Difference between revisions of "Part:BBa K2404018:Design"

 
 
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===Design Notes===
 
===Design Notes===
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This part was put together using Golden Gate cloning, so has the appropriate restriction enzyme recognition sites in it. It is flanked by Type IIS restriction endonuclease recognition sites.
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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
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 +
> Restriction digest at 37C
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- [https://www.neb.com/products/r0535-bsai BsaI] <br>
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- Enzyme buffer <br>
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- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_404002 PDF1.2]  <br>
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- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_K2404001 TSHH] <br>
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- Level 1 plasmid [https://parts.igem.org/Part:BBa_P10503 pGB-A2]
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<br>
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<br>
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> Inactivate enzyme at 80C
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<br>
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<br>
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> Add [https://www.neb.com/products/m0202-t4-dna-ligase T4 ligase]
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<br>
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<br>
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> Transform E.coli and plate onto kanamycin (50ug/ul) plates
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 +
<br>
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<br>
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 +
This part was created using golden gate assembly, and thus has typeIIS restriction enzyme recognition sites flanking it.
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<br>
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Latest revision as of 10:40, 31 October 2017


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