Biology Reference
In-Depth Information
the metabolic flux towards producing the target compound. With synthetic biology tools,
in conjunction with systems metabolic engineering strategies, the engineering of
microorganisms can achieve a high level of efficiency and control to optimize the
production of the target compound.
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In this chapter, we shall explore the fundamentals
of computational synthetic biology, the tools and methods in predicting and developing
synthetic networks at the genetic and pathway levels, and finally discuss the necessity for
a systems-level analysis of the synthetic network to gauge the effect it has on the overall
phenotype of the host cell.
FUNDAMENTAL COMPONENTS OF SYNTHETIC BIOLOGY
Synthetic biology designs synthetic biological networks for the purpose of introducing
specified functions and new capabilities, such as genetic counters used to count the number
of arabinose pulses, and sensors for detecting boundaries between light and dark.
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17
Towards this goal, regulation of genes and their physiological functions have been identified
(e.g. riboswitch, promoter regulations, quorum sensing) and modified for various
applications.
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One such application is the programming of living cells with genetic
parts for strain design. To conceptualize the complex regulation of cellular functions, the
analogy to electronic circuits and computer programing has been widely used.
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17
This
comparison is fitting when observing the logic and decision nodes of gene expression and
control in response to various stimuli and perturbations. Genetic programming is
engineering of the genetic code to implement and configure de novo regulation of cellular
functions.
Boolean logic operators (e.g. NOT, AND, NAND, OR, NOR) are commonly employed
to simulate decision nodes of various biological functions, such as promoter regulation,
riboswitch, and quorum sensing (
Fig. 8.1
).
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Promoter regulation is the most
commonly engineered biological function in synthetic biology.
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Riboswitches are
another function and usually consist of two functional groups: an aptamer and an
expression platform.
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In riboswitches, the aptamer selectively binds to the target ligand
and the expression platform modulates the gene expression. A tandem riboswitch utilizes
two riboswitches to create NOR, AND, and NAND operators. The riboswitches are
influenced independently, and do not interact with each other despite the close proximity
of the active sites. Biological sensors capable of detecting variations in chemical or photon
concentrations and quorum sensing are also employed to send signals to the cell and
influence cellular functions and responses to stimuli.
17
In this section, design of synthetic
biological tools to function as counters, signaling systems to perform specific functions,
and oscillators are discussed.
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Genetic Counters
Genetic counters retain a memory of events or objects, each as a distinct state, and allow for
the accounting of tightly controlled states for the maintenance of metabolism and cellular
growth.
15
Two examples of a genetic counter developed for synthetic biology are the
riboregulated transcriptional cascade (RTC) counter and the DNA invertase cascade (DIC).
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The RTC counter utilizes a transcriptional cascade and has been employed in translational
regulation. Two types of the RTC genetic counter have been developed: the two-counter,
a counter that can count up to two; and the three-counter, a counter that can count up to
three. These RTC counters were designed to count the number of arabinose pulses (two or
three for the respective counters) experienced by the cell. The two-counter is driven by the
constitutive promoter P
Ltet0
1
leading to the expression of the T7 RNA polymerase (RNAP).
This RNAP then binds to the T7 promoter and transcribes the green fluorescent protein
(GFP) gene.
15
The expression for the T7 RNAP and the GFP genes are further regulated by
riboregulators (ribreg), which silence and activate the post-transcriptional expression of the
