Difference between revisions of "Part:BBa K3075002:Design"

Revision as of 00:02, 22 October 2019

TycA^S563A-SpyT-His

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 2289
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 2299
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 1223
Illegal BsaI.rc site found at 2143
Illegal SapI.rc site found at 2499

Design

The following gene construct was designed to enable the cloning and expression of the recombinant protein TycA^S563A-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 enable cloning into pET-19b plasmid 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 system).

Figure 3: Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the TycA^S563A-SpyT gene construct. Image by Linda Chen.

Source

Originated from Brevibacillus parabrevis

@@ Line 16: / Line 16: @@
 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 system).
-[[File:PAM_Annotated_Sequence.png]]
+[[File:TycA_anottated.png]]
 '''Figure 3:''' Sequence annotation of the designed gene constructs for a visual representation of the spatial arrangement of the TycA<sup>S563A</sup>-SpyT gene construct. Image by Linda Chen.