Project

Part:BBa_K3683000:Design

Designed by: Leyi Zheng, Xu Lin Zi Cai, JieYing Gan, Guanghui Sun, Ouqiao Li, Charlotte Zhang, Taylor Khouw Shimizu, Emily Roshan   Group: iGEM20_PYMS_GZ_China   (2020-10-13)


A universal platform with pseodoviruse of SARS-CoV-2


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 3094
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1435
    Illegal NgoMIV site found at 1951
    Illegal NgoMIV site found at 2014
    Illegal NgoMIV site found at 2509
    Illegal NgoMIV site found at 2653
    Illegal NgoMIV site found at 2686
    Illegal NgoMIV site found at 2963
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 647
    Illegal BsaI.rc site found at 898
    Illegal BsaI.rc site found at 2558
    Illegal SapI.rc site found at 915
    Illegal SapI.rc site found at 1596


Design Notes

Production and titration of SARS-CoV-2 S pseudoviruses: to generate and culture either wild type SARS-CoV-2 S or S-D614G variant pseudotyped virus using a pair of cooresponding mutant primaers; Psuedovirus infection and neutralization; Authentic virus neutralization;

Source

The psPAX2 was purchased from addgene, and the pLOVE-Luciferase-EGFP was purchased from GenScript (Nanjing, China), the full length Spike gene (S) from the SARS-CoV-2 (previously 2019-nCoV) strain Wuhan-Hu-1 (GenBank: MN908947) was codon-optimized (sequence shown in Supplementary Table 1), synthesized, and cloned into pCAGGS vector (pCAGGS S(1-1254aa)) using seamless cloning by GenScript. The primers S-D614G-F, 5′-CTGTACCAGGgCGTGAATTGCACCGAGGTGC-3′ and S-D614G-R 5′- TGCAATTCACGcCCTGGTACAGCACGGCCACC-3′ were used to generate S-D614G mutant by PCR-based direct mutagenesis using High-fidelity DNA polymerase Mix (P525, Vazyme) with the following condition: 95℃ 5min, 95℃ 30s, 56℃ 30s, 72℃ 4min for 28 cycles, 72℃ 10min. We next purified the exact size of S-D614G PCR product by gel extraction, then we used Exnase II (C214, Vazyme) to make the linearized product circled. Then circled S-D614G plasmids were transformed into DH5α competent cells, single clones were select to grow recombinant plasmids in culture. The information for these maps are shown in Supplementary map.

References

Wu et al, A new coronavirus associated with human respiratory disease in China, Nature. 2020 Mar;579(7798):265-269. doi: 10.1038/s41586-020-2008-3.

Hu et al, Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2, Genes Dis. 2020 Jul 17. doi: 10.1016/j.gendis.2020.07.006

Hu et al, D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity, BioRxiv, 2020, doi: https://doi.org/10.1101/2020.06.20.161323.

Yang et al, A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity, Nature. 2020 Jul 29. doi: 10.1038/s41586-020-2599-8.

Zhang et al, The D614G mutation in the SARS-CoV-2 spike protein reduces S1 shedding and increases infectivity, bioRxiv. 2020 Jun 12;2020.06.12.148726. doi: 10.1101/2020.06.12.148726.

Ou et al, Emergence of SARS-CoV-2 spike RBD mutants that enhance viral infectivity through increased human ACE2 receptor binding affinity, BioRxiv, 2020, doi: https://doi.org/10.1101/2020.03.15.991844

Grubaugh et al, Making Sense of Mutation: What D614G Means for the COVID-19 Pandemic Remains Unclear, Cell. 2020 Aug 20;182(4):794-795.doi: 10.1016/j.cell.2020.06.040.

Korber et al, Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus, Cell. 2020 Aug 20;182(4):812-827.e19.doi: 10.1016/j.cell.2020.06.043.

Wiersinga et al, Pathophysiology, Transmission, Diagnosis, and Treatment of Coronavirus Disease 2019 (COVID-19): A Review, JAMA. 2020 Aug 25;324(8):782-793. doi: 10.1001/jama.2020.12839.

Baum et al, Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies, Science. 2020 Aug 21;369(6506):1014-1018.doi: 10.1126/science.abd0831.

Hansen et al, Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail, Science. 2020 Aug 21;369(6506):1010-1014. doi: 10.1126/science.abd0827.

Du et al, The spike protein of SARS-CoV--a target for vaccine and therapeutic development, Nat Rev Microbiol. 2009 Mar;7(3):226-36.doi: 10.1038/nrmicro2090.