Difference between revisions of "Part:BBa K3075003:Design"
 
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__NOTOC__
 
<partinfo>BBa_K3075003 short</partinfo>
 
<partinfo>BBa_K3075003 short</partinfo>
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<partinfo>BBa_K3075003 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3075003 SequenceAndFeatures</partinfo>
  
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===Design===
 
===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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The following gene construct was designed to enable the cloning and expression of the recombinant protein LXYL-p1-2-SpyT-His within a T7 expression system.
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Additions to the gene are as follows:
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:*'''Gibson forward and reverse overhangs''' – complementary overhangs outside the protein coding region enables cloning into pET19b via Gibson assembly
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:*'''Hexahistidine peptide tag''' – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.
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Additionally, GSG linkers are included between the peptide sequences. This flexible linker was designed to permit individual unhindered protein folding of each component (enzyme and tag).
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[[File:LXYL_anottated.png]]
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'''Figure 1:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the LXYL-p1-2-SpyT-His gene construct. Image by Linda Chen.  
  
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Image
 
  
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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).
 
  
 
===Source===
 
===Source===
  
 
Originated from ''Lentinula edodes'' (Shiitake mushroom)
 
Originated from ''Lentinula edodes'' (Shiitake mushroom)

Latest revision as of 00:52, 22 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 cloning and expression of the recombinant protein LXYL-p1-2-SpyT-His within a T7 expression system.

Additions to the gene are as follows:

  • Gibson forward and reverse overhangs – complementary overhangs outside the protein coding region enables cloning into pET19b via Gibson assembly
  • Hexahistidine peptide tag – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.

Additionally, GSG linkers are included between the peptide sequences. This flexible linker was designed to permit individual unhindered protein folding of each component (enzyme and tag).

LXYL anottated.png

Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the LXYL-p1-2-SpyT-His gene construct. Image by Linda Chen.


Source

Originated from Lentinula edodes (Shiitake mushroom)