Part:BBa_K3205000

A device for detection benzyl alcohol by producing GFP.

Pare is a AreR-regulated σ54-dependent promoter in Acinetobacter sp. Strain ADP1 and induced by benzyl alcohol. We built a device for feeling benzyl alcohol signal by fusing the promoter with gfp and verified the function in E. coli.

Usage and Biology

AreR is a member of the NtrC/XylR regulatory protein family, and controls the expression of the areCBA genes in Acinetobacter sp. Strain ADP1. This σ54-type enhancer protein can bind to the repeat sequence upstream of the Pare promoter after activated by benzyl alcohol. In the case of cofactor-induced DNA looping, a closed promoter complex will be formed with RNA polymerase to turn on the downstream genes transcription. In this part, we express AreR with J23102 constituent promoter, use Pare to regulate GFP expression. A significant difference in fluorescence intensity were observed under different concentrations of benzyl alcohol. This part can be used for the detection of benzyl alcohol. You can also replace gfp with other genes for other signaling pathways that require benzyl alcohol induction.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 66
  Illegal BglII site found at 712
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 673
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2772
  

Functional Parameters

We transformed this part into DH5α and pick three monoclonals from the same plate as parallel repeat, 10ml LB medium containing 170μg/mL chloramphenicol was used and the seed culture were grown at 37°C while shaking at 200 RPM overnight. Dilute the seed fluid to 10mL with LB at a rate of 1:100. Culture the cells as above describe until OD₆₀₀=0.3, then different concentrations of benzyl alcohol were added and each concentration set three parallel repeats. Shake the bacteria culture bottle to make the benzyl alcohol fully dissolved, add the sample to 96-well plate and cultivate in Synergy H1 hybrid multi-mode reader with 37°C, 180cpm. The OD₆₀₀ value and fluorescence value were determined every 30 minutes for 8 hours. Through data processing, the standard fluorescence value of each hole was obtained, and Figure 1. shows the results of three parallel repeat experiments.

Figure 1: Induced GFP with benzyl alcohol. A. Kinetic curves of fluorescence intensity induced by different concentrations of benzyl alcohol. B. Fluorescence intensity induced by benzyl alcohol of different concentrations for 8h. The error bars are obtained from three repeat experments.