Difference between revisions of "Part:BBa K4767012"

 
 
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<partinfo>BBa_K4767012 short</partinfo>
 
<partinfo>BBa_K4767012 short</partinfo>
  
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Uses a factor Pars(BBa_K4767001) ,strong RBS(BBa_J34801), DNA binding transcriptional repressor arsR(BBa_J15101), luxR(&#9651;2-162)(BBa_K4767000) TT(BBa_B0015) ,Plux&#8544;(BBa_R0062) and mtrC (BBa_K3102020).This composite part is used in the iron reduction experiment and MFC.
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Uses a factor P<i><sub>ars</sub></i>(BBa_K4767001) ,strong RBS(BBa_J34801), DNA binding transcriptional repressor <i>arsR</i>(BBa_J15101), <i>luxR</i>(△2-162)(BBa_K4767000) TT(BBa_B0015) ,P<i><sub>luxI</sub></i>(BBa_R0062) and <i>mtrC</i> (BBa_K3102020).This composite part is used in the iron reduction experiment and MFC.
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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In order to construct a positive feedback circuit which does not require 3OC6HSL produced by LuxI, we engineered LuxR by deleting 2-262 amino acids in the N-terminal domain (AHL binding domain) and reserving a C-terminal domain with the function of activating transcription, obtaining a resulting regulator LuxR(△2-162).  LuxR(△2-162) can active the gene transcription driven by the <i>lux</i> promoter in the absence of AHL. To construct the amplifier, we cloned <i>gfp</i> and LuxR(Δ2-162) behind the <i>lux</i> promoter. In this design, LuxR(Δ2-162) functions in a positive feedback loop as it can bind to the P<i><sub>luxI</sub></i>promoter and activate its own transcription.
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<center>https://static.igem.wiki/teams/4767/wiki/part/img-1176.png</center>
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After the modification of the LuxR(Δ2-162), we applied the amplifier into the arsenic-response system we build. The transcriptional activator, LuxR(Δ2-162), was used to replace <i>cymA</i> as reporter, and it was regulated by the <i>arsR</i>-P<i><sub>ars</sub></i> circuit, while <i>cymA</i> together with a second LuxR(Δ2-162) was placed under the promoter P<i><sub>luxI</sub></i>, which was activated by LuxR(Δ2-162). When arsenic is present, it activates the expression of LuxR(Δ2-162) in the first circuit, which turns on the expression of <i>cymA</i> and LuxR(Δ2-162) from the following circuit. The second LuxR(Δ2-162) activates its own expression as well as that of <i>cymA</i> and forms a positive feedback loop to enhance the output signal from <i>cymA</i>. These two parts work together as the arsenic EAS-based sensor with the positive feedback amplifier. Our results suggested that, compared with the sensor without positive feedback, the one with a positive feedback amplifier functions well in enhancing the iron reduction rates, increasing the detection range, and improving sensitivity. By introducing the positive feedback amplifier into the arsenic sensor, the output signal was enhanced so much that the specificity of the sensor toward arsenic was also significantly increased.
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<center>https://static.igem.wiki/teams/4767/wiki/part/img-1177.png</center>
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<center>Fig. Iron reduction rates in different arsenic levels. Plus sign refers to the strains with the amplifier.</center>
  
 
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<partinfo>BBa_K4767012 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4767012 SequenceAndFeatures</partinfo>
  
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===References===
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Xiaoqiang Jia, Bu Rongrong, Zhao Tingting, et al. Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection[J]. Applied and environmental microbiology, 2019, 85(11): 1.
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Nistala, G.J., et al., A modular positive feedback-based gene amplifier. J Biol Eng, 2010. 4: p. 4.
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 08:26, 8 October 2023


Pars-RBS-arsR-RBS-luxR(△2-162)-TT-PluxⅠ-RBS-luxR(△2-162)-RBS-mtrC -TT

Uses a factor Pars(BBa_K4767001) ,strong RBS(BBa_J34801), DNA binding transcriptional repressor arsR(BBa_J15101), luxR(△2-162)(BBa_K4767000) TT(BBa_B0015) ,PluxI(BBa_R0062) and mtrC (BBa_K3102020).This composite part is used in the iron reduction experiment and MFC.


Usage and Biology

In order to construct a positive feedback circuit which does not require 3OC6HSL produced by LuxI, we engineered LuxR by deleting 2-262 amino acids in the N-terminal domain (AHL binding domain) and reserving a C-terminal domain with the function of activating transcription, obtaining a resulting regulator LuxR(△2-162). LuxR(△2-162) can active the gene transcription driven by the lux promoter in the absence of AHL. To construct the amplifier, we cloned gfp and LuxR(Δ2-162) behind the lux promoter. In this design, LuxR(Δ2-162) functions in a positive feedback loop as it can bind to the PluxIpromoter and activate its own transcription.

img-1176.png

After the modification of the LuxR(Δ2-162), we applied the amplifier into the arsenic-response system we build. The transcriptional activator, LuxR(Δ2-162), was used to replace cymA as reporter, and it was regulated by the arsR-Pars circuit, while cymA together with a second LuxR(Δ2-162) was placed under the promoter PluxI, which was activated by LuxR(Δ2-162). When arsenic is present, it activates the expression of LuxR(Δ2-162) in the first circuit, which turns on the expression of cymA and LuxR(Δ2-162) from the following circuit. The second LuxR(Δ2-162) activates its own expression as well as that of cymA and forms a positive feedback loop to enhance the output signal from cymA. These two parts work together as the arsenic EAS-based sensor with the positive feedback amplifier. Our results suggested that, compared with the sensor without positive feedback, the one with a positive feedback amplifier functions well in enhancing the iron reduction rates, increasing the detection range, and improving sensitivity. By introducing the positive feedback amplifier into the arsenic sensor, the output signal was enhanced so much that the specificity of the sensor toward arsenic was also significantly increased.

img-1177.png
Fig. Iron reduction rates in different arsenic levels. Plus sign refers to the strains with the amplifier.

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 270
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1543
    Illegal BsaI.rc site found at 929

References

Xiaoqiang Jia, Bu Rongrong, Zhao Tingting, et al. Sensitive and Specific Whole-Cell Biosensor for Arsenic Detection[J]. Applied and environmental microbiology, 2019, 85(11): 1.

Nistala, G.J., et al., A modular positive feedback-based gene amplifier. J Biol Eng, 2010. 4: p. 4.