Difference between revisions of "Part:BBa K3075001: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 DBAT<sup>G38R/F301V</sup>-SnoopT within a T7 expression system.
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The following gene construct was designed to enable the cloning and expression of the recombinant proteins DBAT<sup>G38R/F301V</sup>-SnoopT-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
−
:*Hexahistidine peptide[MT1] tag – high affinity to Ni2+ - enables protein purification using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.
+
:*'''Hexahistidine peptide[MT1] 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 for additional fluidity to allow individual, unhindered protein folding of each component (enzyme and tag).
 
Additionally, GSG linkers are included between the peptide sequences for additional fluidity to allow individual, unhindered protein folding of each component (enzyme and tag).
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[[File:DBAT_seq.png]]
 
[[File:DBAT_seq.png]]
  
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'''Figure 1:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the DBAT<sup>G38R/F301V</sup>-SnoopT 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 DBAT<sup>G38R/F301V</sup>-SnoopT-His gene construct. Image by Linda Chen.
  
  

Revision as of 00:39, 22 October 2019


DBATG38R/F301V-SnoopT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 28
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 771
    Illegal PstI site found at 826
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

The following gene construct was designed to enable the cloning and expression of the recombinant proteins DBATG38R/F301V-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 enables cloning into pET19b via Gibson assembly
  • Hexahistidine peptide[MT1] 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 for additional fluidity to allow individual, unhindered protein folding of each component (enzyme and tag).

DBAT seq.png

Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the DBATG38R/F301V-SnoopT-His gene construct. Image by Linda Chen.


Source

Originated from Taxus cuspidata (Japanese yew)