Team:BGU ISRAEL/Description
Description
Many date the invention of plastic by Alexander Parkes (Parkesine) as far as 1856, back then plastic was made almost entirely of cellulose, a natural substance found in plants. With the advancements made in the field of polymer chemistry during the 20th century, many new polymers were introduced into industry and are known to us today as Polystyrene, Polyethylene and Polyethylene terephthalate (PET). Plastic immediately gained popularity, being cheap, durable and easily molded into different solid shapes, and can be even used as an ink in 3D printers. However, one of its best features was found to be one of its greatest drawbacks, the durability of plastic makes it virtually non-degradable. An average bottle of mineral water takes roughly half a millennium to decompose, thus, leading to a global accumulation of plastic waste. Many ideas were considered in dealing with plastic waste such as burning plastic or burying it, but these solutions are considered damaging to the environment due to plastics toxicity. Since the introduction of plastics, some microbial communities or species have evolved to successfully degrade plastics, however, from an evolutionary point of view, probably due to the relatively short period of exposure to plastics, they are yet to be efficient in plastic biodegradation.
Our goal as the Ben-Gurion University IGEM team is to overcome this evolutionary hurdle by devising several approaches using synthetic biology tools for efficient plastic biodegradation. In addition, we plan to utilize the high energy stored in PET molecules, for electricity production. In order to achieve that, four courses of action were chosen:
- An Organism Evolution Approach: Since plastic is a new synthetic polymer introduced recently to the environment by mankind, not many organisms have adapted to utilizing it as a carbon source, and those that have, have yet to perfect that ability. Hence, one of our approaches is to use an organism which has developed a "solution" for the utilization of plastic, and try to improve that "solution" using methods of experimental evolution and serial passaging. We have chosen to improve the bacterium Rhodococcus ruber that was isolated by Prof. Alex Sivan from our university and has been found to have a polyethylene degradation ability.
- A Protein Engineering Approach: The 2nd approach we have adopted is the engineering of the LC-Cutinase protein.
LC-Cutinase is an enzyme discovered from an unknown organism in leaf-branch compost, and has been found to be one of the most efficient enzymes in breaking down PET polymers into degradable products.
Based on the LC-Cutinase structure that was solved in 2012, we have chosen to use a rational mutagenesis approach for its improvement.
In this approach, we made various mutations using an algorithm that compares the sequence of the original protein with that of other homologous proteins and then chooses a set of mutations.
The algorithm then calculates the differences in free energy (ΔΔG) for each of the mutants compared to the free energy of W.T. protein. Using this method we received 4 different variants that we plan to test for improvements in activity and stability. -
Genetic Engineering of Metabolic Pathways: We plan to insert two metabolic pathways to the soil bacterium Pseudomonas putida using genetic engineering. The two pathways will utilize the two PET degradation products - terephthalate and ethylene glycol. The terephthalate degradation pathway, derived from a strain of Commamonas, terminates in protocatechuate, which our chosen bacterium is able to utilize as a carbon source.
The ethylene glycol degradation pathway is derived from E. coli strain MG1655 and terminates in glycolate - also a material which our bacterium can utilize as a carbon source.
This way, we hope to achieve a full degradation of the two PET products and with it to drive the PET biocatalysis reaction by LC-Cutinase forward.
We have chosen to work with Pseudomonas putida, which is a gram-negative bacterium that has a diverse metabolism, including the ability to degrade organic solvents, especially protocatechuate, a toxic substance for most bacteria, which is the product of the first enzymatic cascade for the breaking down of terephthalate.
Our goal is to genetically engineer P. putida so it will contain three plasmids which will encode for three essential components: the ethylene glycol metabolic pathway; a membrane transporter that will carry the terephthalate molecule into the cell and the necessary genes for its degradation. - Microbial Fuel Cells - Since PET is a polymer that contains a lot of energy in its carbon-carbon bonds, excess energy released by our engineered microorganisms from the carbon-carbon bond degradation will be harnessed and utilized in a microbial fuel cell, leading to an efficient and energy producing, rather than consuming, degradation of PET.
