Continuous Cell-Free Replication and Evolution of Artificial Genomic DNA in a Compartmentalized Gene Expression System

Abstract

In all living organisms, genomic DNA continuously replicates by the proteins encoded in itself and undergoes evolution through many generations of replication. This continuous replication coupled with gene expression and the resultant evolution are fundamental functions of living things, but they have not previously been reconstituted in cell-free systems. In this study, we combined an artificial DNA replication scheme with a reconstituted gene expression system and microcompartmentalization to realize these functions. Circular DNA replicated through rolling-circle replication followed by homologous recombination catalyzed by the proteins, phi29 DNA polymerase, and Cre recombinase expressed from the DNA. We encapsulated the system in microscale water-in-oil droplets and performed serial dilution cycles. Isolated circular DNAs at Round 30 accumulated several common mutations, and the isolated DNA clones exhibited higher replication abilities than the original DNA due to its improved ability as a replication template, increased polymerase activity, and a reduced inhibitory effect of polymerization by the recombinase. The artificial genomic DNA, which continuously replicates using self-encoded proteins and autonomously improves its sequence, provides a useful starting point for the development of more complex artificial cells.

Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System

“…Recently, we found that the replication efficiency of circular DNA spontaneously improves through long-term replication due to Darwinian evolution. 18 A similar long-term replication would allow the evolution of highly replicable template DNA encoding tRNA. Another advantage of in vitro evolution is the improvement of tRNA sequences.…”

“…All components of the laboratory-made PURE system were prepared as previously described. 18 EF-Tu was purified using a nickel column. For EF-Tu repurification, purified EF-Tu was 10-fold diluted with a stringent buffer [50 mM HEPES−KOH (pH 7.6), 1 M KCl, 10 mM MgCl 2 , 15% glycerol, 1 mM DTT, and 1% Triton X-100] and allowed to stand on ice for 1 h. The solution was injected into to the HisTrap column (Thermo Fisher Scientific) and washed with the same buffer, omitting Triton X-100.…”

A four-track perspective for bottom-up synthetic cells

“…To face the difficulty of achieving high degrees of integration, an evolutionary approach has been proposed (Abil and Danelon, 2020). Directed evolution strategies should be considered as well (Sakatani et al, 2018;Okauchi and Ichihashi, 2021), especially when connected to adaptive responses. From the architectural viewpoint, complexification can be achieved via multiple compartmentalization.…”

Efficient multi-gene expression in cell-free droplet microreactors

“…However, protein expression levels were low, which was not surprising as cell-free reactions require very high quantities of template DNA for efficient protein production [ 24 , 25 ]. The most obvious solution to this problem, namely the amplification of single DNA molecules directly within a transcription-translation droplet, turned out to be surprisingly difficult as reaction chemistries and temperatures are not entirely compatible [ 26 – 28 ]. The current work-around is a two-step approach consisting of in-droplet Polymerase Chain Reaction, followed by micro-injection or droplet merging into separately prepared cell-free droplets [ 29 – 32 ].…”