Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Iskakova, Madina; Szaflarski, Witold; Dreyfus, Marc; Rèmme, Jaanus; Nierhaus, Knud H.

doi:10.1093/nar/gkl462

Cited by 60 publications

(70 citation statements)

References 33 publications

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“…The protein synthesis rate observed over a few hours in batch mode reactions can be maintained a few days in continuous mode. This experiment demonstrated that the viability of the translation machinery in vitro is in the range of the doubling time of a cell-free reaction (30). For a cell-free reaction initially composed of 10 mg∕mL of proteins, the synthesis of 10 mg∕mL of proteins would take a few days.…”

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

confidence: 88%

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

Noireaux¹,

Maeda

Libchaber

2011

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

297

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: 88%

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

Noireaux¹,

Maeda

Libchaber

2011

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

297

267

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“…Wir nutzten dabei ein GFP-ReportergenKonstrukt, um mit T7-Polymerase mRNA in situ zu erzeugen, die durch Zell-Lysat aus E.-coli-Zellen direkt in detektierbares grün fluoreszierendes Protein (GFP) umgesetzt wurde. [26] Temperatur und zeitliche Abstimmung der gekoppelten Reaktionen wurden dabei auf ein stabil reproduzierbares Signals hin optimiert (siehe die Hintergrundinformationen).…”

Section: Professor Peter B Dervan Gewidmetunclassified

NMR‐Strukturen von Thiostrepton‐Derivaten zur Charakterisierung der ribosomalen Bindetasche

et al. 2011

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“…At the same time, the NTPs, energy substrates and amino acids in the reaction mixture are constantly replenished in the reaction compartment. Using this technique, the cell-free expression reaction can be maintained for tens of hours yielding to more than 10 milligrams of protein per milliliter of reaction [28].…”

Section: 1/ the Classical S30-based Cell-free Expression Systemmentioning

confidence: 99%

Cell-Free Synthesis of Macromolecular Complexes

Botte

Deniaud

Schaffitzel

2016

Advanced Technologies for Protein Complex Production and Characterization

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General rightsThis document is made available in accordance with publisher policies. Please cite only the published version using the reference above. Full terms of use are available: http://www.bristol.ac.uk/pure/about/ebr-terms ABSTRACTCell-free protein synthesis based on E. coli cell extracts has been described for the first time more than fifty years ago. To date, cell-free synthesis is widely used for the preparation of toxic proteins, for studies of the translation process and its regulation as well as for the incorporation of artificial or labelled amino acids into a polypeptide chain. Many efforts have been directed towards establishing cell-free expression as a standard method for gene expression, with limited success. In this chapter we will describe the state-of-theart of cell-free expression, extract preparation methods and recent examples for successful applications of cell-free synthesis of macromolecular complexes.3 1/ Introduction -Motivation and challengesThe capacity of cell extracts to synthesize proteins has been shown in the fifties of the last century [1,2], several years before the identification of ribosomes as proteinsynthesizing machines [3]. The cell-free extract was based on the classical S30 fraction obtained by a 30,000 x g centrifugation step at 4°C for 1 hour. Initially, endogenous mRNA was used for in vitro translation [4]. Subsequently, Nirenberg and Matthaei developed a protocol to degrade endogenous messenger RNA present in the cell extract and to add exogenous mRNA [5,6]. The first cell-free protein synthesis (CFPS) from DNA, using a socalled coupled transcription-translation system was developed in the late sixties by the group of Zubay [7]. They used their coupled transcription-translation system to study the regulation of gene expression by the E. coli lactose operon. Most cell-free extract preparation and in vitro translation protocols are based on this protocol [8,9].Significant improvements with respect to protein yields were achieved in the late eighties, in particular by the group of Spirin, which established the use of phage-specific RNA polymerases, SP6 [10] or T7 RNA polymerases [8]. Using these polymerases a high level of a specific mRNA during the in vitro transcription-translation reaction can be achieved and maintained. Importantly, the Spirin laboratory described the first 'continuous' in vitro translation system. It allows for a continuous exchange of small molecules between a 'feeding compartment' providing energy and substrates (amino acids) for the translation reaction and a 'reaction compartment' from which inhibitory reaction products are removed by dialysis [10,11]. In a continuous set-up, the in vitro translation reaction can continue for several hours or even days, compared to 40-60 minutes using the classical reaction set-up.This allows obtaining significantly increased yields: for instance 6 mg chloramphenicol Cell-free expression has thus several very attractive applications, related to the expression of toxic proteins, rapid production of small q...

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Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

Cited by 60 publications

References 33 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

NMR‐Strukturen von Thiostrepton‐Derivaten zur Charakterisierung der ribosomalen Bindetasche

Cell-Free Synthesis of Macromolecular Complexes

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