Difference between revisions of "Part:BBa K3075001:Design"

 
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<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
  
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===Design===
  
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===Design Notes===
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The following gene construct was designed to enable the recombinant expression of the DBATG38R/F301V protein within an ''E. coli'' chassis (Figure 3). Gibson forward and reverse overhangs were added the 5’ and 3’ ends for cloning via Gibson Assembly. The SnoopTag sequence was added to the C-terminal to enable the conjugation of the DBATG38R/F301V protein to SnoopCatcher containing beta-prefoldin arms of the assemblase scaffold. A hexa-histidine tag was added to the C-terminal end to enable the purification of the protein once expressed by immobilised metal affinity chromatography (IMAC) via a nickel-NTA column. Each gene component was separated by a GSG linker sequences to increase the flexibility between each peptide and prevent steric hindrance of protein folding.
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Design
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The “Gibson fwd overhang - DBATG38R/F301V - GSG - SnoopTag - GSG -6XHisTag - Gibson reverse overhang” gBlock sequence (Figure 1) was codon optimised for E. coli chassis and synthesised as a single gene fragment by Integrated DNA Technologies (IDT).
  
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Image
  
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===Source===
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Figure 1: Sequence annotation of DBATG38R/F301V-SnoopT-His gBlock contains the DBAT gene (blue) with two mutations G38R and F301V (yellow), SnoopTag (pink) and a hexahistidine tag (grey) separated by GSG linkers (silver), TAA stop codon (brown) enclosed within Gibson forward and reverse overhangs (orange).
  
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Source
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===Source===
  
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===References===
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Originated from ''Taxus cuspidata'' (Japanese yew)

Revision as of 03:51, 19 October 2019


DBATG38R/F301V-SnoopT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 28
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 1000
    COMPATIBLE WITH RFC[1000]

Design

The following gene construct was designed to enable the recombinant expression of the DBATG38R/F301V protein within an E. coli chassis (Figure 3). Gibson forward and reverse overhangs were added the 5’ and 3’ ends for cloning via Gibson Assembly. The SnoopTag sequence was added to the C-terminal to enable the conjugation of the DBATG38R/F301V protein to SnoopCatcher containing beta-prefoldin arms of the assemblase scaffold. A hexa-histidine tag was added to the C-terminal end to enable the purification of the protein once expressed by immobilised metal affinity chromatography (IMAC) via a nickel-NTA column. Each gene component was separated by a GSG linker sequences to increase the flexibility between each peptide and prevent steric hindrance of protein folding.

The “Gibson fwd overhang - DBATG38R/F301V - GSG - SnoopTag - GSG -6XHisTag - Gibson reverse overhang” gBlock sequence (Figure 1) was codon optimised for E. coli chassis and synthesised as a single gene fragment by Integrated DNA Technologies (IDT).

Image

Figure 1: Sequence annotation of DBATG38R/F301V-SnoopT-His gBlock contains the DBAT gene (blue) with two mutations G38R and F301V (yellow), SnoopTag (pink) and a hexahistidine tag (grey) separated by GSG linkers (silver), TAA stop codon (brown) enclosed within Gibson forward and reverse overhangs (orange).

Source

Originated from Taxus cuspidata (Japanese yew)