Part:BBa_K1492000

Clickable Outer Membrane Protein x

CPX(Circularly Permuted OmpX) is a membrane protein. This biobrick has a TAG codon which can be used to introduce any Non Natural Amino acid outside the cell. The biobrick also has a HA-tag for screening

Usage and Biology

CPX, or Circularly permuted OmpX, was developed as a bacterial display methodology for N- and C- terminal display. It has been shown to enable rapid screening of very large peptide libraries with high precision and efficiency. OmpX possesses four extracellular loops, with loops 2 and 3 forming a semi rigid β-sheets protruding from the cell surface. The native N- and C-termini were fused together by a GGSG linker, and the newly formed N and C termini reside on the cell surface.

Figure 1: Structure of OmpX alongside a topological depiction of OmpX and CPX

CPX was created by Rice et al(1) as a scaffold for peptide libraries. Rice et al added a part at the N terminus that is used for the random mutation needed for Peptide Libraries. Furthermore Rice et al shows that CPX can easily be overexpressed without effecting the cell growth or cell viability. This makes CPX a very useful protein for any type of displaying.

(1) Rice, J. J.. "Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands." Protein Science 15.4 (2006): 825-836. Print.

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

Team TU_Eindhoven 2014 tried to create a membrane anchor to which molecules could covalently bind. With this protein it is possible to bind anything to the membrane by using a bio-orthogonal “click” reaction. To test the functionality of the protein several assays were done. For all the assays we used the following vectors pET29a COMPx (Membrane Protein) and pEVOL-PylT-2xPylRS. (tRNA, tRNA synthetase). Both vectors were transformed into BL21(DE3) strain. Colonies of this transformation were grown on LB. After culturing, glycerol stocks were made. All the assays were done by culturing the bacteria from this glycerol stock. For all the protocols of TU Eindhoven 2014 see: http://2014.igem.org/Team:TU_Eindhoven -> Notebook -> protocols