Difference between revisions of "Part:BBa K3765013"

Latest revision as of 18:00, 20 October 2021

OpdA_Signal+OpdA+22aaL+SpyTag002+sfGFP+10xHis

CDS of fusion protein consisting of SpyTag002 linked to OpdA (phosphotriesterase) and sfGFP, which can covalently bind to SpyCatcher/SpyCatcher002. In our project, the protein encoded by this part is designed to get anchored to a SpyCatcher002-dCBD fusion protein.

Usage and Biology

Contains the native 22 amino acid OpdA signal peptide for extracellular secretion. Contains a 10xHis Tag attached at the C terminus for purification by Ni-NTA affinity chromatography. Purification can also be done by the Spy&Go system using the SpyTag-SpyDock interaction (Khairil Anuar INA et al, Nat Commun. 2019). Also contains an sfGFP module which can be used for measuring expression levels in vivo, or for assaying the binding extent of the SpyTag002/SpyCatcher002 pair. The OpdA enzyme is a phosphotriesterase which degrades organophosphates. To preserve folding and activity, it has been spaced out from the other modules using a flexible 22 amino acid linker.

A pair of AgeI sites have been added on either side of the SpyTag002 module for it to be easily removed in case any other anchoring strategy needs to be employed. The AgeI site after digestion followed by ligation also provides a site of linearization for a plasmid carrying this insert, so that virtually any module can be cloned between the OpdA enzyme and the sfGFP by Gibson Assembly, InFusion Cloning etc. A pair of KpnI sites have been added on either side of the sfGFP module for it to be removed if reporter characteristics are not required, which can help in making the protein less bulky.

Fig 1. Feature Map

Reference: Khairil Anuar INA, Banerjee A, Keeble AH, Carella A, Nikov GI, Howarth M. Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox. Nat Commun. 2019 Apr 15;10(1):1734. doi: 10.1038/s41467-019-09678-w.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1920
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 1219
Illegal AgeI site found at 1282
1000
COMPATIBLE WITH RFC[1000]

@@ Line 9: / Line 9: @@
 A pair of AgeI sites have been added on either side of the SpyTag002 module for it to be easily removed in case any other anchoring strategy needs to be employed. The AgeI site after digestion followed by ligation also provides a site of linearization for a plasmid carrying this insert, so that virtually any module can be cloned between the OpdA enzyme and the sfGFP by Gibson Assembly, InFusion Cloning etc. A pair of KpnI sites have been added on either side of the sfGFP module for it to be removed if reporter characteristics are not required, which can help in making the protein less bulky.
-[[File:T--IISc-Bangalore--images--Map_C2.png|thumb|center|900px|Fig 2. Feature Map]]
+[[File:T--IISc-Bangalore--images--Map_C2.png|thumb|center|900px|Fig 1. Feature Map]]
 '''Reference:''' Khairil Anuar INA, Banerjee A, Keeble AH, Carella A, Nikov GI, Howarth M. Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox. ''Nat Commun.'' 2019 Apr 15;10(1):1734. doi: 10.1038/s41467-019-09678-w.
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