Team:CCA San Diego/Future Research

Future Studies

The applications and detection of degradation of the Polycyclic Aromatic Hydrocarbons described -- Phenanthrene and Fluorene -- would be greatly improved with the implementation of a quantifiable reporter plasmid designed to test the products of the synthetic pathway. Such a reporter would detect the presence of salicylate and phthalate, the compounds that are created as a result of those studied in this paper respectively. These components will be further degraded by most host cells, but can be quantified before cellular processes carry out the rest of the degradation pathway. Two plasmids fragments were hypothesized to use RFP as a quantifiable reporter molecule in an immunoassay in the presence of Salicylate and Phthalate. The MarR translational repressor from the MarRAB operon in E. Coli was used to bind to a specific sequence on the fragment to allow transcription only in the presence of Salicylate in solution. The theorized phtR translational repressor from the Mspyr1_01380 operon in Mycobacterium gilvum was identified along with an associated unknown hypothetical protein, optimized, and left in its constitutive promoter. The operon Mspyr1_01400 that is regulated by the phtR translational repressor has been removed and replaced by an RFP gene.

Figure 1. Pthalate Reporter Construct

Figure 2. Salicylate Reporter Construct

Further quantification, proof of concept, and implementation into the degradation pathway must be performed before this system is used in testing the efficiency of the two PAH degradation pathways.
Additional research needs to be done to make the catabolic PAH pathways span over a wider range of bacterium to allow for greater changes in external temperature and chemical factors when used in pump and treat methods of remediation. In future research, the catabolic pathways will be cloned into a RK2 plasmid to be used in various strains of bacteria for the purpose of gene augmentation. Further characterization of degradation products will be performed using methods of thin layer chromatography and immune-detection. In addition, promoter regulated by intermediates of the pathways will be cloned with reporter genes to evaluate PAH degradation efficiency.

Canyon Crest Academy iGEM 2017 CC;