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).

Figure 1. Laccase Activity Monitoring

3. Laccase 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).

Figure 2. E. coli BL21 strain overexpressing laccase that degrades BaP

Catechol 1,2-dioxygenase

1. Biobrick DNA length verification

2. Catechol 1,2-dioxygenase 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).

Figure 3. Time series analysis

Cell Viability Experiment with Voltage

This experiment is designed to investigate the BL21 viability when voltage is applied. This is necessary for the voltage sensor experiments in our project. The protocol of the following experiment can be found here.(hyperlink to https://2013.igem.org/Team:Hong_Kong_CUHK/protocol)

(a) (b)

(c) (d)

(e)

Figure 5. (a) Spread plate result with 0V applied. (b) Spread plate result with 9V applied. (c) Spread plate result with 18V applied. (d) Spread plate result with 27V applied. (e) Spread plate result with 45V applied.

From these results, it can be said that by applying a continuous DC Voltage for 24 hours to the samples do not kill the cell. Colonies are growing in all plates. Decreasing quantity of colonies is only due to the increasing dilution factor.