Coding
P+A

Part:BBa_K3682008:Design

Designed by: YOU-CHENG LEE   Group: iGEM20_NYMU-Taipei   (2020-10-15)


pepP sequence +hACE2 RBD


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3155
    Illegal PstI site found at 1644
    Illegal PstI site found at 2587
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3155
    Illegal PstI site found at 1644
    Illegal PstI site found at 2587
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3155
    Illegal BglII site found at 260
    Illegal BglII site found at 1860
    Illegal BamHI site found at 2425
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3155
    Illegal PstI site found at 1644
    Illegal PstI site found at 2587
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3155
    Illegal PstI site found at 1644
    Illegal PstI site found at 2587
    Illegal AgeI site found at 1243
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1363
    Illegal SapI site found at 1650


Design Notes

We originally chose to use Gibson assembly to connect the fragments. However, because we put the GGSGGS linker on the primer of ACE2 instead of customizing one, the PCR results remain low yield. Therefore, We changed our method to linear ligation and customized the GGSGGS linker. Also, in order to let BL21 strain E.coli express the fusion protein, we put it into a bacterial expression backbone at the same time when the three fragments are doing linear ligation. The results of Restriction enzyme digestion of the plasmid indicated the success of our plasmid construction.


Fig1. Restriction enzyme digestion results of pepP-GGSGGSLinker-ACE2 construct

Source

hACE2 RBD: We cloned the part from a plasmid we bought at addgene.org (id 145033) Linker: We customized it from a biotechnology company pepP sequence: We cloned it from E.coli K12 strain

References