Difference between revisions of "Part:BBa K3075001:Design"
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===Design=== | ===Design=== | ||
| − | 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. | + | 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: | ||
| − | :*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[MT1] | + | :*'''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). | ||
| 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<sup>G38R/F301V</sup>-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-His gene construct. Image by Linda Chen. |
Revision as of 00:39, 22 October 2019
DBATG38R/F301V-SnoopT-His
Assembly Compatibility:
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 771
Illegal PstI site found at 826 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 771
Illegal PstI site found at 826 - 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 28
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 771
Illegal PstI site found at 826 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 771
Illegal PstI site found at 826 - 1000COMPATIBLE 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).
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)