Generator

Part:BBa_K103018:Design

Designed by: Michael Lower   Group: iGEM08_Warsaw   (2008-10-09)

OmpA_linker_omega_linker under Plac


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1081
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 875


Design Notes

Preparation of BBa_K103018 is in details described [http://2008.igem.org/wiki/index.php?title=Team:Warsaw/JSTest&num=8&arg0=29_September_2008&arg1=30_September_2008&arg2=1_October_2008&arg3=2_October_2008&arg4=6_October_2008&arg5=7_October_2008&arg6=8_October_2008&arg7=9_October_2008&name=Preparation%20of%20OmpA_linker_omega_linker%20under%20Plac%20(BBa_K103018) here] (entries from Univeristy of Warsaw 2008 iGEM team notebook).

OmpA_linker_omega_linker (under control of lactose promoter) fragment was amplified from [http://2008.igem.org/Wiki/Team:Warsaw/vectors/pACYC177%2BompA-omega-A-alpha pACYC177+OmpA-omega-ΔA-alpha] using primers: 5' TAGAATTCGCGGCCGCTTCTAGAGCTGGCACGACAGGTTTCCC 3' and 5' CCACTAGTACCGGATCCCGAACCACCCCCACCCCCGCTAC 3'. Because obtained fragment contains EcoRI site we've developed Przanowski' method for removing of internal restriction site (described below).

Przanowski' method:

PCR product was digested overnight with EcoRI. Digested fragment was Klenow-blunted and religated with T4 ligase. Product of ligation (without internal EcoRI site) was PCR amplified using primers listed above, digested with EcoRI and BcuI and ligated into pSB2K3

Source

References