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Biochemical Society Transactions (2006) Volume 34, part 2
Synthesizing cellular networks from evolved
ribosome–mRNA pairs
O. Rackham and J.W. Chin1
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
Abstract
We describe cellular engineering for the creation of multiple new cellular modules each composed of an
orthogonal ribosome and orthogonal mRNA. These modules operate independently of the endogenous
ribosome and mRNA. We discuss some of the applications of orthogonal pairs and highlight the expression
of Boolean logic in gene regulation using multiple orthogonal pairs.
Orthogonal translation
The ribosome is a 2.5 MDa molecular machine that converts the information encoded in mRNA into protein [1].
In bacterial cells, the ribosome is most commonly directed to
mRNAs by sequences 5 to the mRNA start codon known
as a Shine–Dalgarno sequence or RBS (ribosome-binding
site) [2]. The interaction between the ribosome and the RBS
is mediated by base pairing between in the MBS (mRNAbinding sequence) in the 16 S rRNA and the RBS (Figure 1a)
[3].
We set out to create new mRNAs and ribosomes with
particular properties that differ from those of the endogenous
pair. We required an RBS that does not direct translation of
downstream genes by endogenous ribosomes. We describe
mRNAs containing RBS sequences with this property as
orthogonal mRNAs (O-mRNAs) [4]. We also required a
new ribosome (O-ribosome) that translates the O-mRNA,
but does not significantly translate any of the thousands of
cellular transcripts bearing cellular RBS sequences.
In the process of natural evolution, new function is believed
to have arisen from old by the duplication of existing sets
of genes and the alteration of the new copies to perform
new functions. We investigated whether we could achieve
an analogous effect on a laboratory timescale in two steps,
each of which filters a population of cells to provide those
containing the orthogonal molecules that we are interested in.
In the first step, we created a library of new potential RBSs by
creating all of the possible combinations of nucleotides at the
nucleotide positions of the existing RBS [4]. This library was
used to replace the RBS upstream of a novel gene fusion. The
gene fusion is between the chloramphenicol acetyltransferase
gene and a uracil phosphoribosyltransferase gene, which
is phenotypically silent under normal growth conditions.
However, it allows the selection for or against its own
expression, and for or against elements that control that expression, upon addition of chloramphenicol or 5-fluorouracil
Key words: directed evolution, gene regulatory circuit, genetic code, protein synthesis, ribosome–
mRNA pair, synthetic biology.
Abbreviations used: FDG, fluorescein di-β-d-galactopyranoside; MBS, mRNA-binding sequence;
O-mRNA, orthogonal mRNA; O-ribosome, orthogonal ribosome; RBS, ribosome-binding site.
1
To whom correspondence should be addressed (email [email protected]).
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Biochemical Society
respectively. By growing the library of RBS mutants on
5-fluorouracil, we were able to select O-mRNA sequences
that are not translated by the endogenous ribosome. The
selected O-mRNAs were combined with library ribosomes
containing mutant 16 S rRNAs and grown on chloramphenicol to find those mutant ribosomes that specifically translate
O-mRNAs. Finally, we were able to show that these ribosomes do not significantly translate cellular transcripts and
do not significantly alter the growth rate of the cells that
contain them. These ribosomes are therefore orthogonal (Oribosomes). Extensions of this approach will find applications
in the engineering of almost any molecular interaction that
can be linked to gene expression. The approach provides a
powerful route to the creation of new cellular modules with
prescribed connections to and insulation from other cellular
functions.
From the two-step selection, we isolated three distinct
types of orthogonal pairs (O-ribosome–O-mRNA pairs) that
differ in their predicted base pairing. We next investigated
the relationship between these pairs, and asked whether the
ribosome from one pair could translate genes placed downstream of the RBS of another pair. Since there are three ribosomes and three binding sites, there are 512 possible topologies for the network describing the interactions between
O-ribosomes and O-mRNAs. To determine the observed relationship between O-ribosomes and O-mRNAs, we created
all of the possible combinations and measured their translational efficiency. We find that O-ribosome-A translates its
cognate O-mRNA-A, but not the non-cognate O-mRNA-C,
and O-ribosome-C translates its cognate O-mRNA-C, but
not the non-cognate O-mRNA-A. Similarly, O-ribosome-B–
O-mRNA-B and O-ribosome-C–O-mRNA-C are mutually
orthogonal (Figure 1b) [4]. These experiments demonstrate
that we have created a group of translational modules that
have the molecular specificities that define independent
function.
