In a very short amount of time, the field of synthetic biology has exploded to become one of the most exciting areas of research in the past several decades. The creation of new DNA bases and de novo organisms are some of the exciting developments we have witnessed over the past several years. Moreover, the field of synthetic biology does not only observe and describe processes of life but also mimics them. A key characteristic of life is the ability to replicate, which means the maintenance of a chemical system. Now, investigators at the Max Planck Institute of Biochemistry generated such a system—able to regenerate parts of its own DNA and protein building blocks.

The findings from the new study were published recently in Nature Communications through an article titled “In vitro self-replication and multicistronic expression of large synthetic genomes.”

In the field of synthetic biology, researchers investigate so-called “bottom-up” processes, which means the generation of life mimicking systems from inanimate building blocks. One of the most fundamental characteristics of all living organisms is the ability to conserve and reproduce itself as distinct entities. However, the artificial “bottom-up” approach to creating a system, which is able to replicate itself, is a great experimental challenge. For the first time, scientists have succeeded in overcoming this hurdle and synthesizing such a system.

“Our system is able to regenerate a significant proportion of its molecular components itself,” explained senior study investigator Hannes Mutschler, PhD, head of the Biomimetic Systems research group at the Max Planck Institute for Biochemistry. He and his research team are dedicated to imitating the replication of genomes and protein synthesis with a “bottom-up” approach. Both processes are fundamental for the self-preservation and reproduction of biological systems. The researchers have succeeded in producing an in vitro system, in which both processes could take place simultaneously.

In order to start this process, the researchers needed a construction manual as well as various molecular machines and nutrients. Translated into biological terms, this means the construction manual is DNA, which contains the information to produce proteins. Proteins are often referred to as molecular machines because they often act as catalysts, which accelerate biochemical reactions in organisms.

In the current study, the researchers optimized an in vitro expression system that synthesizes proteins based on a DNA blueprint. Due to several improvements, the in vitro expression system is now able to synthesize proteins, known as DNA polymerases, efficiently. These DNA polymerases then replicate the DNA using nucleotides.

“Unlike previous studies, our system is able to read and copy comparatively long DNA genomes,” remarked lead study investigator Kai Libicher, a scientist in the Mutschler lab.

“Our in vitro translation system enables self-encoded replication and expression of large DNA genomes under well-defined, cell-free conditions,” the authors wrote. “In particular, we demonstrated 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 the 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.”

The scientists assembled the artificial genomes from up to eleven ring-shaped pieces of DNA. This modular structure enables them to insert or remove certain DNA segments easily. The largest modular genome reproduced by the researchers in the study consists of more than 116,000 base pairs, reaching the genome length of very simple cells.

Apart from encoding polymerases that are important for DNA replication, the artificial genome contains blueprints for further proteins, such as 30 translation factors originating from E. coli. Translation factors are important for the translation of the DNA blueprint into the respective proteins. Thus, they are essential for self-replicating systems, which imitate biochemical processes. In order to show that the new in vitro expression system is not only able to reproduce DNA but is also able to produce its own translation factors, the researchers used mass spectrometry. With this analytic method, they determined the number of proteins produced by the system.

Surprisingly, some of the translation factors were even present in larger quantities after the reaction than added before. According to the researchers, this is an important step towards a continuously self-replicating system that mimics biological processes.

In the future, the research team wants to extend the artificial genome with additional DNA segments. In cooperation with colleagues from the research network MaxSynBio, they want to produce an envelope system that can remain viable by adding nutrients and disposing of waste products. Such a minimal cell could then be used, for example, in biotechnology as a tailor-made production machine for natural substances or as a platform for building even more complex life-like systems.

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