In order to demonstrate that our engineered bacterial cells containing our lipase constructs worked under realistic conditions, we developed a quantitative assay using the substrate p-nitrophenol octanoate, which contains an ester bond chemically identical to those constituting lipid molecules found within fatbergs. When this ester bond is hydrolysed via our lipases, p-nitrophenol octanoate is broken down into p-nitrophenol and octanoic acid. P-nitrophenol is yellow in solution, compared to p-nitrophenol octanoate which is colourless. Consequently, we can monitour the production and accumulation of p-nitrophenol using spectrophotometry as a way to measure lipase activity.
Following the production of a standard absorbtion spectrum for each lipase, individual Lineweaver-Burk plots for each lipase were constructed, from which the kinetic parameters of Km, kcat and Vmax were derived. This assay was also performed for the Thermostable Lipase A (TliA), a previous iGEM part used by the 2014 Sheffield iGEM team, Stuttgart and KAIST iGEM. This was done to fulfill the bronze medal criteria requiring a previous iGEM part to be characterised, which we have since successfully fulfilled, adding our results to the iGEM registry.
Click here to see our results.