Difference between revisions of "Part:BBa K3332048"

 
 
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iNAP is fused at N-terminal with Lpp-OmpA anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of E.coli.
 
iNAP is fused at N-terminal with Lpp-OmpA 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;Lpp-OmpA is an anchor protein from ''E. Coli'', which can anchor its passenger protein to the cell membrane. It is constituted of 1-9 amino acids of Lpp and 46-159 amino acids of OmpA. It has been widely used in cell-surface display.
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&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;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 N terminal with Lpp-OmpA 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="50%" 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 use <partinfo>BBa_K880005</partinfo> to construct the expression system and obtain the composite part <partinfo>BBa_K3332048</partinfo>, which may achieve surface display of iNap on our engineered 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/f/ff/T--XMU-China--XMU-China_2020-J23100_B0034_lpp-ompA-cargo_%28gox%2C_grhpr%2C_inap%29_B0015.png" width="50%" style="float:center">
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        <figcaption>
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:Fig 2. Gene circuit of Lpp-OmpA-iNap
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===Reference===
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<references/>
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Latest revision as of 18:44, 26 October 2020


J23100-RBS-Lpp-OmpA-iNAP-terminator

iNAP is fused at N-terminal with Lpp-OmpA anchoring protein. We use K880005 to construct the expression system and anchor iNAP on the surface of E.coli.


Biology

        Lpp-OmpA is an anchor protein from E. Coli, which can anchor its passenger protein to the cell membrane. It is constituted of 1-9 amino acids of Lpp and 46-159 amino acids of OmpA. It 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 N terminal with Lpp-OmpA 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 use BBa_K880005 to construct the expression system and obtain the composite part BBa_K3332048, which may achieve surface display of iNap on our engineered 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 Lpp-OmpA-iNap


Reference

  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 798
    Illegal XhoI site found at 824
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 564
  • 1000
    COMPATIBLE WITH RFC[1000]