Difference between revisions of "Part:BBa K3033013"

Latest revision as of 09:52, 11 October 2022

Added by BFSU-ICUnited

We promoted BBa_K3033013 to BBa_K4400001, and did experiments to verify the effect of these two components. The experimental results show that the abilities of BBa_K4400001 are better than those of BBa_K3033013.

Assays of whole-cell tyrosinase activity

The tyrosinase activity was assayed according to a previous spec- trophotometric method that measures the conversion of L-DOPA to dopachrome, a red-colored oxidation product. The initial rate of the reaction is proportional to the concentration of the enzymes. To assay the enzyme activity, the cells were centrifuged at 8000 rpm for 10 min after cultivation for 18 h at 150 rpm and 37 ◦C in 100 mL LB medium. Then, sodium phosphate buffered saline (PBS) (pH 7.0) was used to wash and resuspend the cells. An aliquot containing tyrosinase was incubated with 1 mL L-DOPA solution (4 mg/mL) for 5 min at 35 ◦ C, and the optical density at 475 nm was measured with the help of UV-Vis spectrophotometer (DU-800 Nucleic Acids/Protein Analyzer, Beckman Coulter). One unit of enzyme is the amount that catalyzes the trans- formation of 1 μmol of substrate to product per minute. The surface-engineered bacterium E. coli-Tyr was suspended in PBS for 30 days at 25 ◦ C to test the enzyme activity at intervals of 5 days to examine the stability. BPA concentrations were determined using HPLC (Agilent 1200/6460, USA) with reversed-phase Waters ACQUITY BEH C-18 columns, as previously described.

Figure 1

The tyrosinase enzyme activity of BBa_K4400001 was 1.36 ± 0.06 U/g dry weight of the cells, which was not detected in the control group of BBa_K3033013. After the enzyme activity test, the conversion of BPA by the engineered strain was measured . The results showed that conversions at concentrations of 10 nM, 20 nM, 30 nM, 40 nM and 50 nM were catalyzed by BBa_K4400001. The efficiency of the catalysis was increased from 63.54% to 86.37% as BPA concentration decreased from 50 nM to 10 nM, while the control strain could not catalyze the reaction. These results indicate that tyrosinase was suc- cessfully displayed on the cell surface of E. coli with high BPA catalysis activity.

Tyrosinase

Derived from bacillus megaterium and synthesized through IDT. This codes for the enzyme which would be secreted into the extracellular matrix. This part is designed to modify the E.coli to have the ability to secrete tyrosinase from cytosol into extracellular environment. the secreted tyrosinase can convert its substrate tyrosine into L-DOPA which will facilitate the sticky feature of E.coli surface domain.

Sequence and Features