We previously reported assembly and cloning of the synthetic Mycoplasma genitalium JCVI-1.0 genome in the yeast Saccharomyces cerevisiae by recombination of six overlapping DNA fragments to produce a 592-kb circle. Here we extend this approach by demonstrating assembly of the synthetic genome from 25 overlapping fragments in a single step. The use of yeast recombination greatly simplifies the assembly of large DNA molecules from both synthetic and natural fragments.in vivo DNA assembly ͉ genome synthesis ͉ combinatorial assembly ͉ yeast transformation ͉ Mycoplasma genitalium ͉ synthetic biology Y east has long been considered a genetically tractable organism because of its ability to take up and recombine DNA fragments. More than 30 years ago, Hinnen et al. (1) reported the restoration of leucine biosynthesis in Saccharomyces cerevisiae by transformation of a leu2 -strain to LEU2ϩ using a method involving spheroplasts, CaCl 2 , and PEG. Soon after, Orr-Weaver et al. (2) reported mechanistic studies demonstrating that DNA molecules taken up during yeast transformation can integrate into yeast chromosomes through homologous recombination, and that the ends of the linear-transforming DNA are highly recombinogenic and react directly with homologous chromosomal sequences, whereas circular plasmids carrying yeast sequences integrate by a single crossover and only at low frequency. Subsequently, yeast transformation has become an indispensable tool in yeast genetics.Yeast recombination has since been applied to the construction of plasmids and yeast artificial chromosomes (YACs). In 1987, Ma et al. (3) constructed plasmids from two cotransformed DNA fragments containing homologous regions. In another process, called linker-mediated assembly, any DNA sequence can be joined to a vector DNA using short synthetic linkers that bridge the ends (4, 5). Similarly, four or five overlapping DNA pieces can be assembled and joined to vector DNA (4, 6, 7). This work demonstrated that yeast cells can take up multiple pieces of DNA, and that homologous yeast recombination is sufficiently efficient to correctly assemble the pieces into a single recombinant molecule.The limitations of assembly methods in yeast remain unknown. We recently assembled an entire synthetic M. genitalium genome using a combination of in vitro enzymatic recombination in early stages and in vivo yeast recombination in the final stage to produce the complete genome (8). In the first stage, overlapping Ϸ6-kb DNA cassettes were joined four at a time to form 25 Ϸ24-kb A-series assemblies. Three A-series assemblies were then joined to make 1/8 genome Ϸ72-kb B-series assemblies, and then two Bseries assemblies were assembled to make each of the Ϸ145-kb quarter-genome C-series assemblies. We accomplished the final assembly in yeast using three quarter-genome fragments and a fourth quarter fragment that had been cleaved by a restriction enzyme to provide a site for insertion of the vector DNA. Thus, some individual yeast cells have the capacity to simultaneously tak...

There is a general need for engineering of protein-like molecules that organize into geometrically-specific superstructures on molecular surfaces, directing further functionalization to create richly textured, multi-layered assemblies. Here we describe a computational approach whereby the surface properties and symmetry of a targeted surface define the sequence and superstructure of surface-organizing peptides. Computational design proceeds in a series of steps that encode both surface recognition and favorable inter-subunit packing interactions. This procedure is exemplified in the design of peptides that assemble into a tubular structure surrounding single-walled carbon nanotubes (SWNTs). The geometrically-defined, virus-like coating created by these peptides converts the smooth surfaces of SWNTs into highly textured assemblies, with long-scale order, capable of directing the assembly of gold nanoparticles into helical arrays along the SWNT axis.

Mutualisms between species play an important role in ecosystem function and stability. However, in some environments, the competitive aspects of an interaction may dominate the mutualistic aspects. Although these transitions could have far-reaching implications, it has been difficult to study the causes and consequences of this mutualistic–competitive transition in experimentally tractable systems. Here, we study a microbial cross-feeding mutualism in which each yeast strain supplies an essential amino acid for its partner strain. We find that, depending upon the amount of freely available amino acid in the environment, this pair of strains can exhibit an obligatory mutualism, facultative mutualism, competition, parasitism, competitive exclusion, or failed mutualism leading to extinction of the population. A simple model capturing the essential features of this interaction explains how resource availability modulates the interaction and predicts that changes in the dynamics of the mutualism in deteriorating environments can provide advance warning that collapse of the mutualism is imminent. We confirm this prediction experimentally by showing that, in the high nutrient competitive regime, the strains rapidly reach a common carrying capacity before slowly reaching the equilibrium ratio between the strains. However, in the low nutrient regime, before collapse of the obligate mutualism, we find that the ratio rapidly reaches its equilibrium and it is the total abundance that is slow to reach equilibrium. Our results provide a general framework for how mutualisms may transition between qualitatively different regimes of interaction in response to changes in nutrient availability in the environment.

Genetically identical cells in microbial populations often exhibit a remarkable degree of phenotypic heterogeneity even in homogenous environments. Such heterogeneity is commonly thought to represent a bet‐hedging strategy against environmental uncertainty. However, evolutionary game theory predicts that phenotypic heterogeneity may also be a response to negative frequency‐dependent interactions that favor rare phenotypes over common ones. Here we provide experimental evidence for this alternative explanation in the context of the well‐studied yeast GAL network. In an environment containing the two sugars glucose and galactose, the yeast GAL network displays stochastic bimodal activation. We show that in this mixed sugar environment, GAL‐ON and GAL‐OFF phenotypes can each invade the opposite phenotype when rare and that there exists a resulting stable mix of phenotypes. Consistent with theoretical predictions, the resulting stable mix of phenotypes is not necessarily optimal for population growth. We find that the wild‐type mixed strategist GAL network can invade populations of both pure strategists while remaining uninvasible by either. Lastly, using laboratory evolution we show that this mixed resource environment can directly drive the de novo evolution of clonal phenotypic heterogeneity from a pure strategist population. Taken together, our results provide experimental evidence that negative frequency‐dependent interactions can underlie the phenotypic heterogeneity found in clonal microbial populations.