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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 V. fischeri 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 (Cox RS 3rd et al. 2007), singled out for the case study, pTet promoter is normally repressed, due to the presence of tetR gene, from Z1 cassette, integrated in its genome (Lutz R et al. 1997): 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
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The circuit was built assembling aiiA protein generator and luxI translational unit with
The circuit was designed without a terminator element downstream the luxI coding sequence. The lack of a terminator doesn't affect the behaviour of our circuit, since a terminator (
In order to achieve the desired system output, a fine tuning of the whole circuit is required. A deeper understanding of the transcriptional and translational strength of the regulatory elements (promoter+RBS in several combination) and of the kinetics and the activity of the involved enzymes can be exploited to identify a mathematical model able to predict the behaviour of the controlled system, in order to avoid a cost and time expensive combinatorial approach.(Salis HM et al. 2009)
Functional modules
Four basic components of this circuit were identified as crucial to assess the desired circuit behaviour: the promoters pLux and pTet and the enzymes luxI and aiiA.
For each part, a simple measurement system was designed, built and tested, to gather more information about its functioning.
All modules were tested in E.coli MGZ1 strain.
More in detail, the promoters were tested with four different RBSs (RBSX stands for one of these BioBrick parts:
pTet
pLux
AiiA
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Note: Whilst in the final circuit aiiA expression is regulated by pLux promoter, in the measurement system it is driven by pTet promoter in order to avoid interference between inducer and gene product.
LuxI
References
- Cox RS 3rd, Surette MG, Elowitz MB (2007) Programming gene expression with combinatorial promoters. Mol. Syst. Biol. 3:145.
- Lutz R, Bujard H (1997) Independent and tight regulation of transcriptional units in Escherichia coli via the LacR/O, the TetR/O and AraC/I1-I2 regulatory elements. Nucleic Acids Res. 25(6):1203-10.
- Pasotti L, Quattrocelli M, Galli D et al. (2011) Multiplexing and demultiplexing logic functions for computing signal processing tasks in synthetic biology. Biotechnol. J. 6(7):784-95.
- Sleight SC, Bartley BA, Lieviant JA et al. (2010) Designing and engineering evolutionary robust genetic circuits. J. Biol. Eng. 4:12.
- Andersen JB, Sternberg C, Poulsen LK et al. (1998) New unstable variants of green fluorescent protein for studies of transient gene expression in bacteria. Appl. Environ. Microbiol. 64(6):2240-6.
- Salis HM, Mirsky EA, Voight CA (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol.27:946-950.
