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.
  
−
(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.
+
To adapt our BLOOM system to various needs of biomolecule production, we focused on controlling the time interval between the expression of two genes. Our mathematical modeling shows that the most important parameter regulating the temporal difference of our BLOOM system is the protein half-life of the repressors.
 +
<br>
  
−
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.
+
To control the protein half-life, we used ssrA degradation tag. In <i>Escherichia Coli</i>, ssrA-tagged proteins are recognized and efficiently degraded by proteases such as ClpXP or ClpAP. A previous research has identified 12 mutant ssrA tags that promote the degradation of GFP at different rates[1].Therefore, suspecting that there should be more mutants of various degradation efficiencies, we aimed to identify them by mutagenizing WT tag.
 +
<br>
  
−
(figure: Hayamatsu-kun)
+
The wildtype ssrA tag amino acid sequence is AANDENYALAA. 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. Therefore, we chose these three amino acids as the targets for mutagenesis.
 +
<br>
  
−
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).
+
For mutagenesis, 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.  
  
  
 +
                    <div class="figs">
 +
                        <img class="" src="https://2021.igem.org/wiki/images/e/e9/T--Kyoto--ssrA_005.png">
 +
                    </div>
  
−
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)
+
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 protein degradation efficiency of each mutant, GFP fluorescence intensity of each <i>E.coli</i> overnight culture was measured by Qubit, and then compared to that of WT and other mutants.
+
                    <div class="figs">
 +
                        <img class="" src="https://2021.igem.org/wiki/images/5/55/T--Kyoto--ssrA_002_experiment.png">
 +
                    </div>
 +
                                       
 +
Our mutagenesis strategy is as follows:
 +
                    <ol start="1">
 +
                    <li>to J23119 promoter-RBS-GFP insert derived from BBa_K584001, add ssrA tag sequence lacking the three C-terminal amino acids by PCR (first PCR)
 +
                    </li>
 +
                    <li>perform the second PCR on the first PCR product to add the rest of the tag sequence while mutagenizing them with different types of random-base primers listed above.
 +
                    </li>
 +
                    <li>assemble the second PCR product with a linearized pSB4K5-BBa_J04450 backbone by InFront Assembly (a type of homology arm-mediated seamless cloning like In-Fusion) and transform it into E.coli.
 +
                    </li>
 +
                  </ol>
 +
 
 +
<br>
 +
 
 +
We chose a low-copy plasmid pSB4K5 as the backbone to be able to compare the fluorescence intensity by trying to avoid the saturation or variation of GFP intensity of colonies.
 +
 
 +
                    <div class="figs">
 +
                        <img class="" src="https://2021.igem.org/wiki/images/3/36/T--Kyoto--Result_003_ssrA.png">
 +
                    </div>
 +
 
 +
<br>             
 +
As a result of transformation, colonies of various fluorescence intensity were obtained on the LB plates. 72 colonies in total were picked from plates, and cultured overnight in LB media. The images of E.coli cultures were taken under the blue light.
 +
 
 +
                    <div class="figs">
 +
                        <img class="" src="https://2021.igem.org/wiki/images/2/22/T--Kyoto--ssrA_007.png">
 +
                      </div>
 +
 
 +
                    <div class="figs">
 +
                        <img class="" src="https://2021.igem.org/wiki/images/2/26/T--Kyoto--ssrA_011.png">
 +
                      </div>
 +
 
 +
                    <div class="myp">Bacterial cultures of various GFP intensity were observed.
 +
<br>
 +
A10: LB media only, B10: E.coli culture not expressing GFP, B7: E.coli culture expressing non ssrA-tagged GFP, C7: E.coli culture expressing WT ssrA-tagged GFP
 +
 
 +
 
 +
Plasmid was extracted from each bacterial culture by miniprep, and then Sanger-sequenced to  identify ssrA tag sequence. Furthermore, to quantify protein degradation efficiency of each mutant, GFP Fluorescence of each bacterial overnight culture was imaged on Typhoon 9410 (GE healthcare).
 +
 
 +
 
 +
                    <div class="figs">
 +
                        <img class="" src="https://2021.igem.org/wiki/images/3/3b/T--Kyoto--ssrA_004.png">
 +
                    </div>
 +
       
 +
 
 +
Left: GFP fluorescence image of each bacterial overnight culture
 +
Right: sequenced three C-terminal amino acids of each culture
 +
 
 +
The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate of each bacterial overnight culture was imaged for GFP fluorescence, including controls (LB media only, bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non ssrA-tagged GFP (no ssrA tag)).
 +
 +
 
 +
                    <div class="figs">
 +
                        <"https://2021.igem.org/wiki/images/d/d6/T--Kyoto--ssrA_006.png">
 +
                      </div>
 +
 
 +
 
 +
The fluorescence of each culture was quantified by ImageJ as inverted mean gray value. Measured value of each culture was then normalized to the level of the culture expressing non ssrA-tagged GFP (no ssrA tag), and presented as mean ± SD from three biological replicates. WT (LAA) and two mutants reported in a previous research are shown in red boxes.
 +
             
 +
             
 +
We mutagenized ssrA tag by PCR with mixed primers, and successfully obtained a mutant collection of engineered ssrA tag derivatives which show various protein degradation efficiencies.
  
−
(figure: relative GFP intensity)
 
  
 
<br>
 
<br>

Revision as of 19:59, 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


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.


To adapt our BLOOM system to various needs of biomolecule production, we focused on controlling the time interval between the expression of two genes. Our mathematical modeling shows that the most important parameter regulating the temporal difference of our BLOOM system is the protein half-life of the repressors.

