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.

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...

Recombinant in vitro translation systems can regenerate essential protein components that maintain protein synthesis during serial dilution.

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