Composite

Part:BBa_K2404016:Design

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


A TSH antagonist with His-Tags, controlled by the LexA promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 22
    Illegal PstI site found at 888
    Illegal PstI site found at 930
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 22
    Illegal PstI site found at 888
    Illegal PstI site found at 930
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1143
    Illegal XhoI site found at 955
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 22
    Illegal PstI site found at 888
    Illegal PstI site found at 930
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 22
    Illegal PstI site found at 888
    Illegal PstI site found at 930
  • 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 LexA

- 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, and the promoter and terminator isolated from gDNA of E. coli and Agrobacterium tumefaciens , respectively.

References