Part:BBa_K103006

OmpA outer membrane protein A fused to linker; displays proteins on cell surface

One of our basic bricks used to create fusions attached to outer membrane

Usage and Biology This part uses OmpA (outer membrane protein A [http://www.ncbi.nlm.nih.gov/pubmed/17559395?ordinalpos=1&itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_DefaultReportPanel.Pubmed_RVDocSum]) fragment (AAs 46-159) N-terminally fused with lipoprotein signal peptide fragment (AAs 1-9) Proteins fused to Ompa-link are presented on cell's surface Unstructuralized linker (Gly Ser Gly) allows proper folding of both fusion partners In our project Ompa-link is used as outer membrane anchor for our selection system This brick contains our nonstandard restriction sites (NdeI and SacI) that allow 'scarless' cloning and easy creation of translation fusions

This protein structure was predicted using threading and protein fold prediction and may differ from actual one.

Improvement by NEFU-China

Lpp-OmpA, a signal peptide can be used as an outer-membrane-targeting anchor in E. coli. Proteins fused to OmpA-link can be presented on cell surface.

Our team modified the signal peptide sequence to make it more conducive to direct our FABP to anchor to the outer membrane of E.coli. We reduced several amino acids at the N-end of the peptide chain so that the sequence was shorter than that in the Registry of iGEM, BBa_K103006, from University of Warsaw 2008 iGEM team, but the FABP with our signal peptide was expressed in high level. In the meantime, we used codon optimization to further improve the expressed level of FABP. The result showed that expressed levels of FABP with our shorter signal peptides were higher than BBa_K103006 in the Registry of iGEM.

results

E.coli was induced in 37℃ and membrane protein was separated by a speeding centrifuge. The result of western blot showed the difference at the expressed levels of Lpp-ompA-L-FABP in E.coli transformed by our recombinant plasmid and plasmid in registry of iGEM.

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]