The Long Road to a Synthetic Self-Replicating Central Dogma

Abstract

The construction of a biochemical system capable of self-replication is a key objective in bottom-up synthetic biology. Throughout the past two decades, a rapid progression in the design of in vitro cell-free systems has provided valuable insight into the requirements for the development of a minimal system capable of self-replication. The main limitations of current systems can be attributed to their macromolecular composition and how the individual macromolecules use the small molecules necessary to drive RNA and protein synthesis. In this Perspective, we discuss the recent steps that have been taken to generate a minimal cell-free system capable of regenerating its own macromolecular components and maintaining the homeostatic balance between macromolecular biogenesis and consumption of primary building blocks. By following the flow of biological information through the central dogma, we compare the current versions of these systems to date and propose potential alterations aimed at designing a model system for self-replicative synthetic cells.

Figure 1

Basic composition of the reconstituted PURE (protein synthesis using recombinant elements) system necessary for the four major reactions involved in in vitro transcription–translation-coupled protein synthesis. Transcription of input DNA template and aminoacylation of tRNAs initiate translation, while energy currencies are regenerated.

Figure 2

Alterations to the current composition of PURE to improve and/or enable self-regeneration of main macromolecular components of the central dogma apart from ribosomes (see Ribosome Biogenesis in PURE). Current versions of PURE are not capable of synthesizing and assembling functional ribosomes. (1) Tunable promoter strengths are designed to match transcriptional rates for individual genes based on translational requirements. (2) Maturation of tRNAs, for subsequent aminoacylation (3), is ensured by flanking self-cleaving ribozymes such as a 5′ hammerhead ribozyme (HHR) and a 3′ hepatitis delta virus ribozyme (HDV). Mature tRNAs generated in this way directly in PURE could provide the foundation for a PURE-specific reduced tRNA pool, which would alleviate the transcriptional burden in a self-replicating system. (4) Translation levels of proteins can be regulated using tunable RBS strengths, while ribosome reinitiation can be improved by the addition of EF-P. (5) Stalled ribosomes are rescued by EF-4, hence increasing functional protein synthesis rates. (6) Folding of polypeptide chains into their native fold is promoted by protein-folding chaperones such as DnaJ/K and GroES/EL, thereby increasing the effective concentration of active proteins.

Figure 3

Continuous systems for prolonged PURE regeneration setups. (A) Continuous exchange device as described by Jackson et al.: (top) cross-sectional view and (bottom) top view. The PURE reaction is constantly supplied with small molecule components by dialysis from the feeding chambers. (B) Nanoporous continuous exchange setup as developed by Siuti et al. Small metabolites are supplied to the PURE system in the nanoliter container, while excess synthesized proteins are exchanged with the surrounding environment. (C) DNA brush setup as described by Karzbrun et al. The DNA templates, immobilized in silicon, are connected through a diffusive capillary to a channel supplying fresh IVTT reagents (cell extract or PURE). (D) Serial transfers can be used to emulate “generations” of regenerative rounds of the PURE system. After each incubation, a defined amount of the generation is mixed with fresh PURE, lacking predefined essential protein components, and with the DNA encoding for the missing components; the regeneration of the entire system can therefore be interpreted as a function of the regeneration of the missing components. (E) Microfluidic chemostat as developed by Niederholtmeyer et al. and adopted by Lavickova et al. At the left, protein and energy components are mixed in a nanoliter chemostat together with the DNA encoding the protein of interest to be regenerated. A constant volume of fresh PURE components is pumped into the device while an equal amount is extracted from the outlet to achieve steady-state in vitro translation. The right panel is a representation of parallelized chemostat setup as seen in ref (23) to allow for parallel testing of multiple conditions.

Figure 4

Comparing metabolic networks. Standard PURE composition vs hypothetical, self-regenerating PURE augmented with energy regeneration and quality control mechanisms. Elements in solid line boxes [e.g., 10-formyltetrahydrofolate (10-CHO-THF)] cannot be synthesized by the system and therefore must be continuously supplied externally; elements in dashed boxes [e.g., methionyl tRNA-formyltransferase (MTF)] are necessary to kickstart the system and/or can be partially synthesized by the system. (A) DNA (purple) is transcribed into mRNA (blue), which is translated into proteins (red). tRNA aminoacylation is colored pink, and energy regeneration pathways are colored yellow. (B) In a self-replicative PURE, DNA would be replicated in vitro and transcribed into mRNAs, tRNAs (*the scheme presented here does not depict the necessary machinery to generate mature tRNAs), and rRNAs (**we did not include in this scheme the machinery and steps necessary to generate mature rRNAs such as the various types of rRNA modifications found in fully functional ribosomes). Transcribed mRNAs are translated by in vitro synthesized and assembled translational machinery, including ribosomes (***we assume here that ribosomal proteins and rRNAs are synthesized in vitro and contribute efficiently to in vitro ribosome assembly). Along with in vitro synthesis of aaRSs and some TFs as in the minimal PURE, the presented augmented version of PURE would theoretically need to synthesize all of its protein components involved in DNA replication, transcription, translation (in this case, TFs also include EF-Tu), aminoacylation, and energy regeneration. This would also apply to the added modules of mixed acid fermentation (1), mRNA degradation (2), and protein degradation (3).