Difference between revisions of "Part:BBa K2302003"

Revision as of 12:21, 27 October 2017

Lpp-ompA,a peptide that can anchor its fused gene product to the outer membrane.

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.

Usage and Biology

This sequence encodes a functional peptide that allows surface anchoring of its fused gene product. The peptide consists of: (i) the signal sequence and first nine N-terminal amino acids of a mature major lipoprotein of E. coli, (ii) amino acids 46-159 of the outer membrane protein OmpA.[1]

Design

We combined the characteristics of FABP (bind to long-chain fatty acids) and Lpp-OmpA (anchor to the outer membrane of E. coli) in our project. The plasmid pHis contained our restriction sites (HindIII and XbaI). A His tag was added to the C-end of FABP for later western blot to verify the expression of fusion protein Lpp-OmpA-FABP. When the whole fusion protein was expressed, the signal peptidewould lead the FABP to the outer membrane of E. coli and anchor in it. So the Follower E in our project could combine fatty acid by the FABP expressed on the outer membrane of E. coli.

Experiment and Result

In order to make it more conducive to direct the FABP to anchor in the outer membrane of E. coli, our team modified the signal peptide sequence and reduced several amino acids at the N-end of the peptide so that the sequence was shorter than that in the Registry of iGEM, BBa_K103006(https://parts.igem.org/Part:BBa_K103006) , from University of Warsaw 2008 iGEM team, but the FABP with our signal peptides was expressed in high level. In the meantime, we used codon optimization to further improve the expressed level of FABP. E. coli was induced in 37℃ and membrane protein was separated by ultracentrifugation. The result of western blot showed the difference at the expressed levels of Lpp-ompA-L-FABP in E. coli transformed by the recombinant plasmid, indicated that the expressed levels of FABP with our shorter signal peptides were higher than that with BBa_K103006 in the Registry of iGEM.

Link

【1】https://parts.igem.org/wiki/index.php?title=Part:BBa_K103006

【2】http://2017.igem.org/Team:NEFU_China

Sequence and Features