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, while also using non-toxic methods to degrade PET!

“... 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. Ocean plastic is not only hazardous to marine life though - plastics have potential consequences for humans too! Rochman et al. (2015)¹ studied fish from public markets in Makassar, Indonesia and found that 28% of fish contained plastic particles. One type of plastic, in particular, is a source of significant concern: 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). However, because of their size, microplastics present several challenges to researchers:

Plastiback is our solution to gaps in ocean plastic research. Using the power of synthetic biology, we have harnessed the natural ability of Ideonella Sakaiensis to(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 Ideonella Sakaiensis, which is sensitive to PET in that it actually metabolizes it. With Ideonella Sakaiensis’ PET-degrading power, we could begin designing a system to detect PET by the components it breaks down into.

Ideonella Sakaiensis, 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, and thus, we believe they are the best choice of enzyme to use. For our project, we will focus solely on PETase because it produces terephthalic acid, the target of our sensing device.

However, because Ideonella Sakaiensis 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 Ideonella Sakaiensis, we expressed PETase 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, as well as show activity in a pNPB assay.

discuss the secretion systems we used in our PETase compound parts. Look at project design for expalanation of why we want secretion systems and copy/paraphrase here. 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.

Motivation for a Microbial Fuel Cell

Since we had the goal of tracking ocean plastic distribution, we eventually decided on using a microbial fuel cell to convert the terephthalic acid into electricity to produce a signal.

Currently, Microbial Fuel Cell (MFC) technology doesn’t yet produce enough electricity to power anything large, but fortunately our niche application allows a small amount of electricity to make a significant impact. Because oceanographers don’t currently have an efficient and mechanized way to track ocean plastic concentration, the ideal purpose for electricity produced by our MFC is sensing ocean plastic in a floating bioreactor.

How to Create a Microbial Fuel Cell with Terephthalic Acid as a Carbon Source:

In order to create an MFC that utilizes terephthalic acid, we needed to:

• Find a bacteria that metabolizes terephthalic acid as a carbon source
• Find an exogenous mediator that works with our bacteria to shuttle electrons to an anode

*Details of how we achieved this (and other considerations for MFC assembly) can be found on the Project Design Page.

Operating principle of a Microbial Fuel Cell

The answer to the baffling question of whether we can generate an electric signal, using terephthalic acid as the substrate, comes from a relatively novel device called a 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. Then electrons are shuttled onto an anode either by an exogenous mediator or membrane protein.

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).