Difference between revisions of "Part:BBa K1921015"

Revision as of 19:04, 27 October 2017

INPNC

Sequence and Features

Usage

This part is the fusion of N-terminal and C-terminal domain of the ice nucleation protein. Here we established an approach to display PETase on the surface of Escherichia coli (E. coli) using N-terminal and C-terminal of ice nucleation protein as anchoring motif. Bacteria cell surface display means we fix the enzyme onto the out membrane of E.coli. According to the immobilization the enzyme are capable to stay at a proper orientation so that they get more possibilities to combine with the PET. Because the highly hydrophilic C-terminal of INP can combine with the out membrane, the display of our passenger protein, PETase, are allowed to be more stable.Besides, our method solve the problem of the degradation PETase. The enzyme will be stable in the cell surface display system.

Biology

Surface expression of recombinant proteins was first described more than 30 years ago.INP is an OMP that is found in several plant pathogenic bacteria. Our inaK is from Pseudomonas. INP has several unique structural and functional features that make it highly suitable for use in a bacterial surface display system. The specific amino acids of the N-terminal domain are relatively hydrophobic and link the protein to the OM via a glycosylphosphatidylinositol anchor. The C-terminal domain of the protein is highly hydrophilic and exposed to the medium. The central part of INP comprises a series of repeating domains that act as templates for ice crystal formation.

Reference

[1] Shosuke, Yoshida, 1, 2*, Kazumi, Hiraga, 1, Toshihiko, Takehana, 3, Ikuo, Taniguchi, 4, Hironao, Yamaji, 1, Yasuhito, Maeda, 5, Kiyotsuna, Toyohara, 5, Kenji, Miyamoto, 2†, Yoshiharu, Kimura, 4, Kohei, Oda1. A bacterium that degrades and assimilates poly(ethylene terephthalate)[J]. SCIENCE, 2016: 1196-1199

[2]Edwin, van, Bloois1, Remko, T, Winter1, Harald, Kolmar2, and, Marco, W, Fraaije. Decorating microbes: surface display of proteins on Escherichia coli[J]. CELL Press, 2011, 29(2): 79-86

Additional Supplements

The infomation below is updated by TJU_China of iGEM 2017.

Characterization Description

Last year, this part did not submit the corresponding experimental data, we chose this part this year as part of the construction in suface display, while we cannot sure its characteerization. So we did two construction for surface display. One was for E.coli BL21, the other was for Citrobacter rodentium. We used pET28b (with T7 promoter) in E.coli and pACYC184 in C.rodentium (with tetracycline resistance promoter and another promoter gathered from C.rodentium genomePart:BBa_K2328012).

while the performance test. Anchoring the new protein, and supplementing the experimental data of the module, confirm that this anchor timing is available.