Composite

Part:BBa_K4724021

Designed by: Qingqing Zheng   Group: iGEM23_NJTech-China-A   (2023-10-09)
Revision as of 12:55, 10 October 2023 by Moxi977 (Talk | contribs)


IsPETase-Linker-CBM3-6xHisTag

The hydrophobicity of the PET surface prevents IsPETase from binding to PET, which prevents IsPETase from functioning well. By adding a hydrophobic domain CBM3 to IsPETase, the hydrophobic domain of IsPETase can bind to the hydrophobic surface of PET more easily under the force of water molecule movement.

Construction

A recombinant plasmid containing this complex element was constructed using pET-22b(+) as a vector. The recombinant plasmid was obtained by fusing IsPETase with CBM3, a hydrophobic domain with a linker, using the Gibson assembly method. The recombinant plasmid was transformed into an E.coli BL21(DE3) sensory state. Colony PCR was performed using T7 and the post primer of the amplified domain as primers as shown in Fig.1. 1-fig-1.png 

    Fig.1 Colony PCR bands of pET22b-IsPETase-CBM3

Characterization

1. SDS-PAGE After the protein was induced by 1 mM IPTG, we used a nickel column to purify the protein because CBM3 incorporates 6xHis Tag. After the column was equilibrated, 30 mM and 300 mM imidazole buffer were added to rinse the column, and the target protein eluted with 300 mM imidazole buffer was collected. After purification, SDS-PAGE was performed to confirm the successful expression, as shown in Fig.2. <img src="1-fig-2-2-fig-2-6-fig-1.png"> 

    Fig.2 SDS-PAGE of IsPETase-CBM3 supernatant, starch, and purified protein after the introduction of the molecular chaperone pGro7.

M:180kDa Prestained Protein Marker 7-9:IsPETase-CBM3 supernatant, precipitate, and purified enzyme solution (protein size~46kDa) 2. HPLC After protein purification, an enzymatic reaction was performed to measure the enzyme activity. The substrate used was PET powder, which was decomposed into TPA and MHET by IsPETase, and the reaction solution was subjected to high performance liquid chromatography (HPLC), and the 6-min liquid phase result corresponded to TPA and the 8-min liquid phase result corresponded to MHET. The peak area of the product output from the liquid chromatograph was converted into the concentration of the product through a standardized line, as shown in Fig.3. <img scr="1-fig-3a-2-fig-3a-3-fig-3a-4-fig-3a-5-fig-3a-6-fig-3a.png"> <img scr="1-fig-3b-2-fig-3b-3-fig-3b-4-fig-3b-5-fig-3b-6-fig-3b.png"> <img scr="1-fig-3c-2-fig-3c-3-fig-3c-4-fig-3c-5-fig-3c-6-fig-3c.png"> 

    Fig.3 Concentrations of TPA and MHET products of 500 nM IsPETase-CBM3 reacted with PET powder for 48 h at different temperatures. (A) is the product concentration at 30°C, (B) is the product concentration at 37°C, and (C) is the product concentration at 45°C. The product concentration was determined by the reaction of 500 nM IsPETase- CBM3 with PET powder for 48 h at different temperatures.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 790
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 56
    Illegal PstI site found at 933
    Illegal NgoMIV site found at 891
    Illegal AgeI site found at 546
  • 1000
    COMPATIBLE WITH RFC[1000]


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