Difference between revisions of "Part:BBa K3805138:Design"
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For this part, we design two validation experiments | 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. | ||
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| + | 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. | ||
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| + | 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. | ||
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| + | 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. | ||
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| + | [[File:KillingSignal.jpeg|500px|thumb|center|our pathway design]] | ||
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| + | ===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. | |
| − | + | [[File:Ourchanges.jpeg|500px|thumb|center|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 | |
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Latest revision as of 12:32, 21 October 2021
agrBD
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
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.