Part:BBa_K4624400

Native signal peptide of lcc2 laccase from Trametes versicolor

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Biology

N-terminal signal peptide (21 residues) of the Trametes versicolor laccase (lcc2), responsible for efficient enzyme secretion from the white-rot fungus ^[1]. Signal peptide amino acid sequence: MSRFHSLLAFVVASLAAVAHA.

Experimental Design and Results

To integrate the laccase enzyme (BBa_K4624004) into our system, we first had to evaluate the functionality of the native signal peptide on our chassis, E. coli BL21 (DE3). To test its functionality, we designed a construct where the coding sequence of the laccase was fused with the sequence of the fluorescent protein SYFP2 (BBa_K864100) at the 3’-terminal (Fig. 1).

Figure 1: Schematic representation of the level 1 (alpha) construct of the laccase fused with syfp and carrying its native peptide, under the regulation of the AraC/Pbad system.

The sequences of the signal peptide and the laccase were seperated and were both domesticated to the GoldenBraid 2.0 standards using the GoldenBraid Domesticator tool, which removes any internal restriction sites that did not comply with the GoldenBraid grammar and adds the appropriate 4-nt 3’ and 5’ flanking overhangs in order for the inserts to be compatible with our level 0 pUPD2 cloning vector. The native peptide was cloned to a pUPD2 vector and then through digestion-ligation reaction was succesfully assembled into the complete trasncriptional unit (Fig. 2).

Figure 2: Diagnostic digestion of pDGB3α1_pBAD-Na.SP-laccase-syfp-rrnB T1/T7TE with BsaHI, expected bands (bp): 2327, 2049, 1628, 1361, 1291, 610, 423, 185, 163, 81 and 58. Lane 2: pDGB3α1 (no insert).

The isolated plasmid carrying the device was transformed into E. coli BL21 (DE3) chemically competent cells. After O/N incubation at 37^oC, a single colony carrying the construct was used to inoculate 5 ml of LB medium, with the appropriate antibiotic, and the culture was grown O/N at 30^ο and 160 rpm. E. coli BL21 (DE3) cells with the construct containing the syfp2 under the control of the J23118 Anderson promoter and non-transformed E. coli BL21 (DE3) cells were used as negative controls. The next day, O/N cultures were diluted in order to reach the same OD₆₀₀ and the addition of L-arabinose to a final concentration of 100 mM followed. Finally, after a 6h incubation the cultures were centrifuged in order to measure absorbance and fluorescence intensity of the supernatant and the pellet fraction, after cell pellet resuspension ^[2]. Four biological repeats were performed for each condition, the plate was placed into the plate reader and measurements were taken. The results of the normalized fluorescence intensity measurements for each fraction are depicted in Fig. 3.

Figure 3: Normalized fluorescence intensity of the supernatant and pellet fraction for pBAD-Na.SP-laccase-syfp-rrnB T1/T7TE construct after 6h incubation with 1.5% arabinose, (-) control 1: pJ23118-syfp-rrnB T1/T7TE, (-) control 2: non-transformed cells.

From those results we can conclude that the signal peptide from Trametes versicolor is not suitable for laccase secretion from E. coli, since supernatant fluorescence intensity exhibited low values compared with the negative controls.

References

1. Cassland P, Jönsson LJ. Characterization of a gene encoding Trametes versicolor laccase A and improved heterologous expression in Saccharomyces cerevisiae by decreased cultivation temperature. Appl Microbiol Biotechnol. 1999 Sep;52(3):393-400. doi: 10.1007/s002530051537. PMID: 10531652.

2. Linton E, Walsh MK, Sims RC, Miller CD. Translocation of green fluorescent protein by comparative analysis with multiple signal peptides. Biotechnol J. 2012 May;7(5):667-76. doi: 10.1002/biot.201100158. Epub 2011 Sep 20. PMID: 21834133.