Difference between revisions of "Part:BBa K3075000: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 proteins PAM-SnoopT and TycA-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 PAM-SnoopT 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 enable cloning into pET19b plasmid via Gibson Assembly.
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:*'''Gibson forward and reverse overhangs''' – complementary overhangs outside the protein coding region enable cloning into pET19b plasmid 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.
  
  
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[[File:PAM_Annotated_Sequence.png]]
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[[File:PAM_anottated.png]]
  
  
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'''Figure 1:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of PAM-SnoopT and TycA-SpyT gene constructs. 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 PAM-SnoopT gene construct. Image by Linda Chen.
  
 
===Source===
 
===Source===
  
 
Originated from ''Taxus wallichiana var. chinensis''
 
Originated from ''Taxus wallichiana var. chinensis''

Revision as of 23:26, 21 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 cloning and expression of the recombinant protein PAM-SnoopT within a T7 expression system.

Additions to the gene are as follows:


  • Gibson forward and reverse overhangs – complementary overhangs outside the protein coding region enable cloning into pET19b plasmid 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 results in improved fluidity, allowing individual unhindered protein folding of each component (enzyme and tag system).


PAM anottated.png


Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of PAM-SnoopT gene construct. Image by Linda Chen.

Source

Originated from Taxus wallichiana var. chinensis