Difference between revisions of "Part:BBa K4767017"

Latest revision as of 08:50, 8 October 2023

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

Uses a factor P_luxⅠ(BBa_R0062) ,strong RBS(BBa_J34801), luxⅠ(luxI C0061) ,gfp coding region(BBa_E0040), P_tac(M31370), luxR(BBa_C0062), TT(BBa_B0015)and P_bad(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 P_BAD and IPTG-inducible promoter P_tac, respectively. We separately transformed P_luxI-luxR(△2-162)-gfp and P_BAD-luxI-P_tac-luxR-P_luxI-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 P_luxI promoter and activate its own transcription.

As the Figure shows, the cells containing P_BAD-luxI-P_tac-luxR-P_luxI-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 P_luxI 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.

@@ Line 3: / Line 3: @@
 <partinfo>BBa_K4767017 short</partinfo>
-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.
+Uses a factor P<sub><i>luxⅠ</i></sub>(BBa_R0062) ,strong RBS(BBa_J34801), <i>luxⅠ</i>(luxI C0061) ,<i>gfp</i> coding region(BBa_E0040), P<sub><i>tac</i></sub>(M31370), <i>luxR</i>(BBa_C0062), TT(BBa_B0015)and P<i><sub>bad</sub></i>(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-<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 .
+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 P<i><sub>BAD</sub></i> and IPTG-inducible promoter P<i><sub>tac</sub></i>, respectively. We separately transformed P<i><sub>luxI</sub></i>-<i>luxR</i>(△2-162)-<i>gfp</i> and P<i><sub>BAD</sub></i>-<i>luxI</i>-P<i><sub>tac</sub></i>-<i>luxR</i>-P<i><sub>luxI</sub></i>-<i>gfp</i> to the <i>S. oneidensis</i> MR-1 which doesn’t contain the <i>lux</i> 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 <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.
-<center>[[File:BBa_K4767017.jpg]]</center>
+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.
-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.
+<center>https://static.igem.wiki/teams/4767/wiki/part/img-1180.png</center>
+As the Figure shows, the cells containing P<i><sub>BAD</sub></i>-<i>luxI</i>-P<i><sub>tac</sub></i>-<i>luxR</i>-P<i><sub>luxI</sub></i>-<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 P<sub><i>luxI</i></sub> 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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