Part:BBa_K4158008

RBS-AtzR

Cyanuric acid, the final metabolite of atrazine, has a specific LysR-type Transcription regulator (LTTR), AtzR. This part encodes RBS and coding site of AtzR protein. Upon the binding of cyanuric acid, AtzR activates transcription by altering its binding sites to the operon through conformational change. Thus, GFP downstream of the binding region of AtzR on the reporter plasmid (BBa_K4158004) is expressed under the presence of cyanuric acid [1].

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 this part 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

Fig.3. shows the result of SDS-PAGE done with the AtzR-expressing bacterial samples (The abbreviation of the sample names in the figure is shown in Fig. 4.). S12, cell-free extract without protein enrichment, was used as a negative control. From Fig. 3., we could confirm the successful expression of the protein AtzR (35kDa) in the cell-free extracts.  

Fig.3. The result of SDS-PAGE (AtzR)

Fig.4. Abbreviation of the samples of SDS-PAGE

We tested the detection of cyanuric acid in the cell-free reaction, using the AtzR-expressing 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 Atz reporter plasmid (BBa_K4158004) 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.  

Fig.5. The temporal change of the fluorescent values

Fig.6.The comparison of the fluorescence values after 180 min

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
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 415
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 415
  Illegal NotI site found at 701
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 415
  Illegal BamHI site found at 151
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 415
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 415
  
• 1000
  COMPATIBLE WITH RFC[1000]