Biology Reference
In-Depth Information
Membrane components generally serve as functional receptors for extracellular molecular or
physical signals such as metabolites, cytokines, light, and electrical pulses. This will be
discussed in greater detail in the next section. Commonly utilized membrane protein
receptors in synthetic biology belong to two major families
the G-protein coupled
receptors (GPCR)
90
and the tyrosine kinase family.
91
Intracellular components can function to relay signals from activated membrane receptors
and influence gene expression at the transcriptional or translational level. Very often,
intracellular components are already present endogenously within the cells, and need not be
encoded by the synthetic gene circuit; such as the G-proteins associated with the GPCR.
Nevertheless, the transcriptional control element of the synthetic gene circuit must be
responsive to secondary messengers (Ca
2
1
influx, cAMP) activated by the transgenically
expressed membrane receptors. Among the more common transcriptional control elements
utilized in synthetic biology are NFAT,
92
which responds to Ca
2
1
influx, and CRE,
93
which
responds to elevated cAMP levels. Alternatively, the intracellular components encoded by
the synthetic gene circuits may respond directly to small molecules that diffuse into the cells
and activate or repress gene expression. Perhaps the best-known example is the tetracycline
tetO-TetR system developed for mammalian cells.
94
In recent years, there has also been increased interest in synthetic gene circuits that code for
functional RNA molecules.
95
Besides being short-lived transmitters of information from
DNA to proteins, RNA molecules can potentially serve as functional molecules in their own
right, in a manner similar to proteins. Examples of the functionality of RNA molecules are
their roles as short interfering RNA (siRNA) and microRNA in RNA interference,
96
and in
directly binding specific ligands to influence gene expression; that is, RNA aptamer domains
of riboswitches.
97,98
Although either microRNA or siRNA mediates RNA interference in
nature, the expression of short hairpin RNA (shRNA) is preferable for synthetic biology
applications. The reason is that shRNA offers silencing longevity, better delivery options,
and lower costs compared with siRNA or microRNA.
99
The cellular machinery cleaves the
shRNA hairpin structure into siRNA, which then binds to the RNA-induced silencing
complex (RISC) to effect gene silencing.
99
165
RNA structural motifs that can bind to specific ligands (aptamers) have also aroused much
interest in the field of synthetic biology.
95
Of particular interest are riboswitches,
98
which
are
cis
-acting structural motifs within the untranslated portion of an mRNA molecule, and
consist of an aptamer domain linked to an expression platform. Upon binding to its
specific ligand, the aptamer domain effects a structural change in the expression platform,
which in turn affects mRNA transcription, translation, and splicing.
98
A ribozyme is an
RNA molecule with a well-defined tertiary structure that enables it to act like a protein
enzyme in catalyzing biochemical and metabolic reactions within a cell. Because some
ribozymes, such as the hammerhead ribozyme and the hairpin ribozyme, cleave RNA,
there has been increasing interest in utilizing them for antiretroviral therapy.
100,101
An
intriguing possibility is to conjugate ligand-specific aptamer domains to ribozymes to
effect their catalytic function. Indeed, this has already been achieved with hammerhead
ribozymes.
102,103
INDUCTION OF SYNTHETIC GENE CIRCUITS
Synthetic gene circuits can be engineered to be responsive to a diverse array of inductive
stimuli, such as antibiotics,
104
106
orally ingestible food supplements such as vitamins,
amino acids and flavorings,
107
109
specific drugs and medications,
110,111
skin-lotion-
based chemicals that can penetrate the skin,
112,113
endogenous metabolites, hormones
and redox states,
114
116
gaseous chemicals (for example, involving smell),
117,118
and
physical stimuli such as light and electrical pulses.
119,120
These are summarized in
Table 9.2
.
