Phenotypic variation is the phenomenon in which clonal cells display different traits even under identical environmental conditions. This plasticity is thought to be important for processes including bacterial virulence1–8, but direct evidence for its relevance is often lacking. For instance, variation in capsule production in the human pathogen Streptococcus pneumoniae has been linked to different clinical outcomes9–14, but the exact relationship between variation and pathogenesis is not well understood due to complex natural regulation15–20. In this study, we used synthetic oscillatory gene regulatory networks (GRNs) based on CRISPR interference together with live cell microscopy and cell tracking within microfluidics devices to mimic and test the biological function of bacterial phenotypic variation. We provide a universally applicable approach for engineering intricate GRNs using only two components: dCas9 and extended sgRNAs (ext-sgRNAs). Our findings demonstrate that variation in capsule production is beneficial for pneumococcal fitness in traits associated with pathogenesis providing conclusive evidence for this longstanding question.
Novel in vitro platforms based on human neurons are needed to improve early drug testing and address the stalling drug discovery in neurological disorders. Topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons have the potential to become such a testing system. In this work, we build in vitro co-cultured circuits of human iPSC-derived neurons and rat primary glial cells using microfabricated polydimethylsiloxane (PDMS) structures on microelectrode arrays (MEAs). Our PDMS microstructures are designed in the shape of a stomach, which guides axons in one direction and thereby facilitates the unidirectional flow of information. Such circuits are created by seeding either dissociated cells or pre-aggregated spheroids at different neuron-to-glia ratios. Furthermore, an antifouling coating is developed to prevent axonal overgrowth in undesired locations of the microstructure. We assess the electrophysiological properties of different types of circuits over more than 50 days, including their stimulation-induced neural activity. Finally, we demonstrate the inhibitory effect of magnesium chloride on the electrical activity of our iPSC circuits as a proof-of-concept for screening of neuroactive compounds.
As the microelectronics field pushes to increase device density through downscaling component dimensions, various novel micro- and nano-scale additive manufacturing technologies have emerged to expand the small scale design space. These techniques offer unprecedented freedom in designing 3D circuitry but have not yet delivered device-grade materials. To highlight the complex role of processing on the quality and microstructure of AM metals, we report the electrical properties of micrometer-scale copper interconnects fabricated by Fluid Force Microscopy (FluidFM) and Electrohydrodynamic-Redox Printing (EHD-RP). Using a thin film-based 4-terminal testing chip developed for the scope of this study, the electrical resistance of as-printed metals is directly related to print strategies and the specific morphological and microstructural features. Notably, the chip requires direct synthesis of conductive structures on an insulating substrate, which is shown for the first time in the case of FluidFM. Finally, we demonstrate the unique ability of EHD-RP to tune the materials resistivity by one order of magnitude solely through printing voltage. Through its novel electrical characterization approach, this study offers unique insight into the electrical properties of micro- and submicrometer-sized copper interconnects and steps towards a deeper understanding of micro AM metal properties for advanced electronics applications.
Genotype networks are sets of genotypes connected by small mutational changes that share the same phenotype. They facilitate evolutionary innovation by enabling the exploration of different neighborhoods in genotype space. Genotype networks, first suggested by theoretical models, have been empirically confirmed for proteins and RNAs. Comparative studies also support their existence for gene regulatory networks (GRNs), but direct experimental evidence is lacking. Here, we report the construction of three interconnected genotype networks of synthetic GRNs producing three distinct phenotypes in Escherichia coli. Our synthetic GRNs contain three nodes regulating each other by CRISPR interference and governing the expression of fluorescent reporters. The genotype networks, composed of over twenty different synthetic GRNs, provide robustness in face of mutations while enabling transitions to innovative phenotypes. Through realistic mathematical modeling, we quantify robustness and evolvability for the complete genotype-phenotype map and link these features mechanistically to GRN motifs. Our work thereby exemplifies how GRN evolution along genotype networks might be driving evolutionary innovation.
Bottom-up neuroscience utilizes small, engineered biological neural networks to study neuronal activity in systems of reduced complexity. We present a platform that establishes up to six independent networks formed by primary rat neurons on planar complementary metal–oxide–semiconductor (CMOS) microelectrode arrays (MEAs). We introduce an approach that allows repetitive stimulation and recording of network activity at any of the over 700 electrodes underlying a network. We demonstrate that the continuous application of a repetitive super-threshold stimulus yields a reproducible network answer within a 15 ms post-stimulus window. This response can be tracked with high spatiotemporal resolution across the whole extent of the network. Moreover, we show that the location of the stimulation plays a significant role in the networks’ early response to the stimulus. By applying a stimulation pattern to all network-underlying electrodes in sequence, the sensitivity of the whole network to the stimulus can be visualized. We demonstrate that microchannels reduce the voltage stimulation threshold and induce the strongest network response. By varying the stimulation amplitude and frequency we reveal discrete network transition points. Finally, we introduce vector fields to follow stimulation-induced spike propagation pathways within the network. Overall we show that our defined neural networks on CMOS MEAs enable us to elicit highly reproducible activity patterns that can be precisely modulated by stimulation amplitude, stimulation frequency and the site of stimulation.
