The previous stage of biosensor construction that the Denovocastrians 2020 team achieved was only theoretically able to detect the prescence of benzoate. We improved on this by assembling the portion which allows detection of catechol, thereby completing construction of the biosensor. We then proceeded to perform tests which proved that the biosensor works in the way that it was designed. Our team was able to conclusively determine that the CatM transcription factor is affected by the presence of catechol, as it was able to induce mCherry expression. We were also able to show that BenM is affected by the presence of benzoate as it induced green fluorescent protein expression. This demonstrates that our biosensor works and can be used to detect the presence of benzoate (a metabolite of benzene) and catechol (product of benzene degradation). This can be used to identify sites contaminated by BTEX and gauge the efficacy of potential bioremediation strategies.
To prepare for the eventual implementation of our organism into sites contaminated with BTEX, the global expression profile was examined in a Rhodococcus species degrading benzene. Through proteomics we identified proteins whose regulation was upregulated or triggered by the presence of benzene, including oxidoreductases and lipopolysaccharide synthases, as well as the degrading enzymes themselves. Homologous proteins present in strains of E. coli amenable to transformation were identified and will become targets to consider when optimising the organism for BTEX degraded sites.