Difference between revisions of "Part:BBa K3075000:Design"

 
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===Design Notes===
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===Design===
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Design
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The following gene construct was designed to enable the recombinant expression of the PAM 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 PAM 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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Image
  
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===Source===
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Figure 1: Sequence annotation of PAM-SnoopT-His gBlock contains the PAM gene (purple), 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 wallichiana var. chinensis''

Revision as of 04:31, 19 October 2019


PAM-SnoopT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 498
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 498
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 498
    Illegal BamHI site found at 2131
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 498
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 498
    Illegal NgoMIV site found at 393
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

The following gene construct was designed to enable the recombinant expression of the PAM 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 PAM 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.

Image

Figure 1: Sequence annotation of PAM-SnoopT-His gBlock contains the PAM gene (purple), 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 wallichiana var. chinensis