Difference between revisions of "Part:BBa K2404016:Design"

 
 
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===Design Notes===
 
===Design Notes===
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These parts were put together using Golden Gate cloning, and have appropriate restriction enzyme recognition sites as a result. Type IIS recognition sites flank the construct.
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Experimental design for composite part construction:
  
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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_P10000 LexA]  <br>
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- Level 0 plasmid containing [https://parts.igem.org/Part:BBa_P10401 NosT]  <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>
 +
 +
This part was created using golden gate assembly, and thus has typeIIS restriction enzyme recognition sites flanking it.
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<br>
  
  

Latest revision as of 09:53, 31 October 2017


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