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SOLUTION
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The goal of the project is to provide a proof of concept for the design and implementation of an ‘in vivo control system’ in E. coli: CTRL-E. This circuit is realized by assembling BioBrick parts with rational criteria, exploiting the information available for the basic modules (experimental data) to support a model-based approach.
The circuit implementing the negative-feedback loop control is designed with the purpose to keep constant over time the concentration of the cellular signalling molecule 3OC6-HSL (involved in Vibrio fisheri quorum sensing system), by regulating the expression of an enzyme that degrades it.
CTRL-E is composed by two elements: a LuxI (BBa_C0061, 3OC6-HSL synthetase) expression cassette driven by the aTc-inducible pTet promoter and an AiiA (BBa_C0060, autoinducer lactonase) expression cassette driven by the 3OC6-HSL-inducible pLux promoter.
In E.coli MGZ1 strain, singled out for the case study, pTet promoter is normally repressed, due to the presence of tetR gene integrated in its genome: TetR product is able to inhibit the activity of pTet, thereby the 3OC6-HSL production. This allows the modulation of pTet activity by using tetracycline or anhydrotetracyclin (aTc) as inducers. A variation in the inducer concentration in input permits to modify the set-point of the 3OC6-HSL production in output. When a critical amount of signal molecule is reached into the cells, the complex consisting of 3OC6-HSL and its transcriptional factor LuxR (constitutively expressed by pLambda promoter) is able to activate the pLux promoter, that regulates the expression of AiiA lactonase. So the HSL molecule regulates its own production via a negative feed-back loop system.
CTRL-E is composed by two elements: a LuxI (BBa_C0061, 3OC6-HSL synthetase) expression cassette driven by the aTc-inducible pTet promoter and an AiiA (BBa_C0060, autoinducer lactonase) expression cassette driven by the 3OC6-HSL-inducible pLux promoter.
In E.coli MGZ1 strain, singled out for the case study, pTet promoter is normally repressed, due to the presence of tetR gene integrated in its genome: TetR product is able to inhibit the activity of pTet, thereby the 3OC6-HSL production. This allows the modulation of pTet activity by using tetracycline or anhydrotetracyclin (aTc) as inducers. A variation in the inducer concentration in input permits to modify the set-point of the 3OC6-HSL production in output. When a critical amount of signal molecule is reached into the cells, the complex consisting of 3OC6-HSL and its transcriptional factor LuxR (constitutively expressed by pLambda promoter) is able to activate the pLux promoter, that regulates the expression of AiiA lactonase. So the HSL molecule regulates its own production via a negative feed-back loop system.
Circuit design
Text 2 here
Functional modules
Text 3 here
pTet
Text 4 here
pLux
Text 5 here
AiiA
Text 5 here
LuxI
Text 4 here
