Difference between revisions of "Part:BBa K3075002:Design"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3075002 short</partinfo>
 
<partinfo>BBa_K3075002 short</partinfo>
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<partinfo>BBa_K3075002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3075002 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 TycAS653A 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 TycAS653A 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 TycA<sup>S563A</sup>-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 enable cloning into pET-19b 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 flexible linker was designed to permit individual unhindered protein folding of each component (enzyme and tag system).  
  
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Image
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[[File:TycA_anottated.png]]
  
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Figure 1: Sequence annotation of TycAS653A-SpyT-His gBlock contains the TycA gene (orange) with mutation S653A (yellow), 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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'''Figure 3:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the TycA<sup>S563A</sup>-SpyT-His gene construct. Image by Linda Chen.
  
 
===Source===
 
===Source===
  
 
Originated from ''Brevibacillus parabrevis''
 
Originated from ''Brevibacillus parabrevis''

Latest revision as of 00:48, 22 October 2019


TycAS563A-SpyT-His


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2289
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 2299
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1223
    Illegal BsaI.rc site found at 2143
    Illegal SapI.rc site found at 2499


Design

The following gene construct was designed to enable the cloning and expression of the recombinant protein TycAS563A-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 enable cloning into pET-19b 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 flexible linker was designed to permit individual unhindered protein folding of each component (enzyme and tag system).

TycA anottated.png

Figure 3: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the TycAS563A-SpyT-His gene construct. Image by Linda Chen.

Source

Originated from Brevibacillus parabrevis