Difference between revisions of "Part:BBa K3599018"
 
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===Design & Experience===
 
===Design & Experience===
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In iGEM2020 TPR_China Project, an aromatic sensor was constructed using Pa promoter, the eGFP ([[Part:BBa_K3599016|BBa_K3599016]]) and the sfGFP ([[Part:BBa_K3599017|BBa_K3599017]]) were both used as downstream reporters to form the sensor devices. To sense the aromatic compounds, PaaX under [[Part:BBa_K1031744|J23114]] constitutive promoter was used together.
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In iGEM2020 TPR_China Project, an aromatic sensor was constructed using Pa promoter, the eGFP ([[Part:BBa_K3599016|BBa_K3599016]]) and the sfGFP ([[Part:BBa_K3599017|BBa_K3599017]]) were both used as downstream reporters to form the sensor devices. To sense the aromatic compounds, PaaX under [[Part:BBa_K1031744|J23114]] constitutive promoter was used together.<br>
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To characterize the original function of PaaX sensor device, we use 2-Hydroxyphenylacetic Acid (2HA) as the corresponding aromatic compond. According to the biological knowledge, PaaX could sense the intracellular concentrations of phenylacetyl-CoA, of which the chemical structure is semilar with 2HA.
  
 
====The fluorescence of the sensor induced by small molecules====
 
====The fluorescence of the sensor induced by small molecules====
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And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.
 
And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.
 
[[File:T--TPR China--Sensor3.jpg|600px|thumb|center|The fluorescence of the sensor induced by PAN]]<br>
 
[[File:T--TPR China--Sensor3.jpg|600px|thumb|center|The fluorescence of the sensor induced by PAN]]<br>
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===Reference===
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[1] Ferrández, A., Miñambres, B., Garcı́a, B., Olivera, E. R., Luengo, J. M., Garcı́a, J. L., & Dı́az, E. (1998). Catabolism of phenylacetic acid in Escherichia coli characterization of a new aerobic hybrid pathway. Journal of Biological Chemistry, 273(40), 25974-25986. [https://www.jbc.org/content/273/40/25974.short 10.1074/jbc.273.40.25974]<br>
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[2] Ferrández, A., Garcı́a, J. L., & Dı́az, E. (2000). Transcriptional Regulation of the Divergent paaCatabolic Operons for Phenylacetic Acid Degradation inEscherichia coli. <i>Journal of Biological Chemistry</i>, 275(16), 12214-12222. [https://www.jbc.org/content/275/16/12214.short 10.1074/jbc.275.16.12214]

Latest revision as of 15:39, 27 October 2020


Pa


Overview

An induced promoter which could be regulated by transcription factor PaaX (BBa_K1031710).


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]


Design & Experience

In iGEM2020 TPR_China Project, an aromatic sensor was constructed using Pa promoter, the eGFP (BBa_K3599016) and the sfGFP (BBa_K3599017) were both used as downstream reporters to form the sensor devices. To sense the aromatic compounds, PaaX under J23114 constitutive promoter was used together.

To characterize the original function of PaaX sensor device, we use 2-Hydroxyphenylacetic Acid (2HA) as the corresponding aromatic compond. According to the biological knowledge, PaaX could sense the intracellular concentrations of phenylacetyl-CoA, of which the chemical structure is semilar with 2HA.

The fluorescence of the sensor induced by small molecules

Through the corresponding small molecule sensing experiment, except Dmpr sensor, all our sensors have great induction effects on its corresponding aromatic small molecules. Among which NahR-sfGFP was the best! It is 63 times of the negative control.And here we can see that Xyls-sfGFP is 18 times of the Xyls-GFP( BBa_K1031911). And what surprises us is that Paax sensor also has a good response effect to small molecules, which has not been detected before.

The fluorescence of the sensor induced by small molecules

The fluorescence of the NahR sensor induced by small molecules

The fluorescence of the sensor induced by PAN

And here is the fluorescence of different sensors induced by PAN.This data shows the same result that NahR-sfGFP responded best.

The fluorescence of the sensor induced by PAN


Reference

[1] Ferrández, A., Miñambres, B., Garcı́a, B., Olivera, E. R., Luengo, J. M., Garcı́a, J. L., & Dı́az, E. (1998). Catabolism of phenylacetic acid in Escherichia coli characterization of a new aerobic hybrid pathway. Journal of Biological Chemistry, 273(40), 25974-25986. 10.1074/jbc.273.40.25974
[2] Ferrández, A., Garcı́a, J. L., & Dı́az, E. (2000). Transcriptional Regulation of the Divergent paaCatabolic Operons for Phenylacetic Acid Degradation inEscherichia coli. Journal of Biological Chemistry, 275(16), 12214-12222. 10.1074/jbc.275.16.12214