Difference between revisions of "Part:BBa K3075002: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-SnoopTag and TycA-SpyTag 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 TycA-SpyTag within a T7 expression system.
  
 
Additions to the gene are as follows:
 
Additions to the gene are as follows:

Revision as of 23:56, 21 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 TycA-SpyTag 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).

PAM Annotated Sequence.png

Figure 3: 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.


Source

Originated from Brevibacillus parabrevis