Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

Shin, Jinwoo; Noireaux, Vincent

doi:10.1186/1754-1611-4-8

Cited by 215 publications

(333 citation statements)

References 30 publications

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“…Compared to bacteriophage transcription, the structure of bacterium promoters allows for a much larger modularity of transcription, even with a single transcription factor. The recent development of an E. coli cell-free system driven by the endogenous RNA polymerase as efficient as conventional bacteriophage cell-free systems opens previously undescribed possibilities to develop DNA programs (41).…”

Section: Bottom-up Development Of An Artificial Cell: Broad Consideramentioning

confidence: 99%

Development of an artificial cell, from self-organization to computation and self-reproduction

Noireaux¹,

Maeda

Libchaber

2011

Proc. Natl. Acad. Sci. U.S.A.

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295

267

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This article describes the state and the development of an artificial cell project. We discuss the experimental constraints to synthesize the most elementary cell-sized compartment that can self-reproduce using synthetic genetic information. The original idea was to program a phospholipid vesicle with DNA. Based on this idea, it was shown that in vitro gene expression could be carried out inside cell-sized synthetic vesicles. It was also shown that a couple of genes could be expressed for a few days inside the vesicles once the exchanges of nutrients with the outside environment were adequately introduced. The development of a cell-free transcription/translation toolbox allows the expression of a large number of genes with multiple transcription factors. As a result, the development of a synthetic DNA program is becoming one of the main hurdles. We discuss the various possibilities to enrich and to replicate this program. Defining a program for self-reproduction remains a difficult question as nongenetic processes, such as molecular self-organization, play an essential and complementary role. The synthesis of a stable compartment with an active interface, one of the critical bottlenecks in the synthesis of artificial cell, depends on the properties of phospholipid membranes. The problem of a self-replicating artificial cell is a long-lasting goal that might imply evolution experiments.Defining Life Since Schrödinger's classic book What Is Life? (1), the definition of life has always been a puzzle with a variety of partial answers, none really satisfying. One answer is that life is a coded system with mutation and error correction (2). The information is encoded into DNA (the genotype) and the genetic code allows translating this information into proteins (the phenotype). The genetic code is universal, and the genome can support mutations, recombination, duplication, and partial transfer from organisms to organisms. Error correction limits the risk of melting the information into random sequences. This is fine, but life is also metabolism with a permanent absorption and transformation of nutrients from the environment (3). A constant flux of energy is needed to sustain the operation of this living dynamical system out of equilibrium. But life is also self-reproduction (4). Cells originate from preexisting cells by cell division. The DNA genome is replicated, the cell self-reproduces as well as the complete organism. Is self-reproduction a constraint of evolution present at each step and imposing severe design constraints or a possible definition of life? Or is life an emerging phenomenon resulting from complex chemical reactions, developing networks and cycles until, at a certain critical size of this network, life starts as an emerging property (5)? Within this approach, life is a dynamical system where signal to noise is just marginal; a living system positions itself at the edge of chaos. Finally the only generally accepted theme is that life is the result of evolution, natural selection, and adaptation (6), a...

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Section: Bottom-up Development Of An Artificial Cell: Broad Consideramentioning

confidence: 99%

Development of an artificial cell, from self-organization to computation and self-reproduction

Noireaux¹,

Maeda

Libchaber

2011

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

295

267

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“…The complete residue-specific incorporation of Can into a model protein at all Arg positions was recently reported 31 , using a cell-free expression system 32 . A slight modification of the same system enabled site-specific incorporation of different pyrrolysine analogs into a model protein via stop codon suppression 33 .…”

Section: Introductionmentioning

confidence: 99%

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an <em>Escherichia coli</em> Cell-free Transcription-translation System

