Genetic circuits and reaction cascades are of great importance for synthetic biology, biochemistry, and bioengineering. An open question is how to maximize the modularity of their design to enable the integration of different reaction networks and to optimize their scalability and flexibility. One option is encapsulation within liposomes which enables chemical reactions to proceed in well-isolated environments. Here we adapt liposome encapsulation to enable the modular, controlled compartmentalization of genetic circuits and cascades. We demonstrate that it is possible to engineer genetic circuit-containing synthetic minimal cells (synells) to contain multiple-part genetic cascades, and that these cascades can be controlled by external signals as well as inter-liposomal communication without cross-talk. We also show that liposomes containing different cascades can be fused in a controlled way so that the products of incompatible reactions can be brought together. Synells thus enable more modular creation of synthetic biology cascades, an essential step towards their ultimate programmability.

Efforts to recreate a prebiotically plausible protocell, in which RNA replication occurs within a fatty acid vesicle, have been stalled by the destabilizing effect of Mg 2+ on fatty acid membranes. Here, we report that the presence of citrate protects fatty acid membranes from the disruptive effects of high Mg 2+ ion concentrations while allowing RNA copying to proceed, while also protecting single-stranded RNA from Mg 2+ -catalyzed degradation. This combination of properties has allowed us to demonstrate the chemical copying of RNA templates inside fatty acid vesicles, which in turn allows for an increase in copying efficiency by bathing the vesicles in a continuously refreshed solution of activated nucleotides.The RNA world hypothesis suggests that the primordial catalysts were ribozymes (1, 2), while biophysical considerations suggest that the primordial replicating compartments were membranous vesicles composed of fatty acids and related amphiphiles (3, 4). However, the conditions required for RNA replication chemistry and fatty acid vesicle integrity have appeared to be fundamentally incompatible (5) (Fig S1). Both ribozyme catalyzed and nonenzymatic RNA copying reactions require high (50-200 mM) concentrations of Mg 2+ (or other divalent) ions (6), but Mg 2+ at such concentrations destroys vesicles by causing fatty acid precipitation.We developed a screen for small molecules that protect oleate fatty acid vesicles from disruption by Mg 2+ . We used two assays to monitor the leakage of either a small charged molecule (calcein) or a larger oligonucleotide, allowing us to distinguish between increased (Fig. S5 and S6). In the presence of chelated Mg 2+ , oleate vesicles remained intact but exhibited a modest increase in the permeability of a small polar molecule ( Fig. 1 and S7) and an even smaller increase in the leakage of an oligonucleotide. In terms of vesicle stabilization, citrate was one of the most effective chelators of Mg 2+ .We also examined the stability of model protocell membranes made of myristoleic acid: glycerol monomyristoleate 2:1 and from the more prebiotically reasonable decanoic acid: decanol: glycerol monodecanoate 4:1:1. Citrate-chelated Mg 2+ caused only a small amount of leakage from these vesicles, and the stabilizing effect of citrate was seen for both calcein and oligonucleotides ( Fig. 1 and S8-S13).We then asked if these chelators were compatible with the Mg 2+ catalysis of non-enzymatic template-directed RNA primer extension. We measured the rate at which an RNA primer was elongated when annealed to an oligonucleotide with a templating region of C nucleotides, in the presence of excess activated G monomer, guanosine 5′-phosphor(2-methyl)imidazolide (2MeImpG) (Fig. 2). We examined citric acid, EDTA, NTA and a weakly stabilizing chelator (isocitric acid). In the presence of 50 mM unchelated Mg 2+ , the primer-extension reaction proceeded at a rate of 1.4 h 1 , compared to 0.03 h −1 in the absence of Mg 2+ ions. The addition of 4 equivalents of EDTA or NTA resulted in co...

The advent of Darwinian evolution required the emergence of molecular mechanisms for heritable variation of fitness. One model for such a system involves competing protocell populations, each consisting of a replicating genetic polymer within a replicating vesicle. In this model, each genetic polymer imparts a selective advantage to its protocell by, for example, coding for a catalyst that generates a useful metabolite. Here, we report a partial model of such nascent evolutionary traits in a system consisting of fatty acid vesicles containing a dipeptide catalyst, which catalyzes the formation of a second dipeptide. The newly-formed dipeptide binds to vesicle membranes, imparting enhanced affinity for fatty acids, thus promoting vesicle growth. The catalyzed dipeptide synthesis proceeds with higher efficiency in vesicles than in free solution, further enhancing fitness. Our observations suggest that, in a replicating protocell with an RNA genome, ribozyme-catalyzed peptide synthesis might have been sufficient to initiate Darwinian evolution.