Composite

Part:BBa_K2314115

Designed by: Yuqing Chen   Group: iGEM17_OUC-China   (2017-10-22)


Ice nuclear protein and monomer streptavidin fusion protein(INP-MSA)

This part encoding ice nuclear protein and monomer streptavidin, which make monomer streptavidin can be displayed on the surface of Escherichia coli.J23106 is a constitutive promoter from iGEM part registration. Ice nuclear protein(INP) is a kind of surface display system, which can display the protein on its N terminator.And monomer sterptavidin(mSA) can be linked with biotin in nature but has less squence, because it just has quarter part of sterptavdin. This feature makes our circuit more compact. Parts J23100 through J23119 are a family of constitutive promoter parts isolated from a small combinatorial library. And J23106 is a part of this family. INP is frequently used to develop whole-cell biocatalysts, primarily because: (i) INP does not appear to be hampered by the size of the passenger; and (ii) INP is compatible with the translocation and surface display of proteins that contain multiple cofactors as well as disulfide-bond-containing passengers. Monomeric streptavidin, mSA, corresponding to one subunit of wild type (wt) strepta- vidin tetramer. The monomer was designed by homology modeling, in which the streptavidin and rhizavidin sequences were combined to engineer a high affinity binding pocket containing residues from a single subunit only.
We use CLSM Observation to confirm the connection between E.coli and Saccharomyces cerevisiae. To describe the express of INP-mSA, we use biotin-sterptavidin to do the WesternBlot experiment.Also we use TEM to confirm the connection between E.coli and Saccharomyces cerevisiae.

<img src="T--OUC-China--pro2-2.png"/>
Fig.2 Western blot analysis using HRP-biotin to evaluate J23106-INP-mSA(JIM) expression. lane1, the outer membrane of JIM; lane2, the intimal protein of JIM; lane3,the cytoplasmic protein of JIM; lane4, the outer membrane of DH5α; lane5, the intimal protein of DH5α; lane6, the cytoplasmic protein of DH5α.

<img src="T--OUC-China--pro2-4.png"/>
Fig 2.4 Expression level of INP-mSA fusion protein under different conditions.( I ) the single cell fluorescence values from E.coli DH5α cell expressing JIM fusion and DH5α without a plasmid under 0 ℃ incu-bation for 2h. ( II ) the fluorescence values of E.coli DH5α cell expressing JIM fusion and DH5α under 30 ℃.

The image showed that E. coli containing the Jim plasmid successfully expressed INP-MSA fusion protein. Compared “JIM” with “negative control”, we found that the difference is nearly 3-fold. The fluorescence value showed that after 2 hours incubation under 30 ℃ conditions the Rhodamine-biotin can better binding to mSA. We speculate that there may be an ice dye incubation that causes ice nucleation proteins to form crystals that inhibit the combination of fluorescent dyes and the mSA of fusion proteins. [2]


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 426
    Illegal AgeI site found at 1120
    Illegal AgeI site found at 1180
  • 1000
    COMPATIBLE WITH RFC[1000]


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