Difference between revisions of "Part:BBa K3815015"

Revision as of 18:19, 21 October 2021

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 with LGA. Refer to BBa_M0050 for the biological function of the tag.

Sequence and Features

Result

For a flexible control of protein half-life, we aimed to obtain a collection of mutant tags with various degradation efficiencies. 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.

(figure: Tamura-san)

(figure: Morita-san) We chose a low-copy plasmid pSB4K5 as the backbone to be able to compare the fluorescence intensity by avoiding the saturation or variation of GFP intensity of colonies.

It was reported that the three amino acids at the C terminus of the tag (LAA in wildtype) have a great impact on the degradation rate of tagged protein [need reference]. Therefore, we chose these three amino acids as the targets for mutagenesis.

(figure: Hayamatsu-kun)

We used a primer containing three random bases at either one of the target three C-terminal amino acids (XAA, LXA, LAX), or nine random bases at all of the target three amino acids (XXX). We also included a primer containing wildtype sequence (LAA), and two reported mutants (AAV & ASV) for controls (need reference).

As a result of transformation, colonies of various fluorescence intensity were obtained. 72 colonies were picked and cultured overnight in LB media containing Kanamycin, and the image was taken in a 96-well plate under the blue light.

(figure: 96-well plate)

To quantify protein degradation efficiency of each mutant, GFP fluorescence intensity of each E.coli overnight culture was measured by Qubit, and then compared to that of WT and other mutants.

(figure: relative GFP intensity)

Part collection of mutant ssrA tags

In our mutagenesis experiments, we identified 17 mutant tags which show various protein degradation efficiencies (refer to Result). The other mutant tags are listed below.

Improvement from an Existing Part

This is a part improved from BBa_K1051206. In this part, three C-terminal amino acids LAA are replaced with LGA, resulting in a reduced protein degradation efficiency. Therefore, this part show an increased half-life of fused proteins compared to WT. This part, together with the other mutants described in the part collection above, consists of a large repertoire of protein degradation tags of various efficiencies (refer to Result), which can be applied for fine-tuning of many kinds of synthetic systems that require a precise control of proteins.

Reference