Difference between revisions of "Team:Harvard BioDesign/Description"

Plastiback is a plastic-sensing system that gives feedback on where PET, a type of plastic, is located in the ocean. PET is a durable plastic, with multiple uses, including making the bottles for many of the drinks we enjoy.

While PET pollutes both the land and sea, the majority of PET litter ends up in the ocean (Cressey, 2016). When plastic enters the ocean, it becomes difficult to clean up and even more difficult to study. For researchers who are investigating the scope and map of where PET is, Plastiback presents a novel and sustainable way to track ocean PET. For the earth, Plastiback uses non-toxic methods to degrade PET. And for you, Plastiback has got your back!

“... More than 5 trillion plastic pieces, weighing more than 250,000 tonnes float on the surface of the world’s oceans…” (Cressey, 2016).

Plastic in the ocean has long been a problem for the environment. It is estimated that 80% of plastic debris in the ocean is from a land-based source, such as landfills, wastewater, coastal recreational activities, and littering (Li et al., 2016). The other 20% is ocean-based debris, mostly from the commercial fishing industry discarding fishing gear into the ocean (Li et al., 2016). When plastic litters the ocean, it can be ingested by numerous marine organisms from whales to plankton, causing health complications to individuals, and possibly affecting the ecosystem overall. Plastic also poses a threat to marine organisms that get entangled and die, either to suffocation, injury caused by entanglement, or reduced fitness. However, plastic is not only hazardous to marine life, but a certain type of plastics has potential consequences for humans: microplastics.

“Microplastics” was a term first coined by Richard C. Thompson of Plymouth University in the 2004 paper “Lost at Sea: Where is All the Plastic?” to refer to pieces of plastic less than 5mm across. Since then, the study of microplastics and their effect on the environment have been pursued by marine scientists. Because they are small, they are able to be ingested by more organisms. Additionally, they are able to accumulate harmful chemicals such as dichlorodiphenyltrichloroethane (DDT), an insecticide banned by the U.S. in 1972. (Law and Thompson, 2014). [Include statistic on seafood contamination] However, because of their size, microplastics present several challenges to researchers.

(Then introduce our solution to sensing plastic: Plastiback! A bacteria-based feedback plastic sensing device. What a mouthful, lol.)

Cressey, D. "The Plastic Ocean." Nature 536 (2016): 263-65

Law, K. L. and R. C. Thompson. 2014. "Microplastics In The Seas". Science. 345.6193 (2014): 144-145.

Li W.C., Tse H.F., Fok L. 2016. Plastic waste in the marine environment: a review of sources, occurrence and effects. Sci. Total Environ., 566-567 (2016), pp. 333–349

Thompson, R. C. 2004. "Lost At Sea: Where Is All The Plastic?". Science. 304.5672 (2004): 838-838.

In order to sense PET, we needed a microbe that was sensitive to PET in some way. We found our ideal candidate in Idonella Sakenesis, which is sensitive to PET in that it actually metabolizes it. With Idonella Sakenesis’ PET-degrading power, we could begin designing a system to detect PET - by the components it breaks down into.

Idonella Sakenesis, discovered by Oda group at Kyoto Institute of Technology in 2016, produces two enzymes responsible for the breakdown of PET: PETase and MHETase.

PETase breaks down PET into the compound MHET, or mono(2-hydroxylethyl)terephthalic acid and terephthalic acid. MHETase breaks down MHET into terephthalic acid and ethylene glycol. While there are other enzymes that have the capacity to break down PET, PETase and MHETase are solely dedicated to the role of PET degradation.

However, because Idonella Sakenesis is a fairly new bacteria to the lab, it would require extra tuning to culture and engineer. To harness the power of PETase, we used E. Coli as our chassis, an iGEM favorite and reliable organism.

Though we are using the PETase enzyme from Idonella Sakenesis, we expressed PETase solely in E. Coli. An E. Coli based genetic system is useful because E. Coli is a well-researched chassis organism that has multiple available strains for protein expression optimization.

