Part:BBa_K1694002
Lpp-OmpA-N
Introduction
Lpp-OmpA protein is the expression system of outer membrane protein, which consists of the 20 amino acids of signal sequence, the 9 N-terminal amino acids of the lipoprotein (Lpp) and the residual 46-159 amino acids of the OmpA. The lipoprotein (Lpp) is the most abundant protein on the outer membrane, which possesses the function of targeting to the outer membrane. The OmpA domain constitutes eight-stranded β-barrel to construct an anchor on the outer membrane, providing the stable expression of the protein displayed on the outer membrane. By taking advantage of efficient targeting OmpA to the outer membrane, it allows C-terminal fusion of the protein sequence to be displayed out of the outer membrane.
Modifying and Improving the Existed Biobrick
To be mentioned, we modified this commonly used transmembrane protein which has been submitted by Warsaw iGEM team in 2008, BBa_K103006 . The most contributed modification to this biobrick is that we replaced the long GS linker behide the transmembrane protein but we chose a six nucleotides restriction site, NcoI, to make it express remarkably on the outer membrane. This change has two benefits. First, the appearance of NcoI allowed the linked protein, for example scFv in our project, to be easily changed and to be brought outside conveniently (like a cassette). Second, NcoI also can act as an useful linker in fusion protein (between transmembrane protein and the linked protein), this has been proved in our project. Moreover, we also selected the amino acids of OmpA from the forty-sixth till the one hundred fifty-ninth and the function of Lpp-OmpA to bring scFv onto the outer membrane E. coli is completely proved by our experiments.
An alternative version with BamHI site
If you'd like to directly use this part as a vector/backbone for cloning a fusion protein, you should be noticed that an extra NcoI site is present on Cm resistance gene. BBa_K1991004 replaced NcoI site behind LO with BamHI site and provides an alternative choice for cloning a LO fusion protein
The features of Lpp-OmpA expression system
The versatile applications： The diversity of introduced protein connecting to OmpA can provide the multiple functions outside the surface of the E. coli. For instance, in our project, we can change every kind of scFv we desire.
The stabilization of expression： Due to the anchoring capabilities of lipoprotein and outer membrane protein, the Lpp-OmpA expression system can display passenger proteins more efficiently and also performs the strong adaptability to the various passenger protens.
Experiment
After receiving the DNA sequences from the gene synthesis company, we recombined each Lpp-OmpA-N gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each of the Lpp-OmpA-N. The DNA sequence length of the Lpp-OmpA-N is around 400~500 bp. In this PCR experiment, the Lpp-OmpA-N products size should be near at 650~750 bp. Fig.2 showed the correct size of the Lpp-OmpA-N, and proved that we successful ligated the Lpp-OmpA-N sequence onto an ideal backbone.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 388
- 1000COMPATIBLE WITH RFC[1000]
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