Part:BBa_K4624007

Linker peptide-syfp2

Sequence and Features

Assembly Compatibility:

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

  Illegal NotI site found at 22
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage

This composite part can be used to link a coding sequence to the sequence of the fluorescent protein SYFP2 (BBa_K864100).

Experiments

In order to test the secretion efficiency of several signal peptides in E. coli our team designed a construct where the coding sequence of the lcc2 laccase, from the white-rot fungus Trametes versicolor, was fused at 3'-terminal with this reporter module (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/P_BAD system.

To incorporate this part into the GoldenBraid 2.0 cloning method, we prepared it as the B5 part of the coding sequence. To achieve that, the sequence was domesticated, using the GoldenBraid Domesticator tool, which removes any internal restriction sites that did not comply with the GoldenBraid standards and adds the appropriate 4-nt 3’ and 5’ flanking overhangs in order for the inserts to be compatible with the pUPD2 part domestication vector.

Once this process was completed, the construct was combined with other components in order to create the construct we had designed (Fig. 2). The sequences of the signal peptide and the laccase were seperated and were also domesticated to the GoldenBraid 2.0 standards.

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. 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.