Part:BBa_M36039

Our project

The most common ways to assess liver health typically require that a patient must first recognize symptoms of their own malfunctioning liver and then see a doctor for complete diagnosis. In this way, current methods to diagnosis an unhealthy liver put the burden of disease detection on the patient. Here we report efforts to engineer E. coli bacteria to produce the UnaG protein - a recently discovered fluorescent protein that fluoresces when bound to the endogenous ligand bilirubin (a product of hemolysis in the liver). The UnaG protein’s fluorescent signal would then act as a proxy measure of bilirubin and serve as a reporter of liver function. We conducted several experiments to detect the efficacy of our biological device. The first experiment showed that the UnaG protein was secreted effectively into the extracellular media. The second experiment showed that while the UnaG protein alone fluoresced at a higher intensity than the control cells with no added protein, bilirubin did not significantly increase the degree of this fluorescence. Because bilirubin was found to not increase the natural fluorescence of UnaG proteins in E. coli cells, engineering E. coli bacteria to act as a reporter of liver health is not feasible.

Stage I results: Exportation check

This experiment tested whether the Yebf secretion leader properly exported the UnaG protein into the extracellular media. In order ensure robustness in our results, we assumed a p-value at the 0.01 significance threshold for all statistical analysis. We used the two-tailed t-test method of statistical analysis to determine whether various components of the experiment were significantly different from one another. The supernatant of the centrifuged cells with the UnaG plasmid fluoresced at a significantly higher intensity than the supernatant from the control cells with no added plasmid (p-value = 0.008) in the UnaG fluorescent spectrum (498nm-527nm). We also found that the fluorescent intensity in the UnaG spectrum of cells with the UnaG plasmid was significantly lower than the cells with a different plasmid (UnaG::RFP) (p-value = 1.7E-10). This offers further evidence that the UnaG protein is exported (and therefore no longer present in the cells to fluoresce). Our analysis strongly suggests that the UnaG protein was successfully exported into the extracellular media by the Yebf secretion leader.

Stage 2 results: Bilirubin assay

This experiment evaluated whether the addition of bilirubin led to higher fluorescent intensity in the UnaG fluorescent spectrum. Cells that had been incubated with bilirubin for twenty-four hours did not show significantly higher levels of fluorescent intensity in the UnaG fluorescent spectrum than cells with no bilirubin added (p-value = 0.0405).

Final results

We showed that the Yebf secretion leader can be used to export the UnaG protein from an E. coli cell. We also showed that bilirubin did not increase the fluorescent intensity of our UnaG protein (under the specific conditions of this study). Together, these results indicate that it is unlikely our biological device could function effectively as a reporter of liver health.

Location: Stanford University