Difference between revisions of "Part:BBa K4158004"
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We tested the detection of cyanuric acid in the cell-free reaction, using the AtzR extract. The AtzR pre-enriched extract was mixed with S12 (blank extract) with the ratio of 3:7. 320µM of cyanuric acid and 10nM of this reporter plasmid were added to the reaction. Fig.5. shows the temporal change in fluorescence values over 180 minutes of reaction, and Fig.6. shows the comparison of the values at the end of the reaction. In the presence of cyanuric acid, the fluorescence value increased about 3-fold, and cyanuric acid was successfully detected. The leak expression may be due to the fact that AtzR protein is a transcriptional activator, and that transcription is initiated by RNA polymerase even in the absence of cyanuric acid.    
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We tested the detection of cyanuric acid in the cell-free reaction, using the AtzR extract. The AtzR pre-enriched extract was mixed with S12 (blank extract) with the ratio of 3:7. 320µM of cyanuric acid and 10nM of this reporter plasmid were added to the reaction. Fig.3. shows the temporal change in fluorescence values over 180 minutes of reaction, and Fig.4. shows the comparison of the values at the end of the reaction. In the presence of cyanuric acid, the fluorescence value increased about 3-fold, and cyanuric acid was successfully detected. The leak expression may be due to the fact that AtzR protein is a transcriptional activator, and that transcription is initiated by RNA polymerase even in the absence of cyanuric acid.    
  
  
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[[File:Waseda Tokyo The comparison of the fluorescence values after 180 min.png|400px|thumb|center|Fig.5.The comparison of the fluorescence values after 180 min]]
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[[File:Waseda Tokyo The comparison of the fluorescence values after 180 min.png|400px|thumb|center|Fig.3.The comparison of the fluorescence values after 180 min]]
  
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[[File:Waseda Tokyo The temporal change of the fluorescent values.png|400px|thumb|center|Fig.6. The temporal change of the fluorescent values]]
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[[File:Waseda Tokyo The temporal change of the fluorescent values.png|400px|thumb|center|Fig.4. The temporal change of the fluorescent values]]
  
  

Revision as of 02:53, 10 October 2022


PatzR-sfGFP

This part encodes a superfolder GFP coding site and a promoter regulated by AtzR and cyanuric acid (1,3,5-Triazinane-2,4,6-trione). AtzR is a LysR-type Transcription regulator (LTTR) specific to cyanuric acid [1]. Upon the binding of cyanuric acid, GFP downstream of the binding region of AtzR is expressed as AtzR activates transcription by altering its binding sites to the operon through conformational change. Thus, this part works as a reporter plasmid to confirm existence of cyanuric acid.


Fig.1. The mechanism of GFP expression


In order to detect cyanuric acid in the cell-free protein synthesis system in E.coli, we transformed the AtzR-expressing plasmid BBa_K4158008 into BL21(DE3)Star strain and prepared crude extracts which were pre-enriched with the transcription factor, AtzR [2].


Fig.2. Preparation of AtzR pre-enriched extract


We tested the detection of cyanuric acid in the cell-free reaction, using the AtzR extract. The AtzR pre-enriched extract was mixed with S12 (blank extract) with the ratio of 3:7. 320µM of cyanuric acid and 10nM of this reporter plasmid were added to the reaction. Fig.3. shows the temporal change in fluorescence values over 180 minutes of reaction, and Fig.4. shows the comparison of the values at the end of the reaction. In the presence of cyanuric acid, the fluorescence value increased about 3-fold, and cyanuric acid was successfully detected. The leak expression may be due to the fact that AtzR protein is a transcriptional activator, and that transcription is initiated by RNA polymerase even in the absence of cyanuric acid.  


Fig.3.The comparison of the fluorescence values after 180 min
Fig.4. The temporal change of the fluorescent values


Reference

[1] Xiangyang Liu, Adam D. Silverman, Khalid K. Alam, Erik Iverson, Julius B. Lucks, Michael C. Jewett, and Srivatsan Raman. Design of a Transcriptional Biosensor for the Portable, On-Demand Detection of Cyanuric Acid. ACS Synthetic Biology 2020 9 (1), 84-94. 

[2] Adam D. Silverman, Umut Akova, Khalid K. Alam, Michael C. Jewett, and Julius B. Lucks. Design and Optimization of a Cell-Free Atrazine Biosensor. ACS Synthetic Biology 2020 9 (3), 671-677.  


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4
    Illegal XhoI site found at 115
    Illegal XhoI site found at 587
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 977
    Illegal BsaI site found at 1155