Part:BBa_K3016200

Vibrio natriegens' TorA signal peptide containing YGFP gene

This is a composite part containing Vibrio natriegens' TorA Tat signal peptide (BBa_K3016100) fused with YGFP (BBa_K3016600), a slow-bleaching GFP variant.

TorA is a twin-arginine (RR) motif containing signal peptide for periplasmic transport of proteins via the twin-arginine translocation (Tat) pathway. Derived from Vibrio natriegens’ TMAO reductase (torA) gene.

Thus, the YGFP produced by this part should localise into the periplasm.

Note: TorA signal peptide may be prone to inclusion body formation in Escherichia coli (Jong et al., 2017). Unconfirmed in Vibrio natriegens.

Biology

Twin-arginine translocation pathway

The twin-arginine translocation (Tat) pathway is capable of translocating fully folded proteins up to 150 kDa. It also contains a quality control feature of rejecting misfolded proteins. In some cases, disulfide bridge formation is not required for successful translocation. (Alanen et al., 2015)

Translocation using the tat-pathway requires the protein to contain a N-terminal signal peptide with a twin-arginine (RR) motif. The signal peptide is cleaved during the translocation process. A pair of V. natriegens’ native twin-arginine signal peptides identified by Aalto-Helsinki can be found here (TorAand Aminotransferase)

YGFP

YGFP is a slow bleaching GFP variant derived from a GFP mutant, with different spectral characteristics to regular GFP and YFP. YGFP has an excitation maximum at around 500 nm, and emission maximum at around 510 nm. It is not excited at or over 525 nm, which makes it usable in combination with longer wavelength excited fluorescent proteins. YGFP also retains the fluorescence longer compared to e.g. YFP. (Hansen & Atlung, 2011)

Use

This part can be used to easily test protein localisation into the periplasm via the Tat pathway in Vibrio natriegens.

Characterization

Aalto-Helsinki 2019 characterized this part by expressing it in Vibrio natriegens and Escherichia coli DH5a, testing its localisation into the periplasm.

Vibrio natriegens cells expressing TorA-YGFP

Escherichia coli DH5a cells expressing TorA-YGFP

In the images above we see Vibrio natriegens and Escherichia coli DH5a cells expressing TorA-YGFP. Note the polar localisation of fluorescence. This can be a sign of periplasmic localisation of YGFP under osmotic pressure (Sochacki et al., 2011) or inclusion body formation (Jong et al., 2017).

To be certain of successful periplasmic translocation, a cell fractionation experiment was performed on Vibrio natriegens. The periplasmic fraction of TorA-YGFP expressing cells was extracted (PureFrac-protocol) and placed under UV light, image below. The presence of YGFP in the periplasmic fractions can be seen clearly.

Periplasmic fractions of Vibrio natriegens cells expressing TorA-YGFP

These results indicate that the Vibrio natriegens' TorA-YGFP successfully translocates into the periplasm of Vibrio natriegens, and possibly even of Escherichia coli.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 781
  
• 1000
  COMPATIBLE WITH RFC[1000]

References:

Alanen, H. I., Walker, K. L., Suberbie, M. L. V., Matos, C. F., Bönisch, S., Freedman, R. B., ... & Robinson, C. (2015). Efficient export of human growth hormone, interferon α2b and antibody fragments to the periplasm by the Escherichia coli Tat pathway in the absence of prior disulfide bond formation. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1853(3), 756-763.

Jong, W. S., Vikström, D., Houben, D., de Gier, J. W., & Luirink, J. (2017). Application of an E. coli signal sequence as a versatile inclusion body tag. Microbial cell factories, 16(1), 50.

Sochacki, K. A., Shkel, I. A., Record, M. T., & Weisshaar, J. C. (2011). Protein diffusion in the periplasm of E. coli under osmotic stress. Biophysical journal, 100(1), 22-31.

Hansen, F. G., & Atlung, T. (2011). YGFP: a spectral variant of GFP. BioTechniques, 50(6), 411-412. https://doi.org/10.2144/000113691