Difference between revisions of "Part:BBa K3205000"
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<partinfo>BBa_K3205000 short</partinfo>
 
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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 &#963;^54-type enhancer protein can binding 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 gene transcription. In this part, we express AreR with J23102 constituent promoter, regulated GFP expression with Pare. 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 or other designs requiring benzyl alcohol induction. You can also replace GFP with other genes for other signaling pathways that require benzyl alcohol induction.  
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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.
  
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===Usage and Biology===
 
===Usage and Biology===
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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 &#963;54-type enhancer protein can binding 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 gene transcription. In this part, we express AreR with J23102 constituent promoter, regulated GFP expression with Pare. 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 or other designs requiring benzyl alcohol induction. You can also replace GFP with other genes for other signaling pathways that require benzyl alcohol induction.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Functional Parameters===
 
===Functional Parameters===
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We transformed this part into DH5α and pick three monoclonals from the same plate as parallel repeat, 10ml LB medium containing 170ug/ml chloramphenicol was used and the seed culture were grown at 37°C while shaking at 200 RPM overnight. Dilute the seed culture to 10ml with LB(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 hybrid multi-mode reader(Synergy H1) 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 show the result of three parallel repeat experiments.
 
<partinfo>BBa_K3205000 parameters</partinfo>
 
<partinfo>BBa_K3205000 parameters</partinfo>
 
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Revision as of 05:45, 8 October 2019


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 binding 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 gene transcription. In this part, we express AreR with J23102 constituent promoter, regulated GFP expression with Pare. 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 or other designs requiring benzyl alcohol induction. 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 170ug/ml chloramphenicol was used and the seed culture were grown at 37°C while shaking at 200 RPM overnight. Dilute the seed culture to 10ml with LB(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 hybrid multi-mode reader(Synergy H1) 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 show the result of three parallel repeat experiments.