Difference between revisions of "Part:BBa K3332050"

 
 
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iNAP is fused at C-terminal with BrkA anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of E.coli.
 
iNAP is fused at C-terminal with BrkA anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of E.coli.
  
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===Usage and Biology===
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===Biology===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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''.<ref>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.</ref><ref>http://2016.igem.org/Team:TJUSLS_China</ref>
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        <img src="https://2020.igem.org/wiki/images/9/94/T--XMU-China--XMU-China_2020-iNAP_Mechanism.png" width="40%" style="float:center">
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:'''Fig 1.''' Mechanism of iNap on the surface of ''E. coli''
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===Usage===
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;Here, we used <partinfo>BBa_K880005</partinfo> to construct the expression system and obtained the composite part <partinfo>BBa_K3332050</partinfo>, 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.
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        <img src="https://2020.igem.org/wiki/images/c/ca/T--XMU-China--XMU-China_2020-J23100_B0034_inap-brkA_B0015.png" width="35%" style="float:center">
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:'''Fig 2.''' Gene circuit of iNap-BrkA
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===References===
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<references/>
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Latest revision as of 23:31, 27 October 2020


J23100-RBS-iNAP-BrkA-terminator

iNAP is fused at C-terminal with BrkA anchoring protein. We use 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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 333
    Illegal XhoI site found at 359
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 99
    Illegal NgoMIV site found at 1520
    Illegal NgoMIV site found at 1949
    Illegal NgoMIV site found at 2585
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 2553