Alternative performance metricsFor the purposes of characterization of a particular biocircuit testing environment, in vitro or in vivo, there are any number of performance metrics that may be used. We have chosen to use integrated mRNA and final protein concentration since they intuitively represent the total transcriptional and translational capacity of a system. Other metrics, such as the mRNA and protein production rates and end times, are complementary to those used in this work and may be particularly informative depending on the specific circuit or system requirement.In Figs. S16 and S17 we show the maximum deGFP and MGapt production rates as a function of reporter concentration under different conditions. Using these measures, stark differences between simple one-stage gene expression and expression from the two-stage T7 cascade can be seen; for example, at low reporter concentrations, the maximum production rate of deGFP in the cascade is considerably larger than the one-stage rate, even when the strongest promoter is used. The cascade deGFP rates are also relatively flat with respect to reporter concentration. There is also a clear effect of NTPs on peak rates when a cascade is used versus simple expression. In the former case, additional NTPs provide for a significant increase in the maximum protein and mRNA production rates, whereas NTPs have little to no effect on maximum production rates for the Pr-deGFP-MGapt construct.The deGFP production end time t end,T L is shown for all conditions in Fig. S18. t end,T L is between 330 and 350 minutes for simple expression in the 'linear' regime, but as high as ∼580 minutes in some of the conditions and concentrations tested. The end time is a particularly good measure of system capacity when extended performance is required.

Flavin-binding LOV domains are blue-light photosensory modules that are conserved in a number of developmental and circadian regulatory proteins in plants, algae, and fungi. LOV domains are also present in bacterial genomes, and are commonly located at the amino termini of sensor histidine kinases. Genes predicted to encode LOV-histidine kinases are conserved across a broad range of bacterial taxa, from aquatic oligotrophs to plant and mammalian pathogens. However, the function of these putative prokaryotic photoreceptors remains largely undefined. The differentiating bacterium, Caulobacter crescentus, contains an operon encoding a two-component signaling system consisting of a LOV-histidine kinase, LovK, and a single-domain response regulator, LovR. LovK binds a flavin cofactor, undergoes a reversible photocycle, and displays increased ATPase and autophosphorylation activity in response to visible light. Deletion of the response regulator gene, lovR, results in severe attenuation of cell attachment to a glass surface under laminar flow, whereas coordinate, low-level overexpression of lovK and lovR results in a light-independent increase in cell-cell attachment, a response that requires both the conserved histidine phosphorylation site in LovK and aspartate phosphorylation site in LovR. Growing C. crescentus in the presence of blue light dramatically enhances cell-cell attachment in the lovKlovR overexpression background. A conserved cysteine residue in the LOV domain of LovK, which forms a covalent adduct with the flavin cofactor upon absorption of visible light, is necessary for the light-dependent regulation of LovK enzyme activity and is required for the light-dependent enhancement of intercellular attachment.Caulobacter ͉ LOV domain ͉ photoreceptor ͉ signal transduction ͉ histidine kinase P roteins that serve as detectors of environmental signals are often modular, containing conserved sensory domains that control diverse signaling outputs (1, 2). One such sensory module is the PAS (Per-ARNT-Sim) domain, which is conserved across all kingdoms of life and is capable of specifically binding a wide range of ligands including heme, flavins, p-coumaric acid, citrate, and other small molecules (3). A subclass of PAS domains, known as LOV domains for their role as sensors of light, oxygen, or voltage, commonly bind a flavin cofactor and function to regulate a number of blue light-dependent processes in plants and fungi (4). These photosensory LOV domains signal by means of a unique photocycle in which photon absorption drives the reversible formation of a covalent adduct between the 4a carbon of the flavin isoalloxazine ring and a conserved cysteine residue (5, 6). Adduct formation is followed by a large structural change at the C terminus of the LOV domain that leads to cell signaling (7,8). Beyond plants and fungi, dozens of proteins containing LOV photosensory domains have been identified in bacterial species (4, 9). Examples of bacterial LOV photosensors include LOV-phosphodiesterases, LOV-HTH transcription fa...

