Part:BBa_K1092005:Experience

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Characterization of BBa_K1092005

1. Biobrick DNA length verification

The inserted biobrick DNA length (1542 bp) was confirmed by restriction digestion and agarose gel electrophoresis (Figure 1).

The DNA sequence was further verified by DNA sequencing.

2. Protein activity test by automatic kinetic spectrophotometric assay using o-phenylenediamine

Laccase is capable of oxidizing o-phenylenediamine (1,2-diaminobenzene) during the stopped-flow period. During oxidation, 2,3-diaminophenazin which can be determined spectrophotometrically, will be produced. Therefore, the kinetic spectrophotometric method was used here to detect laccase activity (Huang et al., 1996).

After 10 to 12 hours of incubation at 37 °C, laccase showed maximum activity when 0.3 mM IPTG was used for introducing protein expression (Figure 2).

3. Protein function verification

Empty vector-bearing (EV) and laccase-overexpressing cells (T7-RBS-Laccase) were induced by 0.1 mM IPTG at OD600 = 0.4, followed by 100 mg/L BaP treatment for 24 h. Cells were removed and extracellular BaP was determined by OD389. Values represent the average of triplicate samples where error bars denote S.E.M., and normalized by the OD389 of cells treated with DMSO only. Paired t-test showed a significant difference between EV and laccase-overexpressing cells (p ≤ 0.01) (Figure 3).

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