Difference between revisions of "Part:BBa K2404014:Design"

 
 
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===Design Notes===
 
===Design Notes===
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These sequences were combined using Golden Gate cloning, and have appropriate restriction endonuclease recognition sites as a result. There are Type IIS restriction enzyme sites flanking the construct.
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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_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 LUC+ <br>
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 +
- Level 1 plasmid [https://parts.igem.org/Part:BBa_P10503 pGB-A1]
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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.
 +
<br>
  
  

Latest revision as of 08:50, 31 October 2017


Luc+ gene under control of the LexA promoter


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 15
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 15
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1171
    Illegal BamHI site found at 1017
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 15
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 15
  • 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 LUC+

- Level 1 plasmid pGB-A1

> 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

All parts are isolated from gDNA. The promoter is from E. coli , the CDS from fireflies, and the terminator from Agrobacterium tumefaciens .

References