Part:BBa_K1921011

BrkA

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 313
  Illegal NgoMIV site found at 742
  Illegal NgoMIV site found at 1378
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1346
  

Usage

The anchor of a new autotransportermetthat diated bacterial surface display system BrkAu to Display based on the structure of BrkA. The unique characteristic of BrkA is that the cleaved passenger domain remains adhered on the bacterial surface, which is conducive to keep the displayed exogenous proteins associated onto the cell, thus, the functionalized bacteria can be recycled/re-used conveniently.Now we hope to display our PETase on the surface of E. coli by using it.

Biology

PETase was found from a kind of microorganism(Ideonella sakaiensis 201-F6) living on PET as the main carbon source. It can degrade macromolecular polymers into monomers. PETase is the only enzyme found in bacteria which can degrade PET.
Autotransporter BrkA (Bordetella serum-resistance killing protein A )is from Bordetella pertussis,PETase is the passenger between the signal peptide and the anchor.

Reference

[1] Fang S, Pang X, Tian X, et al. BrkAutoDisplay: functional display of multiple exogenous proteins on the surface of Escherichia coli, by using BrkA autotransporter[J]. Microbial Cell Factories, 2014, 14(1):1-12.

Pre-expression

Figure 1.This is the pre-expression using E.coli BL21 in different inducing condition.

Surface display HPLC results

Figure 2.Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)Brk when induced at 16℃ and 25 ℃ with 0.02mM IPTG.And the last two were induced with 0.09mM IPTG.