Composite

Part:BBa_K3332049

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


J23100-RBS-iNAP-AIDA-terminator

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

Biology

        AIDA is an anchor protein from E. Coli, which 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 AIDA 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_K3332049. We transformed the constructed plasmid into E. coli BL21 (DE3) to verify its successful expression.

Fig 2. Gene circuit of iNap-AIDA


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 figure1.

Fig 1.DNA gel electrophoresis of restriction digest products of iNap-AIDA-pSB1C3 (Xbal 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_K3332049 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 iNap-AIDA cells, we successfully got fluorescent gradient in the presence of different concentrations of NADPH. And when using negative control cells (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.


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 BamHI site found at 2487
    Illegal XhoI site found at 359
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 99
  • 1000
    COMPATIBLE WITH RFC[1000]


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