Difference between revisions of "Part:BBa K3075001:Design"
Line 18: Line 18:
 
[[File:DBAT_seq.png]]
 
[[File:DBAT_seq.png]]
  
−
'''Figure 1:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the DBAT-SnoopT gene construct. Image by Linda Chen.
+
'''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.
  
  

Revision as of 23:50, 21 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 DBAT-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 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 gene construct. Image by Linda Chen.


Source

Originated from Taxus cuspidata (Japanese yew)