Part:BBa_K4624631
AraC/ParaBAD-Na.SP-laccase-syfp2-rrnB T1/T7TE
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1205
Illegal NotI site found at 2810 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1998
Illegal BglII site found at 2430
Illegal BamHI site found at 1144
Illegal XhoI site found at 2030
Illegal XhoI site found at 2663 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2516
Illegal NgoMIV site found at 2669
Illegal AgeI site found at 979
Illegal AgeI site found at 1397 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 961
Illegal SapI.rc site found at 1462
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).
Experimental Design and Results
The sequences of the native 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).
The isolated plasmid carrying the device was transformed into E. coli BL21 (DE3) chemically competent cells. After O/N incubation at 37oC, 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 30ο 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.
From those results we can conclude that the signal peptide from Trametes versicolor is not suitable for laccase secretion in E. coli, since supernatant fluorescence intensity exhibited low values compared with the rest signal peptides and the negative controls.
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.
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