasmids into Alteromonas. To do this we created two separate Level 1 plasmids and then incorporated the MHETase and PETase sequences into them, separately. Following this, we transformed the bacteria to create Level 2 plasmids, this time combining the MHETase and PETase into one plasmid. This Level 2 plasmid was then finally conjugated Alteromonas which was tested in water to test its ability to break down PET plastics. Since natural habitats often have varying conditions, we have decided to take a focus on the efficiency of our PET-degrading pathway under various environments. We have focused on the effects of different temperatures. We have focused on the effects of different temperatures. There has been a search for the most thermostable PETase, because higher temperature increase points of access that the enzyme can use to break down PET plastics.
We utilized PETase’s ability to produce a reaction called depolymerization. This is what allows it to “eat up” plastic and degrade it when needed. PETase is usually discovered in fungi, however, it was first found in a landfill in Pakistan, present in a bacteria named Ideonella sakaiensis 201-F6, which is capable of hydrolyzing PET plastics (Chen et al., 2018). The bacteria, Ideonella sakaiensis, secretes PETase once administered to PET plastics. PET polymers then bind to PETase’s active site, breaking down ester bonds in the plastic. Once these bonds are broken, PET breaks down into 2 sub-products: MHET and BHET (Chen et al., 2018).. MHETase, a hydrolase that was found with PETase, then breaks down MHET even further by cleaving its bonds, making two more sub-products- TPA (trephtalic acid) and EG (ethylene glycol). BHET, TPA, and EG are the final products of this reaction; when the reaction is finished, the mass of PET has been significantly reduced. PETase also possesses the ability to put the leftover PET monomers back together into a different structure, this process is called repolymerization.
Our Inspiration
Plastic pollution is one of the most dire problems that we suffer from. Unfortunately, plastic pollution in marine environments is a global issue. The average plastic bottle can last for 450 years in a marine environment, which only adds to the exponential growth of the world plastic problem (Surfers Against Sewage). Plastic debris with counts of five trillion, coming to a total of around 260,000 tonnes, is floating over the world's ocean surface (Eriksen et al., 2014). Our mentor, Dr. Garza, is well versed in this topic and sparked our interest in the issue; this eventually drove us to tackle the problem. Additionally, San Diego’s is in close proximity to the ocean, and therefore the increased impact of microplastic pollution; this also pushed focus our project on a potential and relatively novel way to degrade the microplastic polyethylene terephthalate (PET).
Mission Statement
We want our iGEM project to shed light on the possible abilities of microplastic-degrading bacteria, or even generate a realistic biological solution to the threat of rising microplastic pollution.
References
Andrady, Anthony L. "Microplastics in the marine environment." ScienceDirect, Aug. 2011, www.sciencedirect.com/science/article/pii/S0025326X11003055. Accessed 7 Oct. 2023.
Chapman, Alyson. "Plastic waste addressed through synthetic biology with NSF grant." Texas A&M University Engineering, 24 Sept. 2021, engineering.tamu.edu/news/2021/09/plastic-waste-addressed-through-synthetic-biology-with-nsf-grant.html. Accessed 7 Oct. 2023.
Chen, Chun‐Chi, et al. “Structural Studies Reveal the Molecular Mechanism of PETase.” FEBS Journal, vol. 285, no. 20, Oct. 2018, pp. 3717–23. EBSCOhost, https://doi.org/10.1111/febs.14612.
Cressey, Daniel. "Bottles, bags, ropes and toothbrushes: the struggle to track ocean plastics." Nature, 17 Aug. 2016, www.nature.com/articles/536263a. Accessed 7 Oct. 2023.
Eriksen, M., Lebreton, L.C.M., Carson, H.S., 2014. Plastic pollution in the world’s oceans: more than 5 trillion plastic pieces weighing over 250,000 tons afloat at sea. PloS One 9 (12), e111913.
Maity, Writtik, et al. "Emerging Roles of PETase and MHETase in the Biodegradation of Plastic Wastes." Springer Link, 1 Apr. 2021,link.springer.com/article/10.1007/s12010-021-03562-4. Accessed 7 Oct. 2023.
Maqsood, Tariq, et al. "Pyrolysis of plastic species: A review of resources and products." ScienceDirect, Oct. 2021, www.sciencedirect.com/science/article/abs/pii/S0165237021002813. Accessed 7 Oct. 2023.
"Plastic pollution: facts & figures." Surfer's Against Sewage, www.sas.org.uk/plastic-pollution/plastic-pollution-facts-figures/. Accessed 9 Oct. 2023.
"What are Microplastics?" National Ocean Service, 26 Jan. 2023, oceanservice.noaa.gov/facts/microplastics.html#:~:text=Most%20plastics%20in%20the%20ocean,through%20waterways%20into%20the%20ocean. Accessed 7 Oct. 2023.
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/, https://www.alamy.com/thermometer-icon-glass-bulb-with-mercury-measuring-instrument-for-air-temperature-and-body-temperature-isolated-vector-symbol-on-a-white-background-image357361044.html