Part:BBa_K1092003:Experience

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Characterization of BBa_K1092003

1. Biobrick DNA length verification

The inserted biobrick DNA length (936 bp) was confirmed by restriction digestion and agarose gel electrophoresis (Figure 1).

The DNA sequence was further verified by DNA sequencing.

2. Protein function verification by time series degradation assay

Catechol 1,2-dioxygenase is resposible for phenol degredation (Naiem et al., 2011). Before it is integrated into our PAHs degrdation system, hydroquinone, also known as benzene-1,4-diol or quinol, which is an aromatic organic compound belonging to the phenol family, was used as a substrate to test Catechol 1,2-dioxygenase expression and activity.

Time series analysis were performed. Empty vector-bearing (EV) and dioxygenase-overexpressing cells (T7-RBS-Dioxygenase) were induced by 0.3 mM IPTG at OD600 = 0.4, followed by 100 mg/L hydroquinone treatment. Cells were removed and extracellular hydroquinone was determined by OD298 at several intervals. Values represent the average of triplicate samples; error bars denote S.E.M. Paired t-test showed a significant difference in hydroquinone degradation trends between EV and dioxygenase-overexpressing cells (p ≤ 0.05) (Figure 2).

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