Difference between revisions of "Part:BBa K3447107"
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This part contains a luxR gene, which constitutes the AHL sensing system. By connecting the sfGFP gene downstream, we were able to verify the expression intensity of this part. | This part contains a luxR gene, which constitutes the AHL sensing system. By connecting the sfGFP gene downstream, we were able to verify the expression intensity of this part. | ||
− | < | + | The combination of AHL with the luxR protein synthesized by the luxR gene in bacteria forms the Luxl-LuxR complex, which activates the strong expression of the Pr<i>lux</i> promoter and thus regulates the expression of a series of downstream genes.<br> |
+ | ===Characterization=== | ||
+ | LuxR can bind to AHL specifically, which is encoded by luxI, thereby activating the expression of genes after Pr<i>lux</i>. In our project, to verify the inducing effect of AHL, the digestion and agarose gel electrophoresis of luxI and luxR-Pr<i>lux</i>-sfGFP were performed by a standard protocol (Fig. 1A). | ||
+ | As shown in Fig. 1B, the fluorescence of the group with AHL standards shows an obvious dose-dependent manner at a series of AHL concentrations, and the fluorescence intensity induced by our AHL-containing bacteria supernatant was equivalent to that of 0.633 μmol/L standard AHL solution.<br> | ||
+ | [[Image: Fig. 1 AHL can induce the downstream expression.jpg|thumb|center|500px|<b>Fig. 1 AHL can induce the downstream expression.</b> (A) Digestion and electrophoresis of luxI and luxR-Pr<i>lux</i>-sfGFP. (B) Fluorescence intensity of sfGFP under different concentration of AHL. <i>E. coli</i> DH5α was transformed with the designed plasmid, cultured for 8 hrs, and diluted OD<i>600</i> to 0.1. Then the standards of gradient concentration and AHL-containing bacteria supernatant (which had been pre-cultured for 12 hrs) were added into the medium. The emission intensity at 528 nm was measured at the excitation wavelength of 485 nm. The same measurement has been done 12 hrs later. The experiment was performed three times in triplicate. ***, P < 0.001 from respective control using Student’s t test.]]<br> | ||
+ | ==<b>Design</b>== | ||
+ | ===Design Notes=== | ||
+ | We added some synonymous mutations to avoid part rules.<br> | ||
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+ | |||
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+ | ===Source=== | ||
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+ | We found this sequence data in GenBank.<br> | ||
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+ | ===References=== | ||
===Usage and Biology=== | ===Usage and Biology=== | ||
Revision as of 10:22, 27 October 2020
Validation of AHL response system
This part contains a luxR gene, which constitutes the AHL sensing system. By connecting the sfGFP gene downstream, we were able to verify the expression intensity of this part.
The combination of AHL with the luxR protein synthesized by the luxR gene in bacteria forms the Luxl-LuxR complex, which activates the strong expression of the Prlux promoter and thus regulates the expression of a series of downstream genes.
Characterization
LuxR can bind to AHL specifically, which is encoded by luxI, thereby activating the expression of genes after Prlux. In our project, to verify the inducing effect of AHL, the digestion and agarose gel electrophoresis of luxI and luxR-Prlux-sfGFP were performed by a standard protocol (Fig. 1A).
As shown in Fig. 1B, the fluorescence of the group with AHL standards shows an obvious dose-dependent manner at a series of AHL concentrations, and the fluorescence intensity induced by our AHL-containing bacteria supernatant was equivalent to that of 0.633 μmol/L standard AHL solution.
Design
Design Notes
We added some synonymous mutations to avoid part rules.
Source
We found this sequence data in GenBank.
References
Usage and Biology
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1489
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 985