Difference between revisions of "Part:BBa K3805138:Design"
 
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===experiment1：Toggle Switch Verification===
 
===experiment1：Toggle Switch Verification===
  
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To verify the Bi-stable Switch, we replaced our aimed protein gene with GFP sequence. When a certain amount of exogenous AIP was applied to the bacterial fluid, red fluorescence(mCherry) was observed under the microscope. We then applied a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and plotted the corresponding curve.
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To verify the Toggle Switch, we replaced our aimed protein gene with GFP sequence. When a certain amount of exogenous AIP was applied to the bacterial fluid, red fluorescence(mCherry) was observed under the microscope. We then applied a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and plotted the corresponding curve.
  
 
[[File:Ourchanges.jpeg|500px|thumb|center|we replaced our aimed protein gene with GFP sequence]]
 
[[File:Ourchanges.jpeg|500px|thumb|center|we replaced our aimed protein gene with GFP sequence]]
  
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===experiment2：Measurement of AIP generation===
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===experiment2：Verification of AIP generation===
  
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According to our pathway design , 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
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According to our pathway design , 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 verify the concentration of AIP, you need to measure the appearance of mCherry
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The experimental design was divided into two steps.
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Step 1: Obtaining standard data for AIP
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Add a specified amount of AIP solution to the guard's bacterial solution and measure the amount of mcherry
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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
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Latest revision as of 12:32, 21 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

The mechanics of our toggle switch

The guards are actually workers when no AIP is present through our elaborately designed toggle switch. On the cellular membrane of guard lies the AIP receptor AgrC. In the absence of AIP, the guard work as ordinary workers producing aimed protein with Ptrc-2 promoter. However, once bound to AIP, AgrC will phosphorylate AgrA, switching guards to killer mode. Phosphorylated AgrA will activate P2 promoter[1] who will initiate the expression of LacI. Afterwards, LacI will inhibit the Ptrc-2 promoter in return[3], eventually suppressing the synthesis of aimed protein.

In comparison of workers and cheaters, guards can’t bear the metabolic burden of synthesizing proteins and killing signal at the same time. However, with this toggle switch, guards can not only contribute to the production of aimed product in the absence of cheaters, but also be relieved from aimed product synthesize and focus on exterminating cheaters.

Activated by phosphorylated AgrA, P2 promoter also initiates the synthesis of Ompt-mCherry. Under the guidance of Ompt signal peptide, mCherry fluorescent proteins are secreted out of guards and served as the cheater-killing signal molecule of the platform.

As is mentioned above, mCherry binds specifically to the modified PmrB anti-mCherry that exists only on the cytomembrane of cheaters, therefore eventually induces the death of cheaters.


our pathway design

experiment1：Toggle Switch Verification

To verify the Toggle Switch, we replaced our aimed protein gene with GFP sequence. When a certain amount of exogenous AIP was applied to the bacterial fluid, red fluorescence(mCherry) was observed under the microscope. We then applied a concentration gradient of synthetic AIP to the bacterial fluid to establish an accurate coordination between AIP concentration and mCherry secretion and plotted the corresponding curve.

we replaced our aimed protein gene with GFP sequence

experiment2：Verification of AIP generation

According to our pathway design , 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 verify the concentration of AIP, you need to measure the appearance of mCherry


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.