Biology Reference
In-Depth Information
presence of the aptamer
s cognate ligand. This was rationally designed by creating a linker
between the aptamer and the pT181 regulator that would interfere with pT181 if the
aptamer was not folded properly around theophylline. Since in vitro aptamer selection
through SELEX is a general method for engineering aptamers that bind specific ligands, this
strategy is a versatile method for creating sensors that attenuate transcription of desired
genes. If the use of standard platforms for design of allosteric regulators of orthogonal
expression controllers is successful, then expression circuits may be composed of a set of
homogeneously acting, physically similar elements with simplified models of function. This
is a critical step to permitting effective abstraction of circuit design from the underlying
biophysical descriptions of the network. Such abstraction has been argued to be a significant
driver in the success of other engineering disciplines.
5
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Translation
Once mRNA is produced during transcription, it then proceeds to template the synthesis of
protein. Here, another level of regulation, translational control, may occur. In bacteria,
translational control involves the ribosome binding site (RBS), which if weak or blocked
will prevent protein expression but if strong and unblocked will allow protein expression.
Therefore, RBS-based engineering of translational regulators has become a highly developed
field in prokaryotic synthetic biology.
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At the simplest level, translation initiation is modulated by RBSs of different strength.
Like constitutive promoters, engineered RBSs of defined strengths offer the ability to test
different levels of a gene
s expression, which is particularly useful in the optimization of
expression levels in multigene pathways. For example, Salis et al. recently developed a
biophysical model to capture the idiosyncrasies of the interactions between the RBS, the
5
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-UTR) that contain it, and the downstream gene sequence to
generate specified initiation strengths.
10,36
Using more than 100 predictions coupled with
experimental validation, the authors elegantly show how to design, in silico, RBS sequences
that hit the desired level of expression in
E. coli
. Nonetheless, the authors acknowledge that
the RBS strength calculation model developed has only a
untranslated regions (5
'
'
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47% of predicting an RBS that
achieves protein expression within a two-fold range of the target level. Still, this provides a
good starting point for RBS tuning and the important step of mapping sequence to activity.
B
As with constitutive promoters, the ability to hit particular expression values does not by
itself provide a way to link the expression of multiple genes to form regulatory circuits.
For this, RBSs that are controlled by
trans
elements, acting as
are necessary. It has
recently become clear that the ability to exploit basepairing rules in RNA design makes the
construction of large classes of
trans
regulator RNA parts feasible.
'
wires,
'
For example, in a seminal study, Isaacs et al. showed how to take advantage of RNA
basepairing to engineer artificial riboregulators that activate translation in vivo.
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To do this,
the authors exploit a system where a hairpin structure containing the RBS is used to prevent
translational initiation. However, activation of gene expression can be achieved by
expressing a
trans
-regulatory RNA that hybridizes with part of the hairpin to reveal the RBS
and trigger successful translation of the controlled gene. Empirical rules for RNA
hybridization were then used to design two orthogonal regulators, and a more recent study
from the same laboratory has shown how to generate up to four mutually orthogonal
regulators using an extension of this strategy.
38
In a related approach, Mutalik et al. used the IS10 system, wherein an antisense RNA
molecule hybridizes to the 5
-UTR of the target mRNA in order to sequester the RBS, to create
multiple mutually orthogonal variants of the IS10 antisense repression system.
39
To do this,
the authors first created a series of 23 mutant IS10 pairs rationally designed through varying
base pairs in key regions. They then characterized the repression profile of all 529 interactions
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