Part:BBa_K1921015
INPNC
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 72
Illegal NgoMIV site found at 405 - 1000COMPATIBLE WITH RFC[1000]
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. There is no infomation on anchoring protein for C.rodentium, so we want to test this one (since these two strains both belong to Enterobacteriaceae). We used pET28b (with T7 promoter) in E.coli and pACYC184 (a low copy number plasmid which is repeatedly used in C.rodentium) in C.rodentium (with another promoter gathered from C.rodentium genome Part:BBa_K2328012).
We did western blot experiment for these two kinds of construction. The results are as shown in the figure. We can see that we can get our target protein (INPNC + smURFP, Part:BBa_K2328023) in lane 4, but nothing in the lane 2. It may indicated that this anchoring protein should be induced to be over-expressed to get a more satisfying effect. Or it just indicated that the promoter of C,rodentium or the low copy number plasmid were just not suitable for this anchoring protein.
Figure 1. The result if western blot. Lane 1 is E.coli BL21 with no plasmid. Lane 2 is E.coli BL21 with pET28b. Lane 3 is C.rodentium with no plasmid. Lane 4 is C.rodentium with pACYC184. Lanr 5 is positive control (a protein with His-tag).
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