To control the protein half-life, we used ssrA degradation tag. In Escherichia Coli, ssrA-tagged proteins are recognized and efficiently degraded by proteases such as ClpXP or ClpAP. A previous research has identified 12 mutant ssrA tags that promote the degradation of GFP at different rates[1].Therefore, suspecting that there should be more mutants of various degradation efficiencies, we aimed to identify them by mutagenizing WT tag.

The wildtype ssrA tag amino acid sequence is AANDENYALAA. 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. Therefore, we chose these three amino acids as the targets for mutagenesis.

For mutagenesis, 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. 


                       <img class="" src="T--Kyoto--ssrA_005.png">


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. 


                       <img class="" src="T--Kyoto--ssrA_002_experiment.png">

Our mutagenesis strategy is as follows:

  1. to J23119 promoter-RBS-GFP insert derived from BBa_K584001, add ssrA tag sequence lacking the three C-terminal amino acids by PCR (first PCR)
  2. perform the second PCR on the first PCR product to add the rest of the tag sequence while mutagenizing them with different types of random-base primers listed above.
  3. assemble the second PCR product with a linearized pSB4K5-BBa_J04450 backbone by InFront Assembly (a type of homology arm-mediated seamless cloning like In-Fusion) and transform it into E.coli.


We chose a low-copy plasmid pSB4K5 as the backbone to be able to compare the fluorescence intensity by trying to avoid the saturation or variation of GFP intensity of colonies. 

                       <img class="" src="T--Kyoto--Result_003_ssrA.png">


As a result of transformation, colonies of various fluorescence intensity were obtained on the LB plates. 72 colonies in total were picked from plates, and cultured overnight in LB media. The images of E.coli cultures were taken under the blue light. 

                       <img class="" src="T--Kyoto--ssrA_007.png">

                       <img class="" src="T--Kyoto--ssrA_011.png">
Bacterial cultures of various GFP intensity were observed.


A10: LB media only, B10: E.coli culture not expressing GFP, B7: E.coli culture expressing non ssrA-tagged GFP, C7: E.coli culture expressing WT ssrA-tagged GFP


Plasmid was extracted from each bacterial culture by miniprep, and then Sanger-sequenced to identify ssrA tag sequence. Furthermore, to quantify protein degradation efficiency of each mutant, GFP Fluorescence of each bacterial overnight culture was imaged on Typhoon 9410 (GE healthcare). 


                       <img class="" src="T--Kyoto--ssrA_004.png">


Left: GFP fluorescence image of each bacterial overnight culture Right: sequenced three C-terminal amino acids of each culture

The mutant ssrA tag sequence of each clone was sequenced, and biological triplicate of each bacterial overnight culture was imaged for GFP fluorescence, including controls (LB media only, bacterial culture not expressing GFP (no GFP), and bacterial culture expressing non ssrA-tagged GFP (no ssrA tag)). 


                       <"T--Kyoto--ssrA_006.png">


The fluorescence of each culture was quantified by ImageJ as inverted mean gray value. Measured value of each culture was then normalized to the level of the culture expressing non ssrA-tagged GFP (no ssrA tag), and presented as mean ± SD from three biological replicates. WT (LAA) and two mutants reported in a previous research are shown in red boxes.


We mutagenized ssrA tag by PCR with mixed primers, and successfully obtained a mutant collection of engineered ssrA tag derivatives which show various protein degradation efficiencies.




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.

Name Type Part Name Designer
BBa_K3815015 Tag AANDENYALGA.mutant SsrA degradation tag Alexander Liu
BBa_K3815016 Tag AANDENYAKWA.mutant SsrA degradation tag Alexander Liu
BBa_K3815017 Tag AANDENYALAK.mutant SsrA degradation tag Alexander Liu
BBa_K3815018 Tag AANDENYAHPK.mutant SsrA degradation tag Alexander Liu
BBa_K3815019 Tag AANDENYALAL.mutant SsrA degradation tag Alexander Liu
BBa_K3815020 Tag AANDENYANDK.mutant SsrA degradation tag Alexander Liu
BBa_K3815021 Tag AANDENYANEN.mutant SsrA degradation tag Alexander Liu
BBa_K3815022 Tag AANDENYAYES.mutant SsrA degradation tag Alexander Liu
BBa_K3815023 Tag AANDENYAHAK.mutant SsrA degradation tag Alexander Liu
BBa_K3815024 Tag AANDENYALAS.mutant SsrA degradation tag Alexander Liu
BBa_K3815025 Tag AANDENYATAP.mutant SsrA degradation tag Alexander Liu
BBa_K3815026 Tag AANDENYANAA.mutant SsrA degradation tag Alexander Liu
BBa_K3815027 Tag AANDENYAKLA.mutant SsrA degradation tag Alexander Liu
BBa_K3815028 Tag AANDENYALPA.mutant SsrA degradation tag Alexander Liu
BBa_K3815029 Tag AANDENYALAM.mutant SsrA degradation tag Alexander Liu
BBa_K3815030 Tag AANDENYALAI.mutant SsrA degradation tag Alexander Liu
BBa_K3815031 Tag AANDENYAPHA.mutant SsrA degradation tag Alexander Liu



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

  1. Flynn, J.M., Levchenko, I., Seidel, M., Wickner, S.H., Sauer, R.T., and Baker, T.A. (2001). Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis. Proc. Natl. Acad. Sci. U. S. A. 98, 10584–10589.