Part:BBa_K4767017
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1872
Illegal BamHI site found at 1144 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 979
- 1000INCOMPATIBLE 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.
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