Coding
iNAP-BrkA

Part:BBa_K3332005

Designed by: Shuling Xiao   Group: iGEM20_XMU-China   (2020-10-17)


iNAP-BrkA

iNAP is fused at C-terminal with BrkA anchoring protein. We use BBa_K880005 to construct the expression system and anchor iNAP on the surface of E.coli.


Biology

        BrkA is an anchor protein from Bordetella pertussis having β-barrel structure. It can anchor its passenger protein to the cell membrane and has been widely used in cell-surface display. iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from Thermus aquaticus (T-Rex). Upon NADPH binding, iNap shows apparent fluorescence changes. iNap is fused at C terminal with BrkA so that iNap can be displayed on the surface of E. coli.[1][2]

Fig 1. Mechanism of iNap on the surface of E. coli


Usage

        Here, we used BBa_K880005 to construct the expression system and obtained the composite part BBa_K3332050, which may achieve surface display of iNap on our engineering bacteria. Due to the limited time, we did not get the gene in time. As a result, there is a lack of data about this part. The progress of this part remains in the stage of design.

Fig 2. Gene circuit of iNap-BrkA


References

  1. ↑ Zou Y, Wang A, Shi M, et al. Analysis of redox landscapes and dynamics in living cells and in vivo using genetically encoded fluorescent sensors[J]. Nat Protoc, 2018, 13(10): 2362-2386.
  2. ↑ http://2016.igem.org/Team:TJUSLS_China



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 272
    Illegal XhoI site found at 298
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 38
    Illegal NgoMIV site found at 1459
    Illegal NgoMIV site found at 1888
    Illegal NgoMIV site found at 2524
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2492


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Categories
Parameters
None