Part:BBa_K3633013

A composite part to express laccase induced by IPTG

Description

This is a composite BioBrick composed of promoter T7, RBS B0032m, codon-optimized coding sequence of laccase (cotA) from Bacillus sp. HR03, and a his-tag.

The enzyme laccase has been expressed in E. coli BL21(DE3) and its properties have been discussed by Mohammadian, Fathi-Roudsari, Mollania, Badoei-Dalfard, and Khajeh (2010). It was shown to have the highest activity when expressed at 18℃ and put at 70℃ (Mohammadian et al., 2010).

As an oxidase, laccase has been used to catalyze dye synthesis from plant-derived pigment precursors (Jeon et al., 2010). In the study, 15 phenols and 120 combinations were studied. Three combinations, gallic acid and syringic acid, catechin and catechol, and ferulic acid and syringic acid, were further studied as they were oxidized into brown, black, and red materials, respectively (Jeon et al., 2010). Inspired by the principle of synthetic dyes, it was hypothesized that such combinations could be used as an alternative to direct dying with pigments.

The complete coding sequence for laccase (cotA) from Bacillus sp. HR03 has been included in the GenBank database (reference code: FJ663050.1). iGEM20_Shanghai_SFLS_SPBS optimized the codons based on that sequence, constructed the plasmid pET28b-T7-laccase, and tested the expression and activity of laccase.

Experiments & Results

The plasmid was transformed into E. coli BL21(DE3). 0.1 mM IPTG induction was added when the culture reached an OD600 of 1.0. Then, the shake flask was cultured at 25℃ for 20 h (cultured at 25℃ instead of 18℃ because no more shakers were available in the laboratory). Then, the bacterial solution was centrifuged, the cell pellet was resuspended in PBS (pH=7.2-7.4), and the resuspension was sonicated.

SDS-PAGE

The resuspension after sonication was loaded into an SDS-PAGE. The experiment showed an expected result, with a very evident strap at ~58.4 kDa.

Enzyme Activity Assay

EL - ABTS Chromogenic Reagent kit from Sangon Biotech was used to test laccase activity. ABTS can be oxidized by laccase and result in absorbance peaks at 405 nm and 650 nm. The experiment was carried out according to the procedure given by the kit. Enzyme activity of 10% bacterial solution and 2% bacterial solution was compared to the negative control, pure water. The pigment precursor was oxidized and absorbance increased.

Sequence & 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 NgoMIV site found at 931
  Illegal AgeI site found at 1238
  Illegal AgeI site found at 1394
  
• 1000
  COMPATIBLE WITH RFC[1000]