Part:BBa_K4195088

myc tag-his-pirB

Biology

PirA (a 111-residue protein) and PirB (a 438-residue protein) consist of the binary photorhabdus insect-related (Pir) toxins PirAB^vp. These toxins secreted from Vibrio parahaemolyticus cause an emerging disease called acute hepatopancreatic necrosis disease (AHPND) directly in shrimp aquaculture. PirA was reported to facilitate target-specific recognition by binding to certain ligands on the cell membrane/receptor (1).

Usage and design

In order to test the binding ability of purified receptors that we chose to purified toxins, a Myc-tag (EQKLISEEDL) was fused to the N-terminal of his-PirB. This second label (Myc-tag) was added to distinguish the proteins that were used to incubate with the nitrocellulose membrane from those spotted on the membrane when performing dot blot, since the proteins we purified were all fused with His-tag. We constructed this part on the expression vector pET-28a(+) by Gibson assembly, then transformed the plasmid into E. coli BL21(DE3). The positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

Characterization

1. Identification

After transferring the plasmid into E. coli BL21(DE3), colony PCR was used to verify that the plasmid was correct. The expected result was obtained (1569bp).

Fig. 1 DNA gel electrophoresis of the colony PCR products of BBa_K4195088_pET-28a(+).

Reference

1. S.-J. Lin, K.-C. Hsu, H.-C. Wang, Structural Insights into the Cytotoxic Mechanism of Vibrio parahaemolyticus PirA^vp and PirB^vp Toxins. Marine Drugs. 15, 373 (2017).

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 AgeI site found at 949
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 619