Difference between revisions of "Part:BBa K3805138:Design"
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===Design Notes===
 
===Design Notes===
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We chose to amplify the desired fragment from the Staphylococcus aureus genome, and since the agrB and agrD are very close together in this strain, we amplified this fragment according to a single element.
 
  
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According to our pathway design (below), specific binding of AIP to a protein on the guard cell membrane activates the activity of this membrane protein, AgrC, which drives AgrD phosphorylation and activates the P2 promoter, thereby driving the release of mcherry to the extracellular compartment. Therefore, if you want to test the concentration of AIP, you need to measure the amount of mcherry
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For this part, we design two validation experiments
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 +
===experiment1：Toggle Switch Verification===
 +
 
 +
 
 +
===experiment2：Measurement of AIP generation===
 +
 
 +
According to our pathway design (below), specific binding of AIP to a protein on the guard membrane activates the activity of this membrane protein, AgrC, which drives AgrD phosphorylation and activates the P2 promoter, thereby driving the release of mcherry to the extracellular compartment. Therefore, if you want to test the concentration of AIP, you need to measure the amount of mcherry
 +
 
 +
[[File:Pathway.jpeg|500px|thumb|center|our pathway design]]
  
 
The experimental design was divided into two steps.
 
The experimental design was divided into two steps.
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===References===
 
===References===
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[1] Novick RP, Geisinger E. Quorum sensing in staphylococci. Annu Rev Genet. 2008;42:541-64.
 +
[2] Liang H, Deng X, Bosscher M, Ji Q, Jensen MP, He C. Engineering bacterial two-component system PmrA/PmrB to sense lanthanide ions. J Am Chem Soc. 2013 Feb 13;135(6):2037-9.
 +
[3] Gardner TS, Cantor CR, Collins JJ. Construction of a genetic toggle switch in Escherichia coli. Nature. 2000 Jan 20;403(6767):339-42.

Revision as of 14:27, 20 October 2021


agrBD


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

For this part, we design two validation experiments

experiment1：Toggle Switch Verification

experiment2：Measurement of AIP generation

According to our pathway design (below), specific binding of AIP to a protein on the guard membrane activates the activity of this membrane protein, AgrC, which drives AgrD phosphorylation and activates the P2 promoter, thereby driving the release of mcherry to the extracellular compartment. Therefore, if you want to test the concentration of AIP, you need to measure the amount of mcherry

File:Pathway.jpeg
our pathway design

The experimental design was divided into two steps. Step 1: Obtaining standard data for AIP Add a specified amount of AIP solution to the guard's bacterial solution and measure the amount of mcherry Step 2: Determine the amount of mcherry in the deceiver and fit the actual process to the deceiver AIP yield based on the standard data


Source

This part contains the coding regions for group I agrB and agrD (in that order) from S. aureus strain NCTC4137.

References

[1] Novick RP, Geisinger E. Quorum sensing in staphylococci. Annu Rev Genet. 2008;42:541-64. [2] Liang H, Deng X, Bosscher M, Ji Q, Jensen MP, He C. Engineering bacterial two-component system PmrA/PmrB to sense lanthanide ions. J Am Chem Soc. 2013 Feb 13;135(6):2037-9. [3] Gardner TS, Cantor CR, Collins JJ. Construction of a genetic toggle switch in Escherichia coli. Nature. 2000 Jan 20;403(6767):339-42.