For our project, we knew we wanted to create a novel product in E. coli that would make our biodegradation pathway economical and desirable - in other words high-value. Terpenoids are one of the largest, most variable classes of molecules. Their uses are numerous, including antibiotics, hormones, carotenoids and anti-malarial compounds. Terpenoid extraction via plant tissues presents challenges, including resulting in low yield and being generally difficult (Martin, 1). Our product is a plant steroid which promotes seed germination, cell division, and increases lateral bud growth (Sigma, 1). Using genetically engineered E. coli is a less-expensive, simpler path to its production. We believe that this product is a high-value alternative to biofuel, cosmetic, and pet food options often created from recycled frying oil. We hope that when created in conjunction with these current recycled products, the amount of frying oil waste disposed of in landfills and sewage can be greatly decreased.

In order to increase terpenoid production, E. coli can be fitted with the genes for the mevalonate pathway which directs terpenoid backbone production. Our first plasmid consists of four genes, the pMBI operon, inserted into a puc19 backbone (Figure 1). Our second plasmid consists of three genes, the pMevT operon, inserted into a puc19 backbone (Figure 2). Together, these two plasmids direct E. coli to metabolize the fatty acids of the frying oil via the mevalonate pathway to create dimethylallyl pyrophosphate (DMAPP). These two plasmids combined were worked on for a majority of the summer.

Because each of the operons, pMBI and pMevT, require a number of genes to be combined at once, our team chose to use Gibson Assembly as our main method of construction (see more about our Gibson Assembly Protocol here). This is a challenging method, but is often more successful for inserting multiple pieces into a plasmid backbone at once.

Figure 1: Diagram of pMBI in puc19 Plasmid


Figure 2: Diagram of pMevT in puc19 Plasmid