Worst

Exner

Simone

et al. 2016

JoVE

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The canonical set of amino acids leads to an exceptionally wide range of protein functionality. Nevertheless, the set of residues still imposes limitations on potential protein applications. The incorporation of noncanonical amino acids can enlarge this scope. There are two complementary approaches for the incorporation of noncanonical amino acids. For site-specific incorporation, in addition to the endogenous canonical translational machineries, an orthogonal aminoacyl-tRNA-synthetase-tRNA pair must be provided that does not interact with the canonical ones. Consequently, a codon that is not assigned to a canonical amino acid, usually a stop codon, is also required. This genetic code expansion enables the incorporation of a noncanonical amino acid at a single, given site within the protein. The here presented work describes residue-specific incorporation where the genetic code is reassigned within the endogenous translational system. The translation machinery accepts the noncanonical amino acid as a surrogate to incorporate it at canonically prescribed locations, i.e., all occurrences of a canonical amino acid in the protein are replaced by the noncanonical one. The incorporation of noncanonical amino acids can change the protein structure, causing considerably modified physical and chemical properties. Noncanonical amino acid analogs often act as cell growth inhibitors for expression hosts since they modify endogenous proteins, limiting in vivo protein production. In vivo incorporation of toxic noncanonical amino acids into proteins remains particularly challenging. Here, a cell-free approach for a complete replacement of L-arginine by the noncanonical amino acid L-canavanine is presented. It circumvents the inherent difficulties of in vivo expression. Additionally, a protocol to prepare target proteins for mass spectral analysis is included. It is shown that L-lysine can be replaced by L-hydroxy-lysine, albeit with lower efficiency. In principle, any noncanonical amino acid analog can be incorporated using the presented method as long as the endogenous in vitro translation system recognizes it.

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“…Cell-free protein synthesis inside liposomes has first been performed using cellular extracts derived from cytoplasmic parts of Escherichia coli bacteria (Nomura et al 2003;Noireaux and Libchaber 2004). Despite many advantages, including reduced costs (Jewett and Forster 2010), high yield of protein expression (Caschera and Noireaux 2014) and the possibility to use the endogenous bacterial RNA polymerases (Shin and Noireaux 2010), crude extracts are poorly characterized mixtures containing plenty of undesired components that can interfere with the intended reactions. Alternatively, the protein synthesis using recombinant elements (PURE) system developed by the group of Takuya Ueda (Tokyo University) (Shimizu et al 2001(Shimizu et al , 2005, is a minimal gene expression system reconstituted solely from purified enzymes and cofactors (36 different proteins) for transcription, translation, tRNA aminoacylation, energy regeneration and pyrophosphate hydrolysis.…”

Section: Mimicking Cell Division In Its Most Simple Formmentioning

confidence: 99%

Toward the assembly of a minimal divisome

2014

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The construction of an irreducible minimal cell having all essential attributes of a living system is one of the biggest challenges facing synthetic biology. One ubiquitous task accomplished by any living systems is the division of the cell envelope. Hence, the assembly of an elementary, albeit sufficient, molecular machinery that supports compartment division, is a crucial step towards the realization of self-reproducing artificial cells. Looking backward to the molecular nature of possible ancestral, supposedly more rudimentary, cell division systems may help to identify a minimal divisome. In light of a possible evolutionary pathway of division mechanisms from simple lipid vesicles toward modern life, we define two approaches for recapitulating division in primitive cells: the membrane deforming protein route and the lipid biosynthesis route. Having identified possible proteins and working mechanisms participating in membrane shape alteration, we then discuss how they could be integrated into the construction framework of a programmable minimal cell relying on gene expression inside liposomes. The protein synthesis using recombinant elements (PURE) system, a reconstituted minimal gene expression system, is conceivably the most versatile synthesis platform. As a first step towards the de novo synthesis of a divisome, we showed that the N-BAR domain protein produced from its gene could assemble onto the outer surface of liposomes and sculpt the membrane into tubular structures. We finally discuss the remaining challenges for building up a self-reproducing minimal cell, in particular the coupling of the division machinery with volume expansion and genome replication.

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Efficient cell-free expression with the endogenous E. Coli RNA polymerase and sigma factor 70

Cited by 215 publications

References 30 publications

Development of an artificial cell, from self-organization to computation and self-reproduction

Development of an artificial cell, from self-organization to computation and self-reproduction

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an <em>Escherichia coli</em> Cell-free Transcription-translation System

Toward the assembly of a minimal divisome

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