Nanopores comprise a versatile class of membrane proteins that carry out a range of key physiological functions and are increasingly developed for different biotechnological applications. Yet, a capacity to study and engineer protein nanopores by combinatorial means has so far been hampered by a lack of suitable assays that combine sufficient experimental resolution with throughput. Addressing this technological gap, the functional nanopore (FuN) screen now provides a quantitative and dynamic readout of nanopore assembly and function in the context of the inner membrane of Escherichia coli. The assay is based on genetically encoded fluorescent protein sensors that resolve the nanopore-dependent influx of Ca2+ across the inner membrane of E. coli. Illustrating its versatile capacity, the FuN screen is first applied to dissect the molecular features that underlie the assembly and stability of nanopores formed by the S2168 holin. In a subsequent step, nanopores are engineered by recombining the transmembrane module of S2168 with different ring-shaped oligomeric protein structures that feature defined hexa-, hepta-, and octameric geometries. Library screening highlights substantial plasticity in the ability of the S2168 transmembrane module to oligomerize in alternative geometries, while the functional properties of the resultant nanopores can be fine-tuned through the identity of the connecting linkers. Overall, the FuN screen is anticipated to facilitate both fundamental studies and complex nanopore engineering endeavors with many potential applications in biomedicine, biotechnology, and synthetic biology.
Methods for patterning neurons in vitro have gradually improved and are used to investigate questions that are difficult to address in or ex vivo. Though these techniques guide axons between groups of neurons, multiscale control of neuronal connectivity, from circuits to synapses, is yet to be achieved in vitro. As studying neuronal circuits with synaptic resolution in vivo poses significant challenges, we present an in vitro alternative to validate biophysical and computational models. In this work we use a combination of electron beam lithography and photolithography to create polydimethylsiloxane (PDMS) structures with features ranging from 150 nm to a few millimeters. Leveraging the difference between average axon and dendritic spine diameters, we restrict axon growth while allowing spines to pass through nanochannels to guide synapse formation between small groups of neurons (i.e., nodes). We show this technique can be used to generate large numbers of isolated feed-forward circuits where connections between nodes are restricted to regions connected by nanochannels. Using a genetically encoded calcium indicator in combination with fluorescently tagged postsynaptic protein, PSD-95, we demonstrate functional synapses can form in this region.
We present a stimulate and record paradigm to examine the behavior of multiple neuronal networks with controlled topology in vitro. Our approach enabled us to electrically induce and record neuronal activity from 60 independent networks in parallel over multiple weeks. We investigated the network performance of neuronal networks of primary hippocampal neurons until 29 days in vitro. We introduced a systematic stimulate and record protocol during which well-defined 4-node neural networks were stimulated electrically 4 times per second (4Hz) and their response was recorded over many days. We found that the network response pattern to a stimulus remained fairly stable for at least 12 h. Moreover, continuous stimulation over multiple weeks did not cause a significant change in the stimulation-induced mean spiking frequency of a circuit. We investigated the effect of stimulation amplitude and stimulation timing on the detailed network response. Finally, we could show that our setup can apply complex stimulation protocols with 125 different stimulation patterns. We used these patterns to perform basic addition tasks with the network, revealing the highly non-linear nature of biological networks’ responses to complex stimuli.
Bottom-up neuroscience, which consists of building and studying controlled networks of neurons in vitro, is a promising method to investigate information processing at the neuronal level. However, in vitro studies tend to use cells of animal origin rather than human neurons, leading to conclusions that might not be generalizable to humans and limiting the possibilities for relevant studies on neurological disorders. Here we present a method to build arrays of topologically controlled circuits of human induced pluripotent stem cell (iPSC)-derived neurons. The circuits consist of 4 to 50 neurons with mostly unidirectional connections, confined by microfabricated polydimethylsiloxane (PDMS) membranes. Such circuits were characterized using optical imaging and microelectrode arrays (MEAs). Electrophysiology recordings were performed on circuits of human iPSC-derived neurons for at least 4.5 months. We believe that the capacity to build small and controlled circuits of human iPSC-derived neurons holds great promise to better understand the fundamental principles of information processing and storing in the brain.
The direct delivery of molecules and the sampling of endogenous compounds into and from living cells provide powerful means to modulate and study cellular functions. