Designed genetic circuits



Synthetic biology is the study of genetically engineering new biological pathways that can potentially change the behavior of organisms in useful ways. There are many laboratories in the world which are designing more ambitious and mission critical synthetic biology projects. Synthetic biology has the potential to help us better understand how microorganisms function by allowing us to examine how a synthetic pathway behaves in vivo as compared with simulation [1]. There are also numerous exciting potential applications. The Gates foundation is funding research on the design of pathways for production of anti malarial drugs [2]. Scientists have also been working on modifying bacteria to metabolize toxic chemicals [3,4]. Finally, a number of labs are trying to design bacteria to hunt and kill tumors [5]. Since synthetic biology requires the construction of new, complex genetic networks, all of these efforts would benefit from better Genetic Design Automation (GDA) tools. Genetic networks have been studied theoretically [6-13] and experimentally for many years, usually by focusing on analysis of naturally occurring circuits. Genes and their related protein products can be connected into a variety of synthetic "genetic circuit" configurations. Genetic circuits are biological circuits constructed from DNA. An engineered genetic circuit permits the expression of different genes in response to intracellular conditions and the passage of time as specified by the circuit designer. The circuits are assembled from bits of DNA and inserted into foreign cells using common genetic engineering and DNA cloning techniques. Designed genetic circuits possessing prescheduled cellular function and dynamics enable us to analyze the behavior of systems that can be compared to natural gene networks. Implementation of these tailored circuits into living cells has promising implications on processes and productivity in the biotechnology industry and potentially in medicine. Generating a conceptual design of a synthetic circuit include a modular approach, as championed by the engineering community [14,15,16], and evolutionary design [17]. A conceptual design is defined as specifying how each and every component (e.g., sensors, regulatory factors, outputs) is connected to accomplish the desired circuit function. The next step is constructing the functional circuit. Surprisingly, there exist relatively few strategies for this process given the ever-increasing number of published synthetic circuits, suggesting that genetic circuit construction is currently more of an artistic form than a well-established engineering discipline.


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Studies of gene circuits (both experimental and theoretical) have similarities with many other areas of biological research - the principal aim is to understand the relationship between structure and function. For example, as it will be discussed below, patterns of regulation in elementary gene circuits can be understood in terms of the functional requirements for biosynthesis, anabolism and catabolism [18,19]. For a system, the important design features are those that can confer a great advantage in an ecological, sociological and scientific context [20,21]. This is in contrast to directed evolution and rational improvement of synthetic circuits, in which selection of features is an artifact of engineering.


In order to build synthetic circuits, one needs genetic components that should be well characterized, modular (that is, function similarly in different systems) and act independently of other cellular processes. Early synthetic biology experiments focused on transcriptional regulation components because they are relatively well understood and easy to reconfigure. For example, repressors were used to create feedback loops of various sizes in Escherichia coli in order to understand noise suppression [22], bistability [23,24] and oscillations [25] in circuits of one, two and three repressors, respectively. Similarly, synthetic cascades without feedback provided information on delays[26], noise propagation [27] and sensitivity [28]. Very recently, some of these transcriptional circuit designs have been created and analysed in mammalian systems using newly developed transcriptional regulators [29,30]. In one case, a synthetic bistable switch has been shown to operate in a mouse. It is clear that many natural circuits are fundamentally non-transcriptional. An amazing example is the cyanobacterial circadian clock, the operation of which depends on protein phosphorylation but can be independent of transcription and translation (in contrast to its counterpart in Drosophila) [31]. Thus, it is critical to appropriate other interaction mechanisms, such as protein modification, regulated degradation, and so on, for use in synthetic circuits.


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In electronic circuits, information is transferred through signals that exist in the form of voltages and/or currents. In biological systems, a signal may be a chemical quantity such as protein concentration. Gene expression and the resulting protein concentrations are considered as the output of a genetic circuit. The toggle switch, which designates each protein as either on or off, is an example of a digital circuit. A circuit that tracks and generates continuously varying protein concentrations, as in the feedback regulator, is an example of an analog circuit. A biochemical reaction may serve as a single device in a genetic circuit just as a resistor or capacitor acts as an elementary device in electronic networks. Electronic components must obey certain laws of physics. For example, resistors should obey Ohm's Law and are characterized by a parameter known as resistance (O). Likewise, chemical reactions must follow thermodynamic laws and are characterized by reaction rate constants (Kp/ Kc etc). Table I shows some contrasts between electronic and genetic circuits. Both types of circuits can be either analog or digital and both are inherently noisy, though genetic circuits have much poorer signal-to-noise ratios (SNRs) than typical electronic circuits [32]. The response time of a genetic circuit is too much slow, since it is limited by the rates of the biomolecular reactions involved. However, the ease with which these circuits are interfaced with living systems is an overwhelming advantage that compensates for the slow reaction rates.


