Composite

Part:BBa_K3332047

Designed by: Shengyang Zhang   Group: iGEM20_XMU-China   (2020-10-17)


J23100-RBS-INPNC-iNAP-terminator

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


Biology

        Ice nucleoprotein is an anchor protein from Pseudomonas syringae. It can anchor its passenger protein to the cell membrane. N and C terminal of Ice nucleoprotein, which is named after INPNC, can also anchor passenger protein fused with it to the cell membrane.

        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 INPNC so that iNap can be displayed on the surface of E.coli.[1][2][3]

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


Usage

        Here, we used BBa_K880005 to construct the expression system and demonstrated the effect of INPNC-iNap on the surface of E. coli. We obtained the composite part BBa_K3332047 and transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression and enzyme activity. The positive clones were cultivated.

Fig 2. Gene circuit of INPNC-iNap


Characterization

1.Identification

        After receiving the synthesized DNA, restriction digestion was done to certify that the plasmid was correct, and the experimental results were shown in figure3.

Fig 3. DNA gel electrophoresis of restriction digest products of INPNC-iNap-pSB1C3 (Xba I & Pst I sites)

2. Ability of sensing NADPH

        After culturing our engineering bacteria to OD600=1.8~2.0, we obtained E. Coli with BBa_K3332047 by centrifuging at 4000 rpm. Then, the cell precipitation was washed three times with PBS buffer (pH=8.0), and the final precipitation was resuspended in PBS buffer, which was equal to the volume of the original medium.

        We mixed NADPH solutions half-in-half with washed INPNC-iNap cell precipitation dissolved in PBS buffer (pH=8.0) and measured fluorescence changes in the presence of different concentrations of NADPH. TECAN® Infinite M200 Pro was used to detect fluorescence intensity. The samples were excited in 420 nm and the emission was measured at 528 nm.

        When using bacteria carrying INPNC-iNap, we successfully got fluorescent gradient in the presence of different concentrations of NADPH. And when using E. coli with BBa_K880005, we cannot get any gradient. The results prove that our anchor protein works and iNap can sensing NADPH on the surface of bacteria, which is shown in figure 4.

Fig 4. (a)Fluorescent-Time curve of INPNC-iNap and negative control in the presence of different NADPH concentrations;(b) Fluorescent-Time curve of iNap-AIDA and negative control in the presence of different NADPH concentrations.


Reference

  1. ↑ Van Bloois E, Winter R T, Kolmar H, et al. Decorating microbes: surface display of proteins on Escherichia coli[J]. Trends Biotechnol, 2011, 29(2): 79-86.
  2. ↑ 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
  3. ↑ 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 BglII site found at 62
    Illegal BamHI site found at 1314
    Illegal XhoI site found at 1340
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 472
    Illegal NgoMIV site found at 1080
  • 1000
    COMPATIBLE WITH RFC[1000]


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