Part:BBa_K2271066

mRuby-ePTS1

Usage and Biology

This is a composite part containing the fluorescent protein mRuby targeted to the peroxisome via an enhanced PTS1 described by DeLoache et al. (2016)[1.]. PTS1 is a peroxisomal targeting signal recognized by a receptor called Pex5. Naturally, PTS1 consists of Ser-Lys-Leu (SKL) at the carboxy-terminus. Proteins harboring this C-terminal signal are imported into the peroxisome. The ePTS1 leads to an enhanced import of the taged protein. This part is designed as a peroxisomal marker for S. cerevisiea.[1.] For fluorometric and microscopic applications the optimal excitation of 559 nm and emission of 600 nm is discriped.

Experimental Design and Results

For the validation the S. cerevisiae Strain BY4742 was tranformed with this part. The cells were fixated and microscoped with an Elyra PS microscope. A typical peroxisomal localisation could be validated (Figure 1).

Figure 1 mRuby fused to the ePts1 described by DeLoache et al. (2016) [1.] The mRuby is localised in the peroxisomes of the cells

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 860
  Illegal BamHI site found at 1577
  Illegal XhoI site found at 1613
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

[1] Towards repurposing the yeast peroxisome for compartmentalizing heterologous metabolic pathways
William C. DeLoache, Zachary N. Russ & John E. Dueber
Nat. Commun. 7:11152 doi: 10.1038/ncomms11152 (2016).

[2] A Highly Characterized Yeast Toolkit for Modular, Multipart Assembly (2015)
Michael E. Lee, William C. DeLoache, Bernardo Cervantes, and John E. Dueber
ACS Synth. Biol., 2015, 4 (9), pp 975–986 DOI: 10.1021/sb500366v