Difference between revisions of "Part:BBa K3075001:Design"
Line 2: Line 2:
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K3075001 short</partinfo>
 
<partinfo>BBa_K3075001 short</partinfo>
−
 
 
<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3075001 SequenceAndFeatures</partinfo>
 +
  
 
===Design===
 
===Design===
  
−
The following gene construct was designed to enable the recombinant expression of the DBATG38R/F301V 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 DBATG38R/F301V 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.
+
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).
 +
 
 +
[[File:DBAT_seq.png]]
  
−
The “Gibson fwd overhang - DBATG38R/F301V - GSG - SnoopTag - GSG -6XHisTag - Gibson reverse overhang” gBlock sequence (Figure 1) was codon optimised for E. coli chassis and synthesised as a single gene fragment by Integrated DNA Technologies (IDT).
+
'''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.
  
−
Image
 
  
−
Figure 1: Sequence annotation of DBATG38R/F301V-SnoopT-His gBlock contains the DBAT gene (blue) with two mutations G38R and F301V (yellow), SnoopTag (pink) and a hexahistidine tag (grey) separated by GSG linkers (silver), TAA stop codon (brown) enclosed within Gibson forward and reverse overhangs (orange).
 
  
 
===Source===
 
===Source===
  
 
Originated from ''Taxus cuspidata'' (Japanese yew)
 
Originated from ''Taxus cuspidata'' (Japanese yew)

Revision as of 11:34, 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 DBAT-SnoopT gene construct. Image by Linda Chen.


Source

Originated from Taxus cuspidata (Japanese yew)