Difference between revisions of "Part:BBa K4767017"

 
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<partinfo>BBa_K4767017 short</partinfo>
 
<partinfo>BBa_K4767017 short</partinfo>
  
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Uses a factor Plux&#8544;(BBa_R0062) ,strong RBS(BBa_J34801), lux&#8544;(luxI C0061) ,gfP coding region(BBa_E0040), Ptac(M31370), luxR(BBa_C0062)and Pbad(Ara K808000). This part is an easy BioBrick.
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Uses a factor PluxⅠ(BBa_R0062) ,strong RBS(BBa_J34801), luxⅠ(luxI C0061) ,gfP coding region(BBa_E0040), Ptac(M31370), luxR(BBa_C0062)and Pbad(Ara K808000). This part is an easy BioBrick.
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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This partprovided by Dr. Qiang Tang from University of Science and Technology of China, was used as the control. In this control gene circuit, the expression of LuxI for AHL production and LuxR are controlled by arabinose-inducible promoter PBAD and IPTG-inducible promoter Ptac, respectively. We separately transformed PluxI-<i>luxR</i>(△2-162)-<i>gfp</i> and PBAD-<i>luxI</i>-Ptac-<i>luxR</i>-PluxI-<i>gfp</i> to the <i>S. oneidensis</i> MR-1 which doesn’t contain the <i>lux</i> quorum sensing system .
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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 PluxI promoter and activate its own transcription.
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<center>[[File:BBa_K4767017.jpg]]</center>
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As the Figure shows, the cells containing PBAD-<i>luxI</i>-Ptac-<i>luxR</i>-PluxI-<i>gfp</i> were able to produce fluorescence only when both IPTG and arabinose were added to the culture. It indicated that IPTG-induced expression of LuxR only can active the <i>gfp</i> transcription from PluxI when arabinose-induced LuxI produces AHL. The LuxR(△2-162) group, however, drive the expression of downstream <i>gfp</i> without any inducers. The results suggest that <i>luxR</i>(△2-162) can drive downstream gene of the <i>lux</i> promoter constitutively.
  
 
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<partinfo>BBa_K4767017 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4767017 SequenceAndFeatures</partinfo>
  
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===References===
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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  

Revision as of 08:42, 7 October 2023


Pbad-RBS-luxI-TT-Ptac-luxR-TT-PluxI-gfp-TT

Uses a factor PluxⅠ(BBa_R0062) ,strong RBS(BBa_J34801), luxⅠ(luxI C0061) ,gfP coding region(BBa_E0040), Ptac(M31370), luxR(BBa_C0062)and Pbad(Ara K808000). This part is an easy BioBrick.


Usage and Biology

This partprovided by Dr. Qiang Tang from University of Science and Technology of China, was used as the control. In this control gene circuit, the expression of LuxI for AHL production and LuxR are controlled by arabinose-inducible promoter PBAD and IPTG-inducible promoter Ptac, respectively. We separately transformed PluxI-luxR(△2-162)-gfp and PBAD-luxI-Ptac-luxR-PluxI-gfp to the S. oneidensis MR-1 which doesn’t contain the lux quorum sensing system . 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 PluxI promoter and activate its own transcription.

File:BBa K4767017.jpg

As the Figure shows, the cells containing PBAD-luxI-Ptac-luxR-PluxI-gfp were able to produce fluorescence only when both IPTG and arabinose were added to the culture. It indicated that IPTG-induced expression of LuxR only can active the gfp transcription from PluxI when arabinose-induced LuxI produces AHL. The LuxR(△2-162) group, however, drive the expression of downstream gfp without any inducers. The results suggest that luxR(△2-162) can drive downstream gene of the lux promoter constitutively.

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 1872
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 979
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 3021
    Illegal BsaI.rc site found at 3728
    Illegal SapI site found at 961

References

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