Composite

Part:BBa_K2404022:Design

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


TSH antagonist under control of the GST6 promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 72
    Illegal PstI site found at 1045
    Illegal PstI site found at 1087
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1045
    Illegal PstI site found at 1087
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1300
    Illegal XhoI site found at 1112
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 72
    Illegal PstI site found at 1045
    Illegal PstI site found at 1087
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 72
    Illegal PstI site found at 1045
    Illegal PstI site found at 1087
  • 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 GST6

- Level 0 plasmid containing NosT

- 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