Difference between revisions of "Part:BBa K2302003"

Latest revision as of 15:47, 12 October 2023

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

This Part was Instrumental in the UManitoba_2023 PLAnet Zero Project

Our project focuses on the enzymatic degradation of PLA plastics using MGS0156, Est119, RPA1511, and the PET-degrading TTL enzyme. In the context of contributing to the iGEM community, we present two new enzymes (Est119 and RPA1511) and provide additional documentation to the already existing part BBa_2302003 (Lpp-OmpA anchor). In particular, we investigate the esterase activity of Est119 and RPA1511 and the surface displacement of these two enzymes. The addition of Lpp-OmpA anchors will remove the need for protein purification. This anchor will display the enzymes on the surface of the bacteria allowing them to be in direct contact with PLA, without having to lyse the cells and execute their catalytic functions, facilitating the breakdown of our target plastic material (Figure 1).

Figure 1. Surface displacement of enzymes on bacteria membrane. This part was used to express two different esterases on the surface of E.coli BL21 (DE) for application in PLA plastic degradation.

Full experimental details can be found for its use in BBa_K4949004 and BBa_K4949006. The part works as intended and is suitable for expressing proteins on the cell surface. Further experimental details with exact procedure can be foud in the PLAnet Zero results.

Team's UManitoba conclusion:

The use of Lpp-OmpA as a potential secretion signal and anchor can enhance enzyme catalysis and improve plastic degradation, making it a valuable approach for addressing plastic pollution. Notably, the surface displacement of the enzyme is not uniquely beneficial to just our project. Future iGEM teams can explore this part for different applications such as displacement of enzymes on the outer membrane for whole catalysis assay or enzyme cascade for production of bio-based materials. The surface displacement of enzymes eliminate the need for substrate diffusion through the membrane and thus, this technique is especially useful when reactants are known to not cross the membrane readily.