A challenge of synthetic biology is the creation of cooperative microbial systems that exhibit population-level behaviors. Such systems use cellular signaling mechanisms to regulate gene expression across multiple cell types. We describe the construction of a synthetic microbial consortium consisting of two distinct cell types – an “activator” strain and a “repressor” strain. These strains produce two orthogonal cell-signaling molecules that regulate gene expression within a synthetic circuit spanning both strains. The two strains generated emergent, population-level oscillations only when cultured together. Certain network topologies of the two-strain circuit were better at maintaining robust oscillations than others. The ability to program population-level dynamics through the genetic engineering of multiple cooperative strains points the way towards engineering complex synthetic tissues and organs with multiple cell types.

One challenge for synthetic biologists is the predictable tuning of genetic circuit regulatory components to elicit desired outputs. Gene expression driven by ligand-inducible transcription factor systems must exhibit the correct ON and OFF characteristics: appropriate activation and leakiness in the presence and absence of inducer, respectively. However, the dynamic range of a promoter (i.e., absolute difference between ON and OFF states) is difficult to control. We report a method that tunes the dynamic range of ligand-inducible promoters to achieve desired ON and OFF characteristics. We build combinatorial sets of AraC-and LasR-regulated promoters containing −10 and −35 sites from synthetic and Escherichia coli promoters. Four sequence combinations with diverse dynamic ranges were chosen to build multi-input transcriptional logic gates regulated by two and three ligand-inducible transcription factors (LacI, TetR, AraC, XylS, RhlR, LasR, and LuxR). This work enables predictable control over the dynamic range of regulatory components.

Cells can be programmed to monitor and react to their environment using genetic circuits. Design automation software maps a desired circuit function to a DNA sequence, which requires units of gene regulation (gates) that are simple to connect and behave predictably. This poses a challenge for eukaryotes due to their complex mechanisms of transcription and translation. To this end, we have developed gates for yeast (Saccharomyces cerevisiae) that are connected using RNA polymerase (RNAP) flux as the signal carrier and are insulated from each other and host regulation. They are based on minimal constitutive promoters (~120bp), for which rules are developed to insert operators for DNA-binding proteins. Using this approach, we construct nine NOT/NOR gates with nearly-identical response functions and 400-fold dynamic range. In circuits, they are transcriptionally insulated from each other by placing ribozymes downstream of terminators to block nuclear export of mRNAs resulting from RNAP readthrough. Based on these gates, Cello is used to build circuits to specification with up to 11 regulatory proteins. A simple dynamic model predicts the circuit response over days. Extending genetic circuit design automation to eukaryotes simplifies their incorporation into cellular engineering projects, whether it be to stage processes for bioproduction, serve as environmental sentinels, or guide living therapeutics.

Synthetic microbial consortia have an advantage over isogenic synthetic microbes because they can apportion biochemical and regulatory tasks among the strains. However, it is difficult to coordinate gene expression in spatially extended consortia because the range of signaling molecules is limited by diffusion. Here, we show that spatiotemporal coordination of gene expression can be achieved even when the spatial extent of the consortium is much greater than the diffusion distance of the signaling molecules. To do this, we examined the dynamics of a twostrain synthetic microbial consortium that generates coherent oscillations in small colonies. In large colonies, we find that temporally coordinated oscillations across the population depend on the presence of an intrinsic positive feedback loop that amplifies and propagates intercellular signals. These results demonstrate that synthetic multi-cellular systems can be engineered to exhibit coordinated gene expression using only transient, short-range coupling among constituent cells. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

As synthetic biocircuits become more complex, distributing computations within multi-strain microbial consortia becomes increasingly beneficial. However, designing distributed circuits that respond predictably to variation in consortium composition remains a challenge. Here we develop a two-strain gene circuit that senses and responds to which strain is in the majority. This involves a co-repressive system in which each strain produces a signaling molecule that signals the other strain to down-regulate production of its own, orthogonal signaling molecule. This co-repressive consortium links gene expression to ratio of the strains rather than population size. Further, we control the cross-over point for majority via external induction. We elucidate the mechanisms driving these dynamics by developing a mathematical model that captures consortia response as strain fractions and external induction are varied. These results show that simple gene circuits can be used within multicellular synthetic systems to sense and respond to the state of the population.