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Team:TU Eindhoven/Achievements/Submitted Parts/COMPx
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| - | <img id='Fig1' src="https://static.igem.org/mediawiki/2014/5/5e/TU_Eindhoven_OmpX_CPX.png" width=" | + | <img id='Fig1' src="https://static.igem.org/mediawiki/2014/5/5e/TU_Eindhoven_OmpX_CPX.png" width="575" style="display: inline-block; border: 4px solid #00BAC6; padding: 4px; background: #222; margin-bottom: 10px;"> |
<figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Structure of OmpX alongside a topological depiction of OmpX and CPX.</figcaption> | <figcaption style="font-size:18px;color:#CCCCCC;">Figure 1. Structure of OmpX alongside a topological depiction of OmpX and CPX.</figcaption> | ||
</figure> | </figure> | ||
Revision as of 13:56, 14 October 2014
COMPx
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.
CPX was created by Rice et al1 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.
Gene Design
To integrate a Non-natural amino acid in the sequence of CPX some modifications were made. Within the part in which originally random mutations were induced a codon was mutated into the amber stop codon TAG. With a specific tRNA it is possible to implement a Non-Natural amino acid at the place of the TAG codon. The TAG codon was introduced into the Peptide Library by Site-Directed Mutagenesis. A HA-tag was also added to the C-terminus of the protein using overhang primers to use in characterization. After the modifications the protein was renamed to Clickable Outer Membrane Protein X (COMPx).