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The robust surface adherence property of the aquatic bacterium Caulobacter crescentus permits visualization of single cells in a linear microfluidic culture chamber over an extended number of generations. The division rate of Caulobacter in this continuous-flow culture environment is substantially faster than in other culture apparati and is independent of flow velocity. Analysis of the growth and division of single isogenic cells reveals that the cell cycle control network of this bacterium generates an oscillatory output with a coefficient of variation lower than that of all other bacterial species measured to date. DivJ, a regulator of polar cell development, is necessary for maintaining low variance in interdivision timing, as transposon disruption of divJ significantly increases the coefficient of variation of both interdivision time and the rate of cell elongation. Moreover, interdivision time and cell division arrest are significantly correlated between mother and daughter cells, providing evidence for epigenetic inheritance of cell division behavior in Caulobacter. The single-cell growth/division results reported here suggest that future predictive models of Caulobacter cell cycle regulation should include parameters describing the variance and inheritance properties of this system.

The many successes of synthetic biology have come in a manner largely different from those in other engineering disciplines; in particular, without well-characterized and simplified prototyping environments to play a role analogous to wind-tunnels in aerodynamics and breadboards in electrical engineering. However, as the complexity of synthetic circuits increases, the benefits—in cost savings and design cycle time—of a more traditional engineering approach can be significant. We have recently developed an in vitro ‘breadboard’ prototyping platform based on E. coli cell extract that allows biocircuits to operate in an environment considerably simpler than but functionally similar to in vivo. The simplicity of this system makes it a promising tool for rapid biocircuit design and testing, as well as for probing fundamental aspects of gene circuit operation normally masked by cellular complexity. In this work we characterize the cell-free breadboard using real-time and simultaneous measurements of transcriptional and translational activities of a small set of reporter genes and a transcriptional activation cascade. We determine the effects of promoter strength, gene concentration, and nucleoside triphosphate concentration on biocircuit properties, and we isolate the specific contributions of essential biomolecular resources—core RNA polymerase and ribosomes—to overall performance. Importantly, we show how limits on resources, particularly those involved in translation, are manifested as reduced expression in the presence of orthogonal genes that serve as additional loads on the system.

Bistability plays a central role in the gene regulatory networks (GRNs) controlling many essential biological functions, including cellular differentiation and cell cycle control. However, establishing the network topologies that can exhibit bistability remains a challenge, in part due to the exceedingly large variety of GRNs that exist for even a small number of components. We begin to address this problem by employing chemical reaction network theory in a comprehensive in silico survey to determine the capacity for bistability of more than 40,000 simple networks that can be formed by two transcription factor-coding genes and their associated proteins (assuming only the most elementary biochemical processes). We find that there exist reaction rate constants leading to bistability in ∼90% of these GRN models, including several circuits that do not contain any of the TF cooperativity commonly associated with bistable systems, and the majority of which could only be identified as bistable through an original subnetwork-based analysis. A topological sorting of the two-gene family of networks based on the presence or absence of biochemical reactions reveals eleven minimal bistable networks (i.e., bistable networks that do not contain within them a smaller bistable subnetwork). The large number of previously unknown bistable network topologies suggests that the capacity for switch-like behavior in GRNs arises with relative ease and is not easily lost through network evolution. To highlight the relevance of the systematic application of CRNT to bistable network identification in real biological systems, we integrated publicly available protein-protein interaction, protein-DNA interaction, and gene expression data from Saccharomyces cerevisiae, and identified several GRNs predicted to behave in a bistable fashion.

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations of noncoding RNAs in a single living bacterium. Here, we extend the use of FCS to low concentrations of coding RNAs in single living cells. We genetically fuse a red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is the RFP protein alone. Using this construct, we determine in single cells both the absolute [mRNA] concentration and the associated [RFP] expressed from an inducible plasmid. We find that the FCS method allows us to reliably monitor in real-time [mRNA] down to ∼40 nM (i.e. approximately two transcripts per volume of detection). To validate these measurements, we show that [mRNA] is proportional to the associated expression of the RFP protein. This FCS-based technique establishes a framework for minimally invasive measurements of mRNA concentration in individual living bacteria.

Background: Recent years have seen a dramatic increase in the use of mathematical modeling to gain insight into gene regulatory network behavior across many different organisms. In particular, there has been considerable interest in using mathematical tools to understand how multistable regulatory networks may contribute to developmental processes such as cell fate determination. Indeed, such a network may subserve the formation of unicellular leaf hairs (trichomes) in the model plant Arabidopsis thaliana.