Coding

Part:BBa_K3075003:Design

Designed by: David Downes   Group: iGEM19_UNSW_Australia   (2019-10-07)
Revision as of 16:24, 21 October 2019 by DD6861 (Talk | contribs)


LXYL-P1-2- SpyT-His


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1062
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1462
    Illegal PstI site found at 1499
    Illegal NgoMIV site found at 168
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

The following gene construct was designed to enable the cloning and expression of the recombinant proteins LXYL-SpyT and 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 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).

Lxyl ano.png

Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of LXYL-p1-2-SpyT and DBAT-SnoopT gene constructs. Image by Linda Chen.


Source

Originated from Lentinula edodes (Shiitake mushroom)