Difference between revisions of "Part:BBa K3075003: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 LXYL-p1-2 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 SpyTag sequence was added to the C-terminal to enable the conjugation of the LXYL-p1-2 protein to SpyCatcher containing alpha-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 3: Sequence annotation of LXYL-P1-2-SpyT-His gBlock contains the LXYL-P1-2 gene optimised for E. coli (green), SpyTag (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 ''Lentinula edodes'' (Shiitake mushroom)

Revision as of 04:49, 19 October 2019


LXYL-P1-2- SpyT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1062
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
    Illegal NgoMIV site found at 168
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

The following gene construct was designed to enable the recombinant expression of the LXYL-p1-2 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 SpyTag sequence was added to the C-terminal to enable the conjugation of the LXYL-p1-2 protein to SpyCatcher containing alpha-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 3: Sequence annotation of LXYL-P1-2-SpyT-His gBlock contains the LXYL-P1-2 gene optimised for E. coli (green), SpyTag (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 Lentinula edodes (Shiitake mushroom)