Part:BBa_K1166002

HlyA-tag+Secretion system

Introduction

The alpha-hemolysin system is one of the best-studied type 1 secretion systems (T1SS) of E. coli. The secretory machinery consists of three proteins: HlyB, an ATP binding cassette; HlyD, a membrane fusion protein; and TolC, an outer membrane protein (Su, et al., 2012).

Usage

This is a device that allows a protein to be secreted by means of the alpha-hemolysin secretion system in E. coli. It’s designed so that the only thing that you have to do is to assemble your protein part in the device via the biofusion standard (BBF RFC 23), (Cut the device with EcoRI and XbaI, cut your protein part with EcoRI and SpeI, mix & ligate). This procedure will leave your part in frame with a signal peptide.

Note: It’s important that your part doesn’t contain a stop codon nor a terminator since your protein will be fused at the C-terminus.

References

Su L, Chen S, Yi L, Woodard RW, Chen J, Wu J. (2012). Extracellular overexpression of recombinant Thermobifida fusca cutinase by alpha-hemolysin secretion system in E. coli BL21(DE3). Microb Cell Fact. 11:8

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1550
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1489
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1324
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1306