Difference between revisions of "Part:BBa K4593004"

 
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===Usage and Biology===
 
===Usage and Biology===
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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.
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==Team:BNDS-China 2023==
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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.
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===Construction of characterization plasmid===
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Revision as of 02:32, 10 October 2023


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 883
    Illegal NheI site found at 906
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1294
    Illegal BamHI site found at 2178
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 121