Biology Reference
In-Depth Information
oligonucleotide primers that have 5
0
termini sequence homology (at least 25 bp) to the
corresponding ends of the destination vector. In the case of SLIC and Gibson DNA assembly,
exonuclease activity is used to generate complementary overhangs on the target DNA
fragment and the linearized destination vector, which are then annealed in the absence
(SLIC) or presence (Gibson DNA assembly) of ligase. Another major difference between the
two techniques is that T5 exonuclease is utilized in the case of Gibson DNA assembly,
whereas SLIC utilizes T4 DNA polymerase which displays 3
0
exonuclease activity in the
absence of dNTPs.
51,52
By contrast, in the case of CPEC, no exonuclease activity is utilized to
generate overhangs, and neither are oligonucleotide primers utilized.
53
Instead, both the
target gene sequence and the linearized destination vector are melted into single strands and
subsequently annealed to each other, thus allowing both DNA fragments to prime each other
in the presence of Phusion polymerase.
53
TYPES OF SYNTHETIC GENE CIRCUITS
Overview
The application of engineering and computing principles in synthetic biology has helped develop
a diverse variety of synthetic gene circuits with a panoply of different functions. It is convenient to
compare the functioning of synthetic gene circuits with analogous electronic devices. These are
summarized in
Table 9.1
, and can be broadly classified into genetic switches,
5,54
67
oscillators,
6,54,68
72
filters,
73
79
communication modules,
80
83
and other miscellaneous
synthetic gene circuits such as various digital logic gates.
84
89
Each of these is discussed here in
turn.
Genetic Switches
In electronics, a switch is a device that allows conditional transition from one state to
another, in response to an input signal. Similarly, synthetic genetic switches are artificial
regulatory networks that allow cells to undergo conditional transition between gene
expression states. Although it may appear simple and straightforward to genetically engineer
cells to switch on/off gene expression in response to metabolic, physical, or cytokine-
induced stimuli, the challenge is to achieve a robust bistable transition from one state to
another without the tendency to flip randomly between states as a result of fluctuations
inherent in gene expression. This challenge may be overcome through the incorporation of
positive and negative feedback loops.
5,54
57
Construction of bistable genetic toggle switches
has been reported in bacteria,
5,54,55
as well as in yeast
56
and mammalian cells.
57
Subsequently, more complex genetic switches with tunable,
58
hysteresis,
59,60
and gating
61
functions have also been created. Besides control of transcription and translation, genetic
switches for epigenetic regulation have also been built.
62
162
Perhaps one of the most important applications of genetic switches in synthetic biology is
to function as cellular memory elements. Cellular memory can be defined as a protracted
response to previous exposure to a transient stimulus. The best example of cellular memory
in nature is cell fate decision-making during the process of differentiation, whereby a stem/
progenitor cell makes a permanent and irreversible decision to commit to a particular
somatic lineage. Memory elements of gene regulatory networks are characterized by two
major feedback motifs
mutual inhibition and autoregulatory positive feedback.
63
66
Examples of synthetic memory elements displaying mutual inhibition and autoregulatory
positive feedback include genetic toggle switches
5,55
57
and hysteresis networks,
59,60
respectively. Ajo-Franklin et al.
67
constructed a high-fidelity memory device in yeast based
on transcriptionally controlled autoregulatory positive feedback. This synthetic gene circuit
allowed cells to heritably retain an induced state after responding to a transient stimulus.
This was achieved by expression of an
transcription factor that binds to its
own promoter upon exposure to a transient stimulus. The result is that the
auto-feedback
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auto-feedback
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