Composite

Part:BBa_K3332045

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


J23100-RBS-iNAP-his-tag-terminator

        This is a fusion protein of REX and cpYFP that can be purified with the function of His-Tag on the N-terminal. We use it to construct a new part that can be purified with the function of his-tag on the N-terminal and detect NADPH.


Biology

        iNap is a chimera of circularly permuted YFP (cpYFP) and the NADP(H) binding domain of Rex from Thermus aquaticus (T-Rex). iNap has an excitation peak around 420 nm and one emission peak near 528 nm. Upon NADPH binding, iNap show apparent fluorescence changes. [1][2][3]

Fig 1. iNap mechanism


Usage

        Here, 6His-Tag is fused on the C-Terminal of iNap to help purify it, which can be used in vitro characterization of iNap sensors. Then, coding sequence of target gene iNap-Histag was inserted into an expression vectors with J23100 and RBS(BBa_K880005) to obtain BBa_K3332045. We transformed the constructed plasmid into E. coli BL21 (DE3) to verify its expression.

Fig 2. Gene circuit of iNap with Histag


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 figure 3.

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


2. Purification and Proof of expression

        We used J23100 promoter and RBS (BBa_K880005) to highly express iNap-Histag in E. coli in our composite part BBa_K3332045. Then, we used GE AKTA Prime Plus FPLC System to get purified iNap protein. We found no apparent protein peak in AKTA FPLC System and cannot correct purified protein.

        It is supposed that iNap cannot work normally after fused with 6His-Tag in C-Terminal.


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. ↑ Zhao Y, Hu Q, Cheng F, et al. SoNar, a Highly Responsive NAD+/NADH Sensor, Allows High-Throughput Metabolic Screening of Anti-tumor Agents[J]. Cell Metabolism, 2015, 21(5): 777-789.
  3. ↑ Tao R, Zhao Y, Chu H, et al. Genetically encoded fluorescent sensors reveal dynamic regulation of NADPH metabolism[J]. Nat Methods, 2017, 14(7): 720-728.

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
  • 1000
    COMPATIBLE WITH RFC[1000]


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