Device

Part:BBa_K1919002:Design

Designed by: Shiji Zhao   Group: iGEM16_SCU-China   (2016-09-05)
Revision as of 14:22, 14 October 2016 by Century (Talk | contribs) (→‎Design Notes)

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Constitutive promotor J23106 and downstream CecropinXJ


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 506
    Illegal BamHI site found at 549
    Illegal XhoI site found at 750
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Two steps were needed in order to obtain BBa_K1919002. Step 1, BBa_K1919000 was inserted into pET32a(+). The primers used for PCR are as following:

  • F: 5'-CCGGGATCCATGAACTTCGCTAAAATCCTGTCTT-3' (BamH I site was added)
  • R: 5'-CCGCTCGAGTTATTATTTACCGATAGCTTTA-3' (Xho I site was added)

BamH I/Xho I Double enzymatic cutting was applied for both pET32a(+) and BBa_K1919000.

Step 2, RBS and 5 downstream tags as well as original CecropinXJ was inserted at the downstream of promotor J23106 with the same procedure as step 1. The primers used for PCR are as following:

  • F: 5'-GCTCTAGAGCGAAGGAGATATACATATGAGCG-3' (Xba I site was added)
  • R: 5'-TGCACTGCAGTGCAGACTAGTCGTTATTGCTCAGCGGTGG-3'(Pst I site was added)

Xba I/Pst I double enzymatic cutting was applied for PCR product and Spe I/Pst I double enzymatic cutting was used for BBa_J23106. After enzymatic linkage, the construction of BBa_K1919002 was finished. Then this part was transfered into pSB1C3 backbone.

Source

The sequence of constitutive promotor J23106 comes from biobrick BBa_J23106. RBS and 5 downstream tags come from commercial plasmid pET32a(+). The sequence of CecropinXJ comes from biobrick BBa_K1919000

References