Part:BBa_K4624632

AraC/ParaBAD-NSP4-laccase-syfp2-rrnB T1/T7TE

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NotI site found at 2807
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1995
  Illegal BglII site found at 2427
  Illegal BamHI site found at 1144
  Illegal XhoI site found at 2027
  Illegal XhoI site found at 2660
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1273
  Illegal NgoMIV site found at 2513
  Illegal NgoMIV site found at 2666
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1394
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  Illegal SapI.rc site found at 1459
  

Engineering Cycle 2: Laccase secretion

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 the secretion efficiency, we designed 4 different constructs where the coding sequence of the lcc2 laccase with one of the selected signal peptides (Native signal peptide (BBa_K4624400), NSP4 (BBa_K3606042), PelB (BBa_K208004) and MalE (BBa_K1012004)) was fused with the sequence of the fluorescent protein SYFP2 (BBa_K864100) at the 3’-terminal (Fig. 1).

Figure 1: Schematic representation of the standard level 1 (alpha) construct of the laccase fused with the syfp and carrying one of the selected signal peptides (Na.SP, NSP4, PelB and MalE).

Experimental Design and Results

The sequence of the signal peptide NSP4 (BBa_K3606042) and the laccase (BBa_K4624004) were 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 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 (1) pDGB3α1_pBAD-NSP4-laccase-syfp-rrnB T1/T7TE, with BsaHI, expected bands (bp): (1) 2327, 2049, 1628, 1361, 1291, 625, 423, 185, 163, 81 and 58. Lane 4: 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 ^[1]. 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-NSP4-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.

References

1. 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.