Part:BBa_K4907101

I0500-B0034-KpSP-gfp-B0015

Biology

Kp-SP

Kp-SP is a signal peptide found in Kocuria sp.3-3, which guides target proteins to the extracellular environment through the Sec pathway (1). In previous literature, it has shown remarkable secretion efficiency.

GFP

GFP is a kind of green fluorescence protein, whose excitation wavelength is 488 nm and emission wavelength is 533 nm. When expressed in engineered bacteria, GFP will emit green fluorescence under light of 488 nm wavelength (2).

Usage and Biology

To test whether Kp-SP signal peptide can guide target proteins secret or not, this basic part BBa_K4907002 was constructed which codes the fused protein KpSP-GFP. GFP can be guided into extracellular circumstances by KpSP, then the fluorescence can present the success of secretion. Moreover, to guarantee both KpSP and GFP can fold normally, we add a linker between them.

Characterization

Agarose gel electrophoresis (AGE)

BBa_I0500, BBa_B0034, BBa_K4907002 and BBa_B0015 were assembled to obtain the composite part BBa_K4907101, which are assembled on the expression vector pSB1C3 by standard assembly. The constructed plasmids are transformed into E. coli DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing.

Fig. 1 Colony PCR of BBa_K4907101_pSB1C3 in E. coli DH10βcenter

Fluorescence Intensity Ratio

The plasmid verified by sequencing was successfully transformed into E. coli DH10βE.coli</i> DH10β harboring BBa_K4907100_pSB1C3 (for expressing GFP intracellularly) which has no signal peptide’s coding sequence was also tested to compare the fluorescence intensity.After being cultivated and induced by 0.2% L-arabinose at 37 ℃ 12 hours, 1 mL bacterial liquid was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. As Fig. 2 shows, E. coli DH10βharboring BBa_K4907101_pSB1C3’s fluorescence intensity of supernatant versus bacterial liquid significantly exceeds the other. The result verifies that KpSP can transfer target proteins into the extracellular environment.

<center> Fig. 2 Comparation of the fluorescence intensity ratio (supernatant to bacterial culture) between I0500 set as control group.

Reference

1. Y. Cui et al., Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide. Protein Expression Purif. 129, 69-74 (2017).
2. T. D. Craggs, Green fluorescent protein: structure, folding and chromophore maturation. Chem. Soc. Rev. 38, 2865-2875 (2009).

Sequence and Features

Assembly Compatibility:

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

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

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961