Part:BBa_K4593004
Characterization device for P2 promoter in E.coli
Usage and Biology
Quorum Sensing System (QS System) is a way that certain kinds of bacteria use to detect its population and accordingly regulate its gene expression. S. aureus produces a signal molecule, autoinducing peptide (AIPs), during its growth. AIPs are received by a membrane receptor called AgrC. AgrC then transfers a phosphate group to AgrA, which activates a downstream promoter P2 to express the Agr operon (including AgrBDCA). Simultaneously, the other promoter P3 is activated to express a virulence gene, releasing toxins as a result. When the population of S. aureus reaches a threshold level, the concentration of AIPs raises and AgrC is activated, switching on the whole pathway.
Team:BNDS-China 2023
This part is constructed to verify the competence of P2 promoter, that if it can be successfully induced by AIPs. A common strategy of characterization is applied in our experiment. To reveal that P2 promoter is working, a plasmid containing two pathways is constructed: One is a constructive pathway expressing AgrA and AgrC, activated by lacI promoter; the other is an inductive pathway expressing sfGFP, activated by P2 promoter. As hypothesized, if lactose exists in the environment, AgrA and AgrC are expressed and are able to receive signals from AIPs. Thus, if AIPs once present, fluorescence will be observed; on the contrary, no fluorescence will exist in absence of AIPs. If this is the case, P2 promoter will be capable in later experiments.
Construction of characterization plasmid
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 883
Illegal NheI site found at 906 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1294
Illegal BamHI site found at 2178 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 121
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