In vitro genetic reconstruction of bacterial transcription initiation by coupled synthesis and detection of RNA polymerase holoenzyme. Nucleic Acids Res

New England Biolabs Inc., 240 County Road, Ipswich, MA 01938, USA.
Nucleic Acids Research (Impact Factor: 9.11). 05/2010; 38(13):e141. DOI: 10.1093/nar/gkq377
Source: PubMed


In vitro reconstitution of a biological complex or process normally involves assembly of multiple individually purified protein components. Here we present a strategy that couples expression and assembly of multiple gene products with functional detection in an in vitro reconstituted protein synthesis system. The strategy potentially allows experimental reconstruction of a multi-component biological complex or process using only DNA templates instead of purified proteins. We applied this strategy to bacterial transcription initiation by co-expressing genes encoding Escherichia coli RNA polymerase subunits and sigma factors in the reconstituted protein synthesis system and by coupling the synthesis and assembly of a functional RNA polymerase holoenzyme with the expression of a reporter gene. Using such a system, we demonstrated sigma-factor-dependent, promoter-specific transcription initiation. Since protein synthesis, complex formation and enzyme catalysis occur in the same in vitro reaction mixture, this reconstruction process resembles natural biosynthetic pathways and avoids time-consuming expression and purification of individual proteins. The strategy can significantly reduce the time normally required by conventional reconstitution methods, allow rapid generation and detection of genetic mutations, and provide an open and designable platform for in vitro study and intervention of complex biological processes.

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    • "Cell-free protein expression technology combines these three synthetic approaches. Artificial genetic circuits have been synthesized by cell-free protein expression (8,9), the reconstitution approach resulted in the creation of a minimum protein expression system by purified elements (PURE system) (6) and several biological subsystems have been reconstituted using the PURE system (10,11). However, these attempts have not been successful in reconstructing live cells from defined factors, and many problems remain to be solved, particularly the integration of these studies. "
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    ABSTRACT: Replication of all living cells relies on the multirounds flow of the central dogma. Especially, expression of DNA replication proteins is a key step to circulate the processes of the central dogma. Here we achieved the entire sequential transcription–translation–replication process by autonomous expression of chromosomal DNA replication machineries from a reconstituted transcription–translation system (PURE system). We found that low temperature is essential to express a complex protein, DNA polymerase III, in a single tube using the PURE system. Addition of the 13 genes, encoding initiator, DNA helicase, helicase loader, RNA primase and DNA polymerase III to the PURE system gave rise to a DNA replication system by a coupling manner. An artificial genetic circuit demonstrated that the DNA produced as a result of the replication is able to provide genetic information for proteins, indicating the in vitro central dogma can sequentially undergo two rounds.
    Nucleic Acids Research 06/2013; 41(14). DOI:10.1093/nar/gkt489 · 9.11 Impact Factor
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    • "Affinity purification has become a technique of choice to purify complexes involving one particular protein whose interaction partners are unknown. The latter technique was in particular used to study the mechanism of ordered protein assembly until reconstituting a functional structure in vitro [9]; obtaining such fully active molecular machines has since then been described to be feasible in a cell-free protein synthesis system [10]. "
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    ABSTRACT: The components that enable cells and organisms to fulfill a plethora of chemical and physical reactions, including their ability to metabolize, replicate, repair and communicate with their environment are mostly based on the functioning of highly complex cellular machines which are to a large extent composed of proteins. With the development of MS techniques compatible with the analysis of minute amounts of biological material, it has become more and more feasible to dissect the composition and modification of these protein machineries. Indeed, new purification methods of protein complexes followed by MS analysis together with the genomic sequencing of various organisms - and in particular of crop species - now provide unforeseen insight to understand biological processes at a molecular level. We here review the current state of the art of in vivo protein complex isolation and their MS-based analytical characterization, emphasizing on the tandem affinity purification approach.
    Proteomics 05/2011; 11(9):1824-33. DOI:10.1002/pmic.201000635 · 3.81 Impact Factor
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    • "Lipid synthesis is particularly relevant, and together with phospholipid synthesis, fatty acid synthesis should be considered (for a preliminary report, see Murtas 2009). The study on the cell-free synthesis of transcription factors (Asahara and Chong, 2010), and on a short biosynthetic pathway (UDP-N-acetylglucosamine pathway, by Zhou et al., 2010), point toward the realization of more complex systems by the in vitro gene expression approach. Another interesting direction has been pioneered by Davis and coworkers, who let synthetic cells send a chemical message (ribose-borate complex, synthesized inside the synthetic cell via the formose reaction) to a bacteria population, stimulating a quorum sensing response (Gardner et al., 2009). "
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    ABSTRACT: The recent advent, success and diffusion of synthetic biology (SB) are mainly related to its application as markedly bioengineering-oriented discipline. In addition to this classical view, SB also means "constructive" biology, and it is aimed to the construction of synthetic (artificial, man-made) biological-like systems, at the aim of understanding basic concepts of living systems and of their parts. In the last years, we have investigated lipid vesicles (liposomes) as cell models, by studying different aspects of their general reactivity, from their self-reproduction to the hosting of simple and complex biochemical reactions. In the attempt of modeling simple autopoietic systems by vesicle populations, it was firstly shown that simple vesicles may grow and divide according to physical laws, also revealing an unexpected pattern recognized as a "matrix effect", consisting in the conservation of the average size in a population of self-reproducing vesicles. Semi-synthetic minimal cells, on the other hand, are defined as liposome-based synthetic cells that contain the minimal and sufficient number of macromolecular components in order to be defined as "alive". Clearly, the design and the construction of minimal living cells require the establishment of the minimal number of life criteria. These have been generally described as self-maintenance, self-reproduction and evolution capability. The current experimental approach to semi-synthetic minimal living cells exploits the combination between cell-free protein expression and liposome technology, and it is conceptually based on autopoietic theory. In the FP6 SYNTHCELL project, we have investigated the expression of functional proteins inside lipid vesicles by using a minimal set of enzymes, t-RNAs and ribosomes (PURESYSTEM) at the aim of constructing functional cell models. In this contribution, we will discuss recent experimental advancements in the field of synthetic cell constructions, giving emphasis to their relevance in synthetic biology, self-organization and biocomplexity, and in origins of life studies.
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