To find a strain that best expressed PETase, we investigated several genetic systems and strains, including LEMO21 and SHuffle T7. Among the strains we tested, we found that T7 lysY Iq was the optimal strain to express PETase in. To read more about our genetic design, click here. (link to genetic design page)

Using our T7 strain, we were able to confirm presence of PETase using SDS Page and Western blot, along with a pnPb assay.

discuss the secretion systems we used in our PETase compound parts. What are the strengths and weaknesses of these systems and what did we expect to work best. What did we find to work best? Etc.

talk about the organism a little bit (where it is found, what type of bacteria it is) and why we chose it. Show our growth curves.

When our E. coli metabolizes PET, it produces two primary products: terephthalic acid and ethylene glycol. Ethylene glycol, an antifreeze, can help insulate our bioreactor and maintain the temperature conditions necessary for our bacteria's survival. However, when it comes to terephthalic acid, its usefulness is not readily apparent. This is because the acid is used primarily as an ingredient in the production of PET. What purpose could it serve in the middle of the ocean?

One viable option is to further metabolize terephthalic acid. This brings us to Delftia suruhatensis sp. nov., a species of bacteria capable of using terephthalic acid as a carbon source. The bacteria was first discovered by a Japanese research group (Shigematsu et. al 2003). Armed with the knowledge that terephthalic acid is a contaminant in wastewater, the group decided to visit a wastewater plant with the hopes of finding an organism capable of degrading the acid. The scientists collected activated sludge on site and later attempted to isolate a strain of bacteria from the sample capable of utilizing terephthalate as its sole carbon source. They identified this bacterium as a new species of Delftia.

Although there are other species of bacteria capable of degrading terephthalic acid that our team acquired over the course of a research, we determined through experimental analysis that Delftia was the most efficient.

Motivation:

Despite the PETase and MHETase enzymes synergistically and effectively degrading PET, we wanted to be able to convert the degradation product (terephthalic acid) into something useful. After entertaining a few ideas, including decarboxylating terephthalic acid into the valuable product benzene, we eventually decided on using a microbial fuel cell to convert the terephthalic acid into electricity (to produce a signal) with the help of an exogenous mediator.

Currently, Microbial Fuel Cell technology doesn’t yet produce enough electricity to power anything large, so we needed to find a niche application where the small amount of electricity would make a significant impact. Because oceanographers don’t currently have an efficient and mechanized way to track ocean plastic concentration and sensors don’t use much electricity, we decided that sensing ocean plastic in a floating bioreactor would to most optimized use for our electricity produced.

How we chose bacteria?

In order to use terephthalic acid as the substrate in a microbial fuel cell, the bacteria needs to be able to metabolize terephthalic acid and the membrane of the bacteria has to be permeable to exogenous mediators such as methylene blue. After looking through the literature, we decided to test Bacillus sp. 35889, Delftia tsuruhatensis, Serratia marcescens subsp. Sakuensis, and Bosea minatitlanensis because these were all harvested in regions with high terephthalic acid concentration such as bottle recycling plants and wastewater treatment plants.

talk about how we incorporated MFCs into our project. Why MFCs are cool, etc.

Introduction - Operating principle of a Microbial Fuel Cell

The answer to the baffling question of whether we can generate electricity through organic or inorganic matter, comes from a novel device named the Microbial Fuel Cell. Such a device contains specific bacterial strains, located in its anode compartment, produces electrons from the bacteria’s oxidation of the carbon source or electron donor, present in the fuel cell.

A microbial fuel cell contains bacteria in the anode compartment and produces electrons from the bacteria’s oxidation of the carbon source, which acts as the electron donor.

Most of these electrons get transferred to the anode either by mere “direct contact,” or by a transport mechanism inherently produced by the bacteria or externally through exogenous mediators, discussed below (Logan).

What is the next step in the electron movement within the fuel cell? Once the electrons arrive at the anode, the potential difference between the anode (negative) and the cathode (positive), forces them to flow at the other side of the fuel cell, on the cathode compartment. This happens, of course, through an “external resistance or load,” supplied from the outside of the fuel cell.

However, electrons are not the only charged atomic particles moving around. Protons go through the “cation exchange membrane,” separating the anode and the cathode chambers, onto the cathode compartment, where they meet the electrons and oxygen (since the cathode side is usually left open to air), producing water as the final compound. This directed flow of electrons is what generates a current, or generally speaking electricity, in such a device (Logan).