Part:BBa_K1694002

Lpp-OmpA-N

Introduction

Fig.1 Lpp-OmpA-NcoI

Lpp-OmpA protein is the expression system of outer membrane protein, which consists of the 20 amino acids of signal sequence, the nine 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 scFv. By taking advantage of efficient targeting OmpA to the outer membrane, it allows C-terminal fusion of the scFv sequence to be displayed out of the outer mambrane.

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 to a six nucleotides restriction site, NcoI. This changing has two benefits. First, the appearance of NcoI allowing the linked protein, for example scFv in our project, to be easily changed and to be brought outside conveniently (like a cassette). Furthermore, 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 twenty-five till one hundred thirty-eight to ensure that the displayed linked protein (scFv) can stay closer to the outer membrane and will not swing and move uncontrollably at the outer environment.

The features of Lpp-OmpA expression system

The special restriction site─NcoI： This design avoids the dilemma of the mixed restriction sites.

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, we can change every kinds of scFv we want.

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. The 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.

Fig.2 The DNA sequence length of the Lpp-OmpA-N is around 400~550 bp. In this PCR experiment, the Lpp-OmpA-N products size should be near at 650~800 bp.

Fig.3 Lpp-OmpA-NcoI

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
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 388
  
• 1000
  COMPATIBLE WITH RFC[1000]