The pairs created have several potential applications.
Since the endogenous ribosome is an essential cellular hub,
many mutations are lethal, limiting our ability to create
ribosomes to perform new or specialized functions. However,
the O-ribosomes are not essential and it should therefore
Recombinant DNA Technology for the 21st Century
Figure 1 O-ribosomes and Boolean logic
(a) The RBS–MBS interaction within the 70 S ribosome. The molecular
details are modelled from crystal structures of the Thermus thermophilus
ribosome (Protein Data Bank accession code 1YL4; image created
with PyMOL, http://www.pymol.org). (b) A network of O-ribosomes.
Pairwise ribosome–mRNA interaction strengths, based on IC50 values
for chloramphenicol resistance, are indicated by greyscale intensity. WT,
wild-type. (c) Schematic architecture of an AND logic circuit consisting of
O-ribosome–O-mRNA pairs. Black lines indicate functional connections,
while grey lines indicate components that are isolated from each other.
F, fluorescein.
be possible to diverge and specialize their function further.
This same capacity should make O-ribosomes a good tool for
interrogating structure–function relationships in rRNA.
Finally, the orthogonal system provides a route to a fundamentally new type of gene expression regulation, and this
application is discussed in more detail below.
Boolean logic with O-ribosomes
Transcriptional regulation of gene expression has been
investigated for many years, and biologists have borrowed
the language of logic and circuit design, from mathematics
and engineering, for its description [5]. Gene circuits are
of fundamental importance as they control the co-ordinated
development and regulation of cell and organism function.
The emerging area of synthetic biology seeks to create new
cellular function by building new gene circuits to perform
new functions. With notable exceptions [6,7], work in this
area has focused on creating function from the rearrangement
of existing transcription factors and their binding sites into
novel network topologies [5]. We have explored a fundamentally new way of designing logical regulation of gene
expression using O-ribosomes and O-mRNAs [8]. In one
example of this approach, an AND gate containing OmRNA sequences was designed. The gate is composed of two
O-mRNA sequences: O-mRNA-Aω encodes the ω fragment of β-galactosidase, while O-mRNA-Cα encodes the
α fragment of β-galactosidase. Upon synthesis and assembly
of both fragments into a complete β-galactosidase enzyme
[(α + ω)4 ], cells hydrolyse FDG (fluorescein di-β-D-galactopyranoside) to fluorescein (F), which can be detected fluorimetrically (Figure 1c). Cells containing a plasmid encoding
both O-mRNA-Cα and O-mRNA-Aω were programmed
with wild-type rRNA, rRNA-A or rRNA-C, or rRNAC and rRNA-A together, and the conversion of FDG into
fluorescein was measured. Cells programmed with wildtype rRNA produced background fluorescence, as did cells
programmed with rRNA-A or rRNA-C alone. However,
cells programmed with both rRNA-A and rRNA-C gave
a fluorescent signal 20-fold greater than that of other rRNA
combinations. Similar AND and OR functions have also been
constructed in cells containing other mutual O-ribosomes
and their cognate O-mRNAαs and/or O-mRNAωs. This
demonstrates that multiple mutual O-ribosomes can be
functionally expressed in a single cell, providing inputs in
a post-transcriptional logic function. Overall, our results
indicate how orthogonal molecules and a knowledge of the
non-covalent interactions between them may be used to
synthesize unnatural networks and logic functions in vivo.
Ribosomal regulation of gene expression will allow the
creation of even more complex gene circuits, and extensions
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.
References
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3 Yusupova, G.Z., Yusupov, M.M., Cate, J.H. and Noller, H.F. (2001) Cell 106,
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4 Rackham, O. and Chin, J.W. (2005) Nat. Chem. Biol. 1, 159–166
5 Sprinzak, D. and Elowitz, M.B. (2005) Nature (London) 438, 443–448
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Received 16 January 2006
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