Part:BBa_K3815015
AANDENYALGA. mutant SsrA degradation tag
Usage and Biology
This is an engineered derivative of wildtype ssrA tag from Escherichia coli, where three C-terminal amino acids LAA in WT are replaced to LGA. Refer to BBa_M0050 for the biological function of the tag.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Result
obtained by mutagenizing the WT tag with mixed primers. the repertoire of tags is still not large enough to enable a flexible control of protein half-life. Therefore, we aimed to obtain a collection of mutant tags with various degradation efficiencies by mutagenizing the wildtype ssrA tag. We fused the ssrA tag sequence to GFP while introducing mutations in the tag by random-base primers, and cloned the mutant library of ssrA-tagged GFP into a plasmid vector, so that mutant tags of different activity can be identified by comparing GFP intensity of E.coli transformants.
To quantify its efficiency of protein degradation, GFP fluorescence intensity of E.coli overnight culture bearing a plasmid expressing GFP-mutant ssrA tag fusion protein was measured by Qubit, and then compared to that of WT and other mutants.
Part collection of mutant ssrA tags
In our mutagenesis experiments, we identified # of mutant tags which show various protein degradation efficiencies. The other mutants are
Improvement from an Existing Part
This is a part improved from BBa_K1051206. In this part, three C-terminal amino acids LAA were replaced with LGA, resulting in a reduced protein degradation efficiency. Therefore, this part show an increased half-life of fused proteins compared to the WT. This part, together with the other mutants described in the part collection above, composes a large repertoire for a flexible control of protein half-life.
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