De novo design and synthesis of a 30-cistron translation-factor module

Shepherd, Tyson R.; Du, Liping; Liljeruhm, Josefine; Samudyata, S.; Wang, Jinfan; Sjödin, Marcus; Wetterhall, Magnus; Yomo, Tetsuya; Forster, Anthony

doi:10.1093/nar/gkx753

Cited by 30 publications

(44 citation statements)

References 52 publications

(66 reference statements)

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“…Encouraged by the efficient TTcDR in PURErep, we set out to coreplicate a collection of genes coding for crucial components of the PURE reaction such as the 31 essential E. coli translation factors (TFs). To this end, we probed co-TTcDR of pREP (4.6 kb) together with each one of the three large plasmids pLD1 (30 kb, 13 translation factors -TFs), pLD2 (20 kB, 8 TFs), or pLD3 (23 kb, 9 TFs), which were recently cloned to enable recombinant expression of 30 of the 31 TFs 16 . Indeed, the TTcDR products of all four plasmids (including pREP) showed identical MluI restriction patterns as clonal plasmids conventionally propagated in E. coli ( Fig.…”

Section: Purerep Enables Ttcdr Of Large Multipartite Genomesmentioning

confidence: 99%

“…To explore whether PURErep is generally capable of supporting multicistronic expression from these plasmids, we performed cell-free expression from each individual plasmid in presence of BODIPY-Lys-tRNA Lys , which enables the fluorescent labelling of translation products at lysine residue sites. Using the reported expression patterns for affinity-purified TF ensembles from pLD overexpression experiments 16 , we could assign the majority of the de novo synthesised protein subunits to the to the respective TFs ( Supplementary Fig. 5).…”

Section: Purerep Enables Ttcdr Of Large Multipartite Genomesmentioning

confidence: 99%

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In vitro self-replication and multicistronic expression of large synthetic genomes

et al. 2020

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The generation of a chemical system capable of replication and evolution is a key objective of synthetic biology. This could be achieved by in vitro reconstitution of a minimal selfsustaining central dogma consisting of DNA replication, transcription and translation. Here, we present an in vitro translation system, which enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions. In particular, we demonstrate self-replication of a multipartite genome of more than 116 kb encompassing the full set of Escherichia coli translation factors, all three ribosomal RNAs, an energy regeneration system, as well as RNA and DNA polymerases. Parallel to DNA replication, our system enables synthesis of at least 30 encoded translation factors, half of which are expressed in amounts equal to or greater than their respective input levels. Our optimized cell-free expression platform could provide a chassis for the generation of a partially self-replicating in vitro translation system.

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Section: Purerep Enables Ttcdr Of Large Multipartite Genomesmentioning

confidence: 99%

Section: Purerep Enables Ttcdr Of Large Multipartite Genomesmentioning

confidence: 99%

In vitro self-replication and multicistronic expression of large synthetic genomes

et al. 2020

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“…Cost-effective and modular PURE systems with user-defined compositions can be prepared in the laboratory (Shimizu and Ueda, 2010;Horiya et al, 2017), but the labor-intensive protocol requires ∼36 medium to large scale His-tag and ribosome purification steps (Figure 2A). Thus, different approaches to simplify the protocol have been developed, including Histagging of in vivo enzyme pathways (Wang et al, 2012), microbial consortia (Villarreal et al, 2018), and bacterial artificial chromosomes (Shepherd et al, 2017). The first two systems achieved a 10-20% protein yield compared to the commercial PURExpress (NEB).…”

Section: Recombinant Systemsmentioning

confidence: 99%

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Laohakunakorn

Grasemann

Lavickova

et al. 2020

Front. Bioeng. Biotechnol.

101

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Cell-free systems offer a promising approach to engineer biology since their open nature allows for well-controlled and characterized reaction conditions. In this review, we discuss the history and recent developments in engineering recombinant and crude extract systems, as well as breakthroughs in enabling technologies, that have facilitated increased throughput, compartmentalization, and spatial control of cell-free protein synthesis reactions. Combined with a deeper understanding of the cell-free systems themselves, these advances improve our ability to address a range of scientific questions. By mastering control of the cell-free platform, we will be in a position to construct increasingly complex biomolecular systems, and approach natural biological complexity in a bottom-up manner.

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“…[157] In recent years, Jewett and co-workers achieved a >1000-fold activity improvement in activity by tuning the rRNA transcript stoichiometry, [158] alleviating substrate dependencies, [159] and using additives to manipulate molecular crowding and oxidation levels. [161] In the future, the remaining components might be encoded on other large plasmids or TTcDR genomes. [160] While iSAT is an important step toward liberating TTcDRbased replicators from reliance on externally provided IVT systems, it does currently not include in situ synthesis of translation factors or tRNAs.…”

Section: Toward Full Autonomy: Combining In Vitro Ribosome Synthesis mentioning

confidence: 99%

Templated Self‐Replication in Biomimetic Systems

et al. 2019

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nanostructures and nanopatterns. [13][14][15] The specificity of the molecular recognitions between replicative units is concomitant with the encoding and transmission of information, with the degree of specificity determining the fidelty and capacity of information transfer against competing background interactions in noisy "chemical" environments. [16] However, the ability to replicate in this manner does not imply the ability to transmit additional heritable information-simple replicators can only replicate information related to recognition and template directed autocatalysis as this information is intrinsically linked to phenotype. In contrast to most artificial autocatalytic replicators, living systems (including viruses) store the key instructions for replication on an inheritable genetic system rather than in the native structures of the individual sub-components. This decoupling of phenotype and genotype enables heredity and open-ended evolution, the possibility of an indefinite increase in complexity, as evolutionary innovations aquired by natural selection are anchored in the genotype only. [17,18] The most prominent self-replicating molecules are nucleic acids, which form the fundamental basis for information storage and propagation in biological systems. The ability of polynucleic acids to store information is self-evident, and based upon the ability to form cognate Watson-Crick base pair interactions. Nucleic acid systems may have also formed the first genetic replicators in biology. The RNA world hypothesis postulates a stage in the origin of life in which RNA proliferated before the emergence of DNA and proteins. To do this, RNA must be able to carry out several key roles: information storage, catalysis, and (self-) replication. Multiple examples of catalytic RNAs (ribozymes) are known both from nature and in vitro selection experiments, [19][20][21][22] but there is no fossil evidence of an RNA capable of (self-)replication without the involvement of proteins.While minimal nucleic acid-only systems with replicationlike properties have been identified by directed evolution and engineering, limitations such as low activity, lack of generality, and poor information capacity limit their usefulness in bottomup synthetic biology, except in origin of life research. For the creation of more complex biological in vitro systems, RNA or DNA replication mechanisms involving more powerful protein enzymes are likely to be necessary. Even so, the achievement of self-sustained in vitro self-replication inspired by the central dogma of molecular biology is currently hampered by the requirement to encode and efficiently express the enormous A key characteristic of living systems is the storage and replication of information, and as such the development of self-replicating systems capable of heredity is of great importance to the fields of synthetic biology and origin of life research. In this review, the design and implementation of self-replicating systems in the context of bottom-up synthetic biology is discusse...

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De novo design and synthesis of a 30-cistron translation-factor module

Cited by 30 publications

References 52 publications

In vitro self-replication and multicistronic expression of large synthetic genomes

In vitro self-replication and multicistronic expression of large synthetic genomes

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

Templated Self‐Replication in Biomimetic Systems

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