Difference between revisions of "Part:BBa K3815015"
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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 <i>E.coli</i> transformants.
 
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 <i>E.coli</i> transformants.
  
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[[File:T--Kyoto--ssrA experimental workflow.png|300px|thumb|right|Fig1. ]]
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[[File:T--Kyoto--ssrA_experimental_workflow.png|300px|thumb|right|Fig1. ]]
  
 
(figure: Morita-san)
 
(figure: Morita-san)

Revision as of 17:32, 20 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 to LGA. Refer to BBa_M0050 for the biological function of the tag.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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.

File:T--Kyoto--ssrA experimental workflow.png
Fig1.

(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, we obtained colonies of various fluorescence intensity. 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 # 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.