Plasmid

Part:BBa_K4872011

Designed by: CHAN YING QI   Group: iGEM23_Canton-HS   (2023-08-04)


pGEX-GII.17-VP1


Composite Part: BBa_K4872011

Composite Part: BBa_K4872011 (pGEX-GII.17-VP1)

Construction Design

Plasmid design diagram of pGEX-GII.17-VP1
Figure 1. Plasmid design diagram of pGEX-GII.17-VP1

Plasmid pGEX-GII.17-VP1 is mainly composed of BBa_K4872002 and BBa_K4872003. Correspondingly, for the target gene GII17-VP1, we also used the Tac promoter and Stop codons, and also ligated pGEX-4T-1 as its unloaded load, using zymosynthesis ligation (EcoRI, XhoI) in the process. After that, we transformed the recombinant plasmid pGEX-GII.17-VP1 into E. coli BL21 using heat excitation (Figure 1).

Engineering Principle

GII.17-VP1 gene BBa_K4872002 is the main protein that makes up Norovirus particles.[1] By constructing a recombinant plasmid, GII.17-VP1 can express proteins through IPTG.

Experimental Approach

Construction of plasmid pGEX-GII.17-VP1
Figure 2. Construction of plasmid pGEX-GII.17-VP1

We first amplified the antigen gene GII.17-VP1 using the PCR amplifier (Figure 2A). Then, the GII.17-VP1 fragment and the pGEX-4T-1 plasmid vector were digested with restriction endonucleases EcoRI and XhoI, followed by the ligation using T4 DNA ligase to obtain the recombinant plasmid pGEX-GII.17-VP1 (Figure 2B). As shown in Figure 2C-D, the colony PCR and sequencing results confirmed the successful construction of the plasmid.

SDS-PAGE results of pGEX-GII.17-VP1 protein expression
Figure 3. SDS-PAGE results of pGEX-GII.17-VP1 protein expression (P represents “Precipitation”, S represents “Supernatant”, T represents “Flow through”, W represents “Wash solution”, and E represents “Elutent”)

We inoculated the positive transformant and induced protein expression by IPTG. After obtaining protein lysate, we purified the target protein using GST tags and verified the protein expression and purification results by SDS-PAGE. As shown in Figure 3, we successfully expressed and purified GII.17-VP1 protein (60 kDa).

The GII.17-VP1 protein was successfully expressed and purified in this round of experiments, which is expected to be prepared as a norovirus vaccine. In order to develop this norovirus vaccine, the next plan will be focused on the research of the GII.17-VP1 expression in E. coli Nissle 1917 and more evaluation tests shall be made before the implementation. Meantime, we can continue to construct the recombinant plasmid pGEX-RV-GII.17-VP1 to investigate the potential of the bivalent vaccine against norovirus and rotavirus.

References:

  1. Pogan, R., Weiss, V. U., Bond, K., Dülfer, J., Krisp, C., Lyktey, N., Müller-Guhl, J., Zoratto, S., Allmaier, G., Jarrold, M. F., Muñoz-Fontela, C., Schlüter, H., & Uetrecht, C. (2020). N-terminal VP1 Truncations Favor T = 1 Norovirus-Like Particles. Vaccines, 9(1), 8.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5497
    Illegal BglII site found at 6241
    Illegal BglII site found at 6415
    Illegal BamHI site found at 6308
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2055
    Illegal BsaI site found at 4987
    Illegal BsaI.rc site found at 5011
    Illegal SapI.rc site found at 342
    Illegal SapI.rc site found at 3137


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