Approaches for generating a conceptual design of a designed genetic circuit include a modular approach, as championed by the engineering community, and evolutionary design. A conceptual design is defined as specifying how individual components (e.g., sensors, regulatory factors, outputs) are connected to accomplish the desired circuit function. The next step is constructing the functional circuit. Interestingly, there exist relatively few strategies for this process given the ever-increasing number of published synthetic circuits, suggesting that genetic circuit construction is currently more of an art form than a well-established engineering discipline [33]. In this section, "plug and play" strategy, the "design, then mutate" strategy, and a hybrid of the two etc are shown diagrammatically. Several practical considerations currently limit the utility of this "plug and play" approach. First, design considerations often restrict the pool from which one can select components. In the toggle switch example, one may desire to maintain the same switching effectors (thermal and chemical) that Gardner et al. chose. Second, the function of each component is context dependent; different components may exhibit different interactions and/or sensitivities to inputs when taken out of their natural contexts. Finally, current libraries of well characterized components are sparse. For example, the MIT Parts registry currently reports only around 40 repressors. An alternative replacement of "plug and play" is "design, then mutate." Here, one a priori selects the components that comprise the designed circuit. Rather than swapping out different components, directed evolution is used to manipulate the behavior of these components, and the desired phenotype is obtained via screening or selection. The directed evolution is an appropriate tool for tuning circuits was recognized early in the emergence of the synthetic gene circuit discipline , and the utility of this approach has been demonstrated experimentally via random mutagenesis and screening for functional mutants. However, application of this strategy must be done carefully because the evolutionary search is restricted to the number of mutants that can be screened or selected at each generation, usually 103 to 108.

A circuit must contain (1) Inducer: activates gene expression (2) Repressor: represses gene expression. Mathematics has a great role in designing genetic circuit. An example is given here:-

Derivation of f(y):= Protein y binds gene G: rate constant f k, yG: bound complex.

Protein y unbinds from gene G: rate constant b k.

Equilibrium condition:

Rate of binding = Rate of unbinding, b k / f k = Kd (dissociation constant)

k [y][G] = b k [yG]

[G] = [Gtot] - [yG]

[yG] = [Gtot][y] / (Kd + [y])

Rate of protein synthesis:

f(y) = _ [y] / (Kd + [y])

dy/dt = f(y) - g(y)

Only one stable fixed point .How does one get bistability? So, the answer: Protein dimer regulates gene expression.


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Gardner et al. described a genetic switch, constructed on plasmids that toggled between stable transcriptions from either of two promoters in response to externally provided signals. The plasmid-based circuit was constructed from two promoters and their cognate repressors, arranged so that each promoter can be inhibited by the repressor transcribed by the other promoter [34]. The circuit has two stable states: the ``high'' state, with Plac ON and Ptet OFF so that TetR and GFP are made, and the ``low'' state, with Ptet ON and Plac OFF so that LacI is made. The fluorescent reporter protein GFP, made only in the high state, indicated the state of the system, and was assayed by flow cytometry. Six hour exposure to the Plac inducer IPTG switched the circuit from low to high. Once it is in the high state, the circuit remained in the high state for 30 hours in the absence of inducer. Exposure to the Ptet inducer a Tc switched the circuit back to the stable low state. Elowitz and Leibler constructed a plasmid-based genetic oscillator circuit, termed a ``repressilator'', from components similar to those used by Gardner et al. The repressilator design shown (Fig.5) had used three repressible promoters. Each promoter transcribed the repressor of one of the other promoters. This configuration produced oscillating levels of each repressor protein. A separate reporter plasmid, carrying GFP under the control of the Ptet promoter, responded to the oscillating TetR repressor levels, producing oscillating GFP levels which were measured in single cells by fluorescence microscopy. The period of oscillation was around 160 minutes, nearly 100 minutes longer than the cell cycle, and oscillations persisted for over 600 minutes, or 10 cell cycles. Oscillations stopped when the cells reached stationary phase, demonstrating another interesting deviation from predicted behavior. Used in this manner, simple, well understood circuits could provide a probe into the intracellular environment.


The dream is that well-characterized components can be easily assembled to generate novel genetic regulatory circuits. Synthetic genetic circuits will be useful both as practical devices and as tools for studying properties of genetic circuits. They could be made simpler and easier to model than naturally occurring circuits, facilitating experimental testing of models, as is commonly done in the physical sciences. The reality is that this is hard to accomplish. The components and their assemblies are context dependent: Synthetic circuits do not function outside the cellular context and may behave differently when the context changes. Directed evolution presents a powerful tool for overcoming this problem, but the evolutionary search space can be large. Mathematical modeling can reduce this space to a reasonable size by identifying mutational targets. The modular nature of synthetic gene circuits and the mathematical model permit this reduction. Screening of the circuit for the desired function is necessary. However, screening individual components for altered

function in simple circuits is a subtly different and powerful complementary approach. By evaluating circuit behavior for components with a well-characterized range of functionality, the nominal circuit can be systematically perturbed, permitting model validation and refinement. One question may arise: Is designed genetic circuit related work dangerous? Imaginable hazards associated with designed genetic circuit include:

(a) The accidental release of an unintentionally harmful organism or system

(b) The purposeful design and release of an intentionally harmful organism or system

(c) A future over-reliance on our ability to design and maintain engineered biological systems in an otherwise natural world.

In response to these concerns: (a) work is only done with Biosafety Level 1 organisms and components in approved research facilities, (b) Work should be carried out trained and responsible generation of biological engineers and scientists, (c) learning what is possible using simple test systems. The positive applications are that designed genetic circuits can be used to produce drugs, polyketides, isoprenoids, terpenoid etc. which can be used as anti cancer, anti malarial drugs and can serve a lot of good things for human beings. In my opinion, designed genetic circuits can be used as a tool of gene therapy for some severe diseases like cancer, leukemia, thalassemia etc, but it needs a good effort and sincerity to make it possible for coming future.


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