Part:BBa_K3075004:Design
His-mCerulean3-SnoopT
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 43
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design
The following gene construct was designed to enable to cloning and expression of the recombinant His-mCerulean3-SnoopTag within a T7 expression system (Figure 1).
Additions to the gene are as follows:
- SnoopTag - The SnoopTag has the ability to conjugate to the SnoopCatcher via spontaneous isopeptide bond formation. This allows for the bioconjugation of mCerulean3 to any protein fused to a SnoopCatcher. This is our gold improvement.
- Hexahistidine Tag - The histidine residues has a high binding affinity to Ni2+, this enables the purification of the mCerulean3 protein using Immobilised metal affinity chromatography (IMAC) via a Ni-NTA column.
- Gibson forward and reverse overhangs - 5’ forward and 3’ reverse gibson overhangs are complementary to that of the pET-19b backbone. This allows for the ligation of mCerulean3 into the pET-19b plasmid via Gibson Assembly.
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 tags).
Figure 1:Sequence annotation of the His-mCerulean3-SnoopT gBlock contains the hexahistidine tag (purple), mCerulean3 gene (green) and SnoopTag (pink), separated by GSG linkers (silver), enclosed within 5’ gibson forward and 3’ gibson reverse overhangs (blue). Image produced by Benchling.
New sequence:
Gibson forward overhang - ATG start codon - 6X Histag - GSG - mCerulean3 - GSG - SnoopTag - TGA Stop codon - Gibson reverse overhang