Composite

Part:BBa_K2404018:Design

Designed by: Ryan Coates   Group: iGEM17_Cardiff_Wales   (2017-10-16)


TSH antagonist under control of the PDF1.2 promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1048
    Illegal PstI site found at 1090
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1048
    Illegal PstI site found at 1090
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 412
    Illegal BamHI site found at 1303
    Illegal XhoI site found at 1115
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1048
    Illegal PstI site found at 1090
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1048
    Illegal PstI site found at 1090
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Experimental design for composite part construction:

We conducted a Type IIS restriction enzyme digest using the BsaI enzyme. The reaction mix includes three level 0 plasmid

> Restriction digest at 37C - BsaI
- Enzyme buffer

- Level 0 plasmid containing PDF1.2

- Level 0 plasmid containing TSHH

- Level 1 plasmid pGB-A2

> Inactivate enzyme at 80C

> Add T4 ligase

> Transform E.coli and plate onto kanamycin (50ug/ul) plates



This part was created using golden gate assembly, and thus has typeIIS restriction enzyme recognition sites flanking it.



Source

The CDS was synthesised, the promoter isolated from Arabidopsis thaliana , and the terminator isolated from Agrobacterium tumefaciens

References