Bio-Engineering E.Coli To Degrade Plastic and Save The Baltimore Inner Harbor
Working hard, we managed to successfully clone the Esterase gene into E.coli. Both colony PCR and sequencing were positive for the CE 5 esterase clone. However, Lipase was not successfully cloned and the sequences came up negative and truncated.
Due to the negative results with the lipase, we hypothesized that the lipase enzyme was toxic to the strain of E. coli we were using, causing it to reject the DNA.
We started inducing the positive esterase clone to produce protein, and running the protein produced on a protein gel after purifying on a nickel column. After running multiple protein gels with the esterase, no positive results were found.
Because of this, we hypothesized that the E. coli cells were not producing the esterase because the enzyme was also harmful to the cells.
To bypass the cell issue and test whether or not our constructs could actually produce the protein, we decided to test the constructs and biobricks on their own by translating them using a cell free kit, and then running the product of that on a protein gel. The protein gel results after the cell free kit were inconclusive, possibly because the cell just wasn’t producing enough protein to show up on the gel.
We decided to run the cell free kit products through a FDA enzyme test. Fluorescence was seen, indicating that enzyme was being produced! The results of this test are shown on a bar graph below
Additional tests will need to be done, but the FDA test indicated that our constructs can produce protein, even if the enzymes may not be able to be produced in E. coli
The Baltimore Bio-Crew thanks our sponsors for their generous support of our team that made our project and travel to the Jamboree possible. Thank you!