Part:BBa_K2684000

CotA Laccase of B.subtilis

CotA Laccase is an endospore type protein secreted from B.subtilis Part BBa_K2684000 was improved from previous part BBa_K1336002: https://parts.igem.org/Part:BBa_K1336002. A point mutation has been made to eliminate the EcoRI cutting site so that we can meet the standard of RFC10

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1348
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 286
  

Overview

CotA is a polyphenol oxidase which has capability to decolorize a wide range of dyes, as the catalyst. It has been reported that CotA laccase decomposes dye efficiently even in harsh condition with 3.5% salinity and pH 11.6, which makes it a great catalyzer for dye decomposition.

We isolated CotA gene using PCR from B. subtilis WT 168 genome, and expressed it in E. coli BL21. We quantified the activity of recombinant CotA using a commercial assay kit in which the CotA catalyzes the oxidation of ABTS. When oxidized by laccase, ABTS would turn to a bullish-green color that has an absorbance peak at 420nm.

We tested the enzyme activity of our part based on the weight of our sample. Formula: laccase activity (nmol/min/g) = ΔA /ε(ABTS mmolar extinction coefficient) / d * V (total volume of reaction) / V (volume of sample in the reaction, 0.025mL) / W (sample mass, g) * V (extracted liquid added, 1mL) / T (reaction time, 3 minutes) = 130 *ΔA / W.

For more experimental details, please visit http://2018.igem.org/Team:SHSBNU_China/Experiments

References

Guan, Z.-B., Luo, Q., Wang, H.-R., Chen, Y., & Liao, X.-R. (2018). Bacterial laccases: promising biological green tools for industrial applications. Cellular and Molecular Life Sciences.

“Help:Standards/Assembly/RFC10.” Help:Standards/Assembly/RFC10, parts.igem.org/Help:Standards/Assembly/RFC10.