A central problem in biology is determining how genes interact as parts of functional networks. Creation and analysis synthetic networks, composed well-characterized genetic elements, provide a framework for theoretical modeling. Here, with the use combinatorial method, library networks varying connectivity was generated Escherichia coli . These were encoding transcriptional regulators LacI, TetR, lambda CI, well corresponding promoters. They displayed phenotypic behaviors resembling binary...

Drug efflux machinery inherited asymmetrically In dividing bacterial cells, asymmetric distribution of cell wall constituents occurs between mother cells and their progeny. Asymmetric in a growing population results heterogeneity antibiotic resistance. One consequence is that the presence low levels antibiotic, older tend to live longer than younger cells. Using microfluidic device trap measure Bergmiller et al. showed AcrAB-TolC, main multidrug pump Escherichia coli , clusters at pole (see...

We examine whether the Escherichia coli chromosome is folded into a self-adherent nucleoprotein complex, or alternately confined but otherwise unconstrained self-avoiding polymer. address this through in vivo visualization, using an inducible GFP fusion to nucleoid-associated protein Fis non-specifically decorate entire chromosome. For range of different growth conditions, compact structure that does not fill volume cell, and which moves from new pole cell centre. During rapid growth,...

Bacteria in groups vary individually, and interact with other bacteria the environment to produce population-level patterns of gene expression. Investigating such behavior detail requires measuring controlling populations at single-cell level alongside precisely specified interactions environmental characteristics. Here we present an automated, programmable platform that combines image-based expression growth measurements on-line optogenetic control for hundreds individual Escherichia coli...

Predicting function from sequence is a central problem of biology. Currently, this possible only locally in narrow mutational neighborhood around wildtype rather than globally any sequence. Using random mutant libraries, we developed biophysical model that accounts for multiple features σ70 binding bacterial promoters to predict constitutive gene expression levels We experimentally and theoretically estimated 10-20% sequences lead ~80% non-expressing are one mutation away functional...

The bacterial flagellum is a self-assembling nanomachine. external flagellar filament, several times longer than cell body, made of few tens thousands subunits single protein: flagellin. A fundamental problem concerns the molecular mechanism how grows outside cell, where no discernible energy source available. Here, we monitored dynamic assembly individual flagella using in situ labelling and real-time immunostaining elongating filaments. We report that rate growth, initially ∼1,700 amino...

Abstract Gene regulation relies on the specificity of transcription factor (TF)–DNA interactions. Limited may lead to crosstalk: a regulatory state in which gene is either incorrectly activated due noncognate TF–DNA interactions or remains erroneously inactive. As each TF can have numerous with cis -regulatory elements, crosstalk inherently global problem, yet has previously not been studied as such. We construct theoretical framework analyse effects regulation. find that presents...

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become dominant population of bacteria. Following ascent mutants, densities in these simple communities limited by resources rather than phage. Despite evolution hosts, upon which cannot replicate, is most maintained an apparently stable state Several mechanisms have been put forward account for this result. Here we report results dynamic/evolution...

Characterizing the dynamics of specific RNA levels requires real-time profiling in a single cell. We show that combination synthetic modular genetic system with fluorescence correlation spectroscopy allows us to directly measure real time activity any promoter prokaryotes. Using simple inducible gene expression system, we found induced within bacterium Escherichia coli exhibited pulsating profile response steady input inducer. The deletion an efflux pump key determinant antibiotic...

In many eukaryotic cells going through M-phase, a bipolar spindle is formed by microtubules nucleated from centrosomes. These microtubules, in addition to being "captured" kinetochores, may be stabilized chromatin two different ways: short-range stabilization effects affect close contact with the chromatin, while long-range "guide" microtubule growth towards (e.g., introducing diffusive gradient of an enzymatic activity that affects assembly). Here, we use both meiotic and mitotic extracts...

Fluorescence correlation spectroscopy (FCS) has permitted the characterization of high concentrations noncoding RNAs in a single living bacterium. Here, we extend use FCS to low coding cells. We genetically fuse red fluorescent protein (RFP) gene and two binding sites for an RNA-binding protein, whose translated product is RFP alone. Using this construct, determine cells both absolute [mRNA] concentration associated [RFP] expressed from inducible plasmid. find that method allows us reliably...

Revealing the spectrum of combinatorial regulation transcription at individual promoters is essential for understanding complex structure biological networks. However, computations represented by integration various molecular signals are difficult to decipher in absence simple cis regulatory codes. Here we synthetically shuffle architecture — operator sequences binding activators and repressors a canonical bacterial promoter. The resulting library allows rapid exploration promoter encoded...

Restriction–modification systems are widespread genetic elements that protect bacteria from bacteriophage infections by recognizing and cleaving heterologous DNA at short, well-defined sequences called restriction sites. Bioinformatic evidence shows sites significantly underrepresented in genomes, presumably because bacteriophages with fewer more likely to escape cleavage restriction–modification systems. However, how mutations affect the likelihood of is unknown. Using λ system EcoRI, we...

Which properties of metabolic networks can be derived solely from stoichiometric information about the network's constituent reactions? Predictive results have been obtained by Flux Balance Analysis (FBA), postulating that cells set fluxes within allowed stoichiometry so as to maximize their growth. Here, we generalize this framework single cell level using maximum entropy models statistical physics. We define and compute, for core metabolism Escherichia coli, a joint distribution over all...

Gene expression levels are influenced by multiple coexisting molecular mechanisms. Some of these interactions such as those transcription factors and promoters have been studied extensively. However, predicting phenotypes gene regulatory networks (GRNs) remains a major challenge. Here, we use well-defined synthetic GRN to study in Escherichia coli how network depend on local genetic context, i.e. the neighborhood factor its relative position. We show that one with fixed topology can display...

Understanding the relation between genotype and phenotype remains a major challenge. The difficulty of predicting individual mutation effects, particularly interactions them, has prevented development comprehensive theory that links genotypic changes to their phenotypic effects. We show general thermodynamic framework for gene regulation, based on biophysical understanding protein-DNA binding, accurately predicts sign epistasis in canonical cis-regulatory element consisting overlapping RNA...

Together, copy-number and point mutations form the basis for most evolutionary novelty, through process of gene duplication divergence. While a plethora genomic data reveals long-term fate diverging coding sequences their cis-regulatory elements, little is known about early dynamics around event itself. In microorganisms, selection increased expression often drives expansion mutations, which serves as crude adaptation, prior to divergence refining mutations. Using simple synthetic genetic...