Difference between revisions of "Part:BBa K3075003:Design"
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Additions to the gene are as follows: | 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 | + | :*'''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. | + | :*'''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). | 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). | ||
Revision as of 00:27, 22 October 2019
LXYL-P1-2- SpyT-His
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1462
Illegal PstI site found at 1499 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 1462
Illegal PstI site found at 1499 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1062
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1462
Illegal PstI site found at 1499 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1462
Illegal PstI site found at 1499
Illegal NgoMIV site found at 168 - 1000COMPATIBLE WITH RFC[1000]
Design
The following gene construct was designed to enable the cloning and expression of the recombinant protein LXYL-p1-2-SpyT 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).
Figure 1: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the LXYL-p1-2-SpyT gene construct. Image by Linda Chen.
Source
Originated from Lentinula edodes (Shiitake mushroom)