Cellular logic with orthogonal ribosomes.
ABSTRACT The creation and use of unnatural molecules to control cellular function is a long standing goal of the chemical community, but in general, these efforts have been directed at finding molecules to inhibit or activate a particular molecular target or function, or to elicit a particular phenotype. Here we show that multiple unnatural molecules (orthogonal ribosomes) can be used combinatorially, in a single cell, to program Boolean logic functions. These experiments show how attention to the molecular specificity of noncovalent interactions between unnatural macromolecules allows the synthesis of complex function from the "bottom-up" in living matter.
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ABSTRACT: Genetic code expansion and reprogramming enable the site-specific incorporation of diverse designer amino acids into proteins produced in cells and animals. Recent advances are enhancing the efficiency of unnatural amino acid incorporation by creating and evolving orthogonal ribosomes and manipulating the genome. Increasing the number of distinct amino acids that can be site-specifically encoded has been facilitated by the evolution of orthogonal quadruplet decoding ribosomes and the discovery of mutually orthogonal synthetase/tRNA pairs. Rapid progress in moving genetic code expansion from bacteria to eukaryotic cells and animals (C. elegans and D. melanogaster) and the incorporation of useful unnatural amino acids has been aided by the development and application of the pyrrolysyl-transfer RNA (tRNA) synthetase/tRNA pair for unnatural amino acid incorporation. Combining chemoselective reactions with encoded amino acids has facilitated the installation of posttranslational modifications, as well as rapid derivatization with diverse fluorophores for imaging. Expected final online publication date for the Annual Review of Biochemistry Volume 83 is June 02, 2014. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.Annual review of biochemistry 02/2014; · 29.88 Impact Factor
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ABSTRACT: Biological computation is a major area of focus in synthetic biology because it has the potential to enable a wide range of applications. Synthetic biologists have applied engineering concepts to biological systems in order to construct progressively more complex gene circuits capable of processing information in living cells. Here, we review the current state of computational genetic circuits and describe artificial gene circuits that perform digital and analog computation. We then discuss recent progress in designing gene networks that exhibit memory, and how memory and computation have been integrated to yield more complex systems that can both process and record information. Finally, we suggest new directions for engineering biological circuits capable of computation.Current Opinion in Biotechnology 05/2014; 29C:146-155. · 8.04 Impact Factor
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ABSTRACT: Figure optionsDownload full-size imageDownload high-quality image (53 K)Download as PowerPoint slideCurrent Opinion in Biotechnology. 01/2014; 29:146–155.
Cellular Logic with Orthogonal Ribosomes
Oliver Rackham and Jason W. Chin*
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
Received August 5, 2005; E-mail: email@example.com
We recently described the evolution of several orthogonal
ribosome•orthogonal mRNA pairs (O-ribosome•O-mRNA pairs) in
Escherichia coli.1For each orthogonal pair, the O-ribosome does
not translate cellular mRNAs, and the O-mRNA is not a substrate
for the endogenous ribosome. The specificity of each O-ribosome
results from the incorporation of an orthogonal 16S ribosomal RNA
(O-rRNA) into the ribosome. We have defined the network of
molecular specificities of each O-ribosome, with respect to both
cognate and noncognate orthogonal ribosome binding sites on
mRNA, by considering each pairwise O-ribosome•O-mRNA in-
teraction in isolation. Pairs of O-ribosome•O-mRNA pairs have the
molecular specificities that define mutual orthogonality. For
example, O-ribosome-A translates its cognate O-mRNA-A, but not
the noncognate O-mRNA-C, and O-ribosome-C translates its
cognate O-mRNA-C, but not the noncognate O-mRNA-A. Simi-
larly, O-ribosome-B•O-mRNA-B and O-ribosome-C•O-mRNA-C
are mutually orthogonal (Figure 1). Here we show that subnetworks
of this network graph can be physically realized in a single cell
and allow combinatorial cellular programming of entirely post-
transcriptional Boolean logic functions.
The requirements for the realization of subnetworks are that
multiple distinct ribosome•mRNA pairs can be produced in a
single cell, and that these pairs function independently of other
ribosome•mRNA pairs in this context. The simultaneous expression
of multiple distinct mutant ribosomes in cells has not previously
been demonstrated. It requires the expression and processing of
two ribosomal RNAs from two compatible plasmids. It also requires
that ribosomal proteins are produced from the genome in sufficient
quantities to produce functional ribosomes containing wild-type
ribosomal RNA as well as two functional orthogonal ribosomes,
which each contain a distinct O-rRNA. This is particularly
challenging since excess rRNA may be degraded in vivo,2leading
to a depletion of both wild-type and orthogonal ribosomes.
Moreover, ribosome assembly is not entirely cooperative,3-5and
production of mutant rRNA in excess of the cell’s capacity to
synthesize ribosomal proteins might lead to partially assembled,
and therefore non-functional, wild-type and orthogonal ribosomes.
As a first step toward the simultaneous production of three
ribosomes in the cell (the wild-type ribosome and two O-ribosomes),
O-rRNAs were produced from plasmids of distinct compatibility
groups, and the resulting ribosomes were assayed for function. One
vector for rRNA production has a ColE1 origin of replication and
an ampicillin resistance gene and is present at about 50 copies per
cell. A second vector for rRNA production has an RSF origin of
replication and a kanamycin resistance gene and is present at about
100 copies per cell. We have previously observed that the
production of functional ribosomes incorporating plasmid-encoded
rRNA can be strongly modulated by the sequences flanking the
rRNA transcriptional cassette. To ascertain the effect of plasmid
flanking sequences and plasmid copy number on the activity of
the O-ribosomes incorporating plasmid-encoded rRNA, the transla-
tion of the chloramphenicol acetyl transferase gene (cat) from
O-mRNA-Ccat (a version of cat with the 5′ orthogonal ribosome
binding site C) was measured. Cells containing RSF or ColE1
plasmids encoding rRNA-C confer resistance to chlorampheni-
col, with IC50values of 250 and 150 µg mL-1, respectively, while
O-mRNA-Ccat has an IC50of 10 µg mL-1in the absence of cog-
nate ribosome. Similar results were obtained with other
O-ribosome•O-mRNA pairs. These results demonstrate that highly
active orthogonal ribosomes can be produced from two compatible
plasmids, and that the RSF plasmid leads to a slightly greater
ribosome activity than the ColE1 plasmid, as predicted based on
copy number alone.
To demonstrate that multiple O-ribosomes can be produced in a
single cell, and to begin to address the potential of O-ribosomes
for the expression of Boolean logic, an AND gate containing
O-mRNA sequences was designed. The gate is composed of two
O-mRNA sequences: O-mRNA-Aω directs the synthesis of the ω
fragment of ?-galactosidase, while O-mRNA-CR directs the
synthesis of the R fragment of ?-galactosidase. Upon synthesis and
assembly of both fragments into ?-galactosidase7((R + ω)4), cells
hydrolyze fluorescein di-?-D-galactopyranoside (FDG) to fluores-
cein (F),8which can be detected fluorimetrically (Figure 2a,b).
Cells containing a plasmid encoding both O-mRNA-CR and
O-mRNA-Aω were programmed with either wild-type rRNA,
rRNA-A, rRNA-C, or rRNA-C and rRNA-A together, and the
conversion of FDG to fluorescein was measured. Cells programmed
with wild-type rRNA produce low fluorescence, which is com-
parable to background. This confirms that the orthogonal ribosome
binding sites A and Csdeveloped on the cat genesare portable
and can confer orthogonality to a variety of genes. Cells pro-
Figure 1. Orthogonal ribosome•orthogonal mRNA pairs and their network
of specificities. (a) The sequence of rRNA that interacts with mRNA is
shown (wt is wild-type). Mutations in O-mRNAs and O-rRNAs are shown
in green and blue, respectively.6(b) Pairwise ribosome•mRNA interaction
strengths are indicated by grayscale intensity.
Published on Web 11/22/2005
17584 9 J. AM. CHEM. SOC. 2005, 127, 17584-17585
10.1021/ja055338d CCC: $30.25 © 2005 American Chemical Society
grammed with rRNA-A also produce low fluorescence, as do cells
programmed with rRNA-C. However, cells programmed with both
rRNA-A and rRNA-C give a fluorescent signal 20-fold greater than
that of other rRNA combinations. These data demonstrate that
multiple mutually orthogonal ribosomes can be functionally ex-
pressed in a single cell. Moreover, they show that rRNA-A and
rRNA-C can be used as inputs in a post-transcriptional AND
function. Similar AND functions were also obtained with cells
containing other mutually orthogonal ribosomes and their cognate
O-mRNARs and O-mRNAωs.
Next, we attempted to create a Boolean OR gate. The OR gate
is composed of two O-mRNAs (O-mRNA-AR and O-mRNA-CR),
each of which directs the synthesis of the R fragment of ?-galac-
tosidase (Figure 2c,d). In this system, the ω fragment is constitu-
tively produced from a wild-type ribosome binding site. Cells
programmed with wild-type ribosomes produce a fluorescence
comparable to that observed in the absence of plasmid-encoded R
fragment. Cells programmed with rRNA-A produce a fluorescence
signal 10-fold above background, while cells programmed with
rRNA-C produce a level of fluorescence 15-fold above background.
Cells programmed with both rRNA-C and rRNA-A give a
fluorescent signal more than 50-fold above background. The
increase in fluorescent signal indicates that in this system the ω
fragment is present in excess of the R fragment. When wild-type
ribosome binding sites are used to replace the orthogonal ribosome
binding sites on the mRNA, a similar result is observed. This
suggests that the mismatch in cellular concentration of the ω
fragment and R fragment results from a deficiency in either the
transcription or lifetime of the R fragment mRNA, or degradation
of the R fragment peptide. Overall, these results demonstrate that
rRNA-A and rRNA-C can be used as inputs in a Boolean OR
function. The OR function can also be created using other mutually
orthogonal rRNAs and cognate O-mRNAs.
In conclusion, we have demonstrated that O-ribosomes and
O-mRNAs can be used to create entirely post-transcriptional
combinatorial logic in living cells. The Boolean gates described
require multiple distinct orthogonal ribosomes as inputs and could
not be assembled using the wild-type ribosome since its removal
from the cell is lethal, precluding a value of zero for its input. Our
results begin to demonstrate how unnatural, orthogonal, modular
components and a knowledge of the noncovalent interactions
between components9may be used to synthesize unnatural network
architectures and logical functions in living matter.10Extensions
of our approach may ultimately allow the synthesis of cellular
computers in which signals are carried and specified not by electrical
wires, but rather by molecules with unnatural specificities.11
Supporting Information Available: Experimental procedures and
details of materials. This material is available free of charge via the
Internet at http://pubs.acs.org.
(1) Rackham, O.; Chin, J. W. Nat. Chem. Biol. 2005, 1, 159-166.
(2) Yamagishi, M.; Nomura, M. J. Bacteriol. 1988, 170, 5042-5050.
(3) Stern, S.; Powers, T.; Changchien, L. M.; Noller, H. F. Science 1989,
(4) Recht, M. I.; Williamson, J. R. J. Mol. Biol. 2004, 344, 395-407.
(5) Dodd, J.; Kolb, J. M.; Nomura, M. Biochimie 1991, 73, 757-767.
(6) O-rRNA-A, O-rRNA-B, and O-rRNA-C correspond to O-rRNA-2,
O-rRNA-8, and O-rRNA-9, respectively, in (1).
(7) Ullmann, A.; Jacob, F.; Monod, J. J. Mol. Biol. 1967, 24, 339-343.
(8) Rotman, B.; Zderic, J. A.; Edelstein, M. Proc. Natl. Acad. Sci U.S.A. 1963,
(9) Isaacs, F. J.; Dwyer, D. J.; Ding, C.; Pervouchine, D. D.; Cantor, C. R.;
Collins, J. J. Nat. Biotechnol. 2004, 22, 841-847.
(10) Ashkenasy, G.; Ghadiri, M. R. J. Am. Chem. Soc. 2004, 126, 11140-
(11) Amos, M.; Owenson, G. Cellular Computing; Oxford University Press:
Figure 2. Combinatorial logic with orthogonal ribosomes. (a) The fluorescence generated as a function of ribosome inputs for the AND gate. Fluorescence
is normalized for cell density and time of incubation, as detailed in the Supporting Information. Error bars represent the standard error of at least three
independent trials. (b) Each state of the AND gate. Black lines indicate functional connections, while gray lines indicate components that are insulated from
each other. (c and d) As for (a) and (b), but for the OR gate.
C O M M U N I C A T I O N S
J. AM. CHEM. SOC. 9 VOL. 127, NO. 50, 2005 17585