Part:BBa_K2328032
INPNC + smURFP II + Histag.b
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
Illegal AgeI site found at 969 - 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 smURFP on the surface of many intestinal bacteria using N-terminal and C-terminal of ice nucleation protein as anchoring motif. The protein, smURFP, is a kind of infrared fluorescence protein which can covalently attaches a biliverdin (BV) chromophore without a lyase. It has 642/670-nm excitation–emission peaks, a large extinction coefficient (180,000 M−1cm−1) and quantum yield (18%), and photostability comparable to that of eGFP. It means that it can emit fluorescence without molecular oxygen. So smURFP provide a chance to brighten the strict anaerobic bacteria. However, the BV has a poor ability to cross the cell membrane. So we choose surfacr display to help smURFP combine with BV more easily. And in Bacteria cell surface display means we fix the protein onto the out membrane of intestinal bacteria. According to the characteristic that smURFPs are capable to stay at a proper orientation so that they get more possibilities to combine with the biliverdin. Because the highly hydrophilic C-terminal of INP can combine with the out membrane, the display of our passenger protein, smURFP, are allowed to be more stable. Besides, our method solve the problem of visual observation of intestinal bacteria settled in the intestine.
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. infrared fluorescence protein give a better resolution in living imaging because it can reduce background noise/auto-fluorescence caused by cellular materials and solvent (water), improved tissue depth penetration (up to several cm) and high spatial and temporal resolution within limits.
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
[3] Haque A, Faizi M S, Rather J A, et al. Next generation NIR fluorophores for tumor imaging and fluorescence-guided surgery: A review. [J]. Bioorganic & Medicinal Chemistry, 2017, 25(7):2017.
Results
We did two construction for this surface display. One was for E.coli BL21(Part:BBa_K2328023), 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, ) 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 C.rodentium with no plasmid. Lane 2 is C.rodentium with pACYC184. Lane 3 is E.coli BL21 with no plasmid. Lane 4 is E.coli BL21 with pET28b. Lane 5 is positive control (a protein with His-tag).
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