Coding

Part:BBa_K1694002

Designed by: CHIH-HSUAN HSU   Group: iGEM15_NCTU_Formosa   (2015-09-15)
Revision as of 22:13, 18 September 2015 by LukeChang (Talk | contribs)

Lpp-OmpA-N

Introduction

Lpp-OmpA protein is a 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.

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. showed the correct size of the Lpp-OmpA-N, and proved that we successful ligated the Lpp-OmpA-N sequence onto an ideal backbone.

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.

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]


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