Team:DeNovocastrians/Engineering
The benE gene is known to encode a benzene-H+ symporter protein (Choudary et al., 2017). This gene was a target for our project as it enables bacteria to import benzene/benzoate from their environments to use as a carbon source in their energy generation. We extracted benE genes from Rhodococcus strain 9, Acinetobacter baylyi (ADP1) and Escherichia fergusonii. The genes were successfully ligated into a pTTQ18 plasmid. Of these plasmids, those containing the benE gene from E. fergusonii and Rhodococcus strain 9 were successfully transformed into an E. coli benE knockout strain.
Growth curve in LB + benzoate: The strains transformed with benE were able to grow in a concentration of 15.625mM benzoate. The benE knockout strain and the benE knockout strains transformed with the plasmid only, were unable to grow at the 15.625mM benzoate concentration. This suggested that the benE transporter enables cells to import and utilise benzoate as a carbon source.
HPLC Curve: This result demonstrated that the cells transformed with the benE genes were able to successfully transcribe and translate the transporter, this enabled them to import benzoate from their extra-cellular environment. Compared to the benE knockout cells, the strains containing benE genes (from both Rhodococcus strain 9 and E. fergusonii) had utilised benzoate from their environment as a carbon source, shown here by the reduction in benzoate readings in their associated curves compared to the baseline 15mM benzoate curve. The cells transformed with benE from E. fergusonii imported the highest levels of benzoate compared to the benE knockout cells.
Taken together these results confirm that benE acts as a chemical trans-membrane transporter that enables cells to import benzene/benzoate from their environments. Further, the results suggested that benE may enable cells to survive in higher concentrations of benzene/benzoate, by enabling them to import and use the chemical as a carbon source for energy generation. Therefore, we were able to demonstrate engineering success for the parts BBa_K3694028 (benE E. fergusonii) and BBa_K3694029 (benE Rhod. 9).