Difference between revisions of "Part:BBa K3075003:Design"
 
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===Design===
 
===Design===
  
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The following gene construct was designed to enable the cloning and expression of the recombinant protein LXYL-p1-2-SpyT within a T7 expression system.
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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.
  
 
Additions to the gene are as follows:
 
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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:*'''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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:*'''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).
 
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]]
 
[[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 gene construct. Image by Linda Chen.  
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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.  
  
  

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)