Difference between revisions of "Team:BGU ISRAEL"

BGU-IGEM Team 2016: PLASTICURE

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, three courses of action were chosen:

1. An Organism Evolution Approach – since plastic is a new synthetic polymer introduced to the environment by mankind, only recently, not many organisms have adapted into using it as a carbon source and those that were able to utilize it, have yet to perfect their plastic consumption as well as its utilization as a sole carbon source. Hence, one of our approaches is to use an organism which has adapted into "finding a solution" to use plastic, and try to improve that "solution" using methods such as experimental evolution and serial passaging. We have chosen to improve Rhodococcus ruber that was found to degrade polyethylene.
2. A Protein Engineering Approach – another approach we have adopted, is the engineering of a 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 substrates. Based on the LC-Cutinase structure that was solved in 2012, we have chosen to use a rational mutagenesis approach for our experiment. 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 various mutations and calculates each one of the mutations differences in free energy (DDG) compared to the free energy of WT protein. After the process was completed, we have ended up with 4 different variants other than the WT. We plan to test these variants in order to check if we have succeeded in improving the enzyme’s activity and stability.
3. Genetic Engineering of Metabolic Pathways–we will modify two metabolic pathways using engineered enzymatic cascades which lead to two products, terephthalate and ethylene glycol resulting from the biocatalysis of PET by LC-cutinase. While the terephthalate will be used to produce succinyl co-A and acetyl co-A, the ethylene glycol will be transformed into malate by using a metabolic pathway that already exists in E. coli. This way, our engineered bacteria will degrade PET and will transform the electrons released from PET degradation into energy by using PET as the carbon source. 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. Moreover, Pseudomonas putida contains most of the enzymes required for the first enzymatic cascade required for breaking down terephthalate. Our goal is to genetically engineer Pseudomonas putida so it will contain two plasmids which will encode for three essential components: secretion of LC-cutinase that is required for the initial degradation of PET to its monomers; a membrane transporter that will carry the monomers into the cell and the necessary enzymatic cascades that will further degrade the monomers into TCA cycle metabolites.
4. Microbial Fuel Cells - Since PET is a polymer that contains high energy bonds in its carbon-carbon bonds, excess energy released by our engineered microorganisms from carbon-carbon bond degradation will be harnessed and utilized in microbial fuel cells devices, this way plastic biodegradation will be converted into energy.