Part:BBa_K3447107
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
Jilin_China 2021
Charaterization of Pasr promoter with fluorescence intensity measurement
This year, based on the previous part BBa_K3447107(Jilin_China,2020), we characterized this part again and added new documentation to it.
We obtained the fluorescence data by measuring the fluorescence level for 11 hours. As shown in the Fig1, after the induction of AHL, the response effect reached the maximum at about 5 hrs, and reached a plateau after 7 hrs from the beginning. The concentration of AHL in our supernatant were between that of 10 and 100 nM standard solutions.
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