Difference between revisions of "Part:BBa K3075000:Design"
 
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<partinfo>BBa_K3075000 short</partinfo>
 
<partinfo>BBa_K3075000 short</partinfo>
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<partinfo>BBa_K3075000 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3075000 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 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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The following gene construct was designed to enable the cloning and expression of the recombinant protein PAM-SnoopT-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 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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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).
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[[File:PAM_anottated.png]]
  
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Image
 
  
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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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'''Figure 1:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of PAM-SnoopT-His gene construct. Image by Linda Chen.
  
 
===Source===
 
===Source===
  
 
Originated from ''Taxus wallichiana var. chinensis''
 
Originated from ''Taxus wallichiana var. chinensis''

Latest revision as of 00:42, 22 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-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 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-His gene construct. Image by Linda Chen.

Source

Originated from Taxus wallichiana var. chinensis