Part:BBa_K2328023
INPNC + smURFP I + 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 a fusion protein of the N-terminal of ice nucleation protein and smURFP. smURFP (small ultra-red FP) is an far-infrared fluorescent protein. Through this method, we can anchor smURFP on the out membrane of Escherichia coli, then we can use this typical Escherichia coli to connect with biliverdin. Prokaryote surface display system method is mature enough. INPNC is frequently used to surface display, primarily because: (i) INPNC does not appear to be hampered by the size of the passenger; and (ii) INPNC is compatible with the translocation and surface display of proteins that contain multiple cofactors as well as disulfide-bond-containing passengers.
Biology
smURFP (small ultra-red FP) is an far-infrared fluorescent protein. It is desirable for our in vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP. surface expression of recombinant proteins was first described more than 30 years ago. Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. INPNC is an OMP that is found in several plant pathogenic bacteria. . 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. However, the N-terminal domain appears to be the only prerequisite for successful targeting and surface-anchoring. In the 3’end of the smURFP we also added his-tag so that we can testify whether the smURFP is expressed or not by using confocal.
Reference
[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.
Results
We did two construction for this surface display. One was for E.coli BL21, the other was for Citrobacter rodentium (Part:BBa_K2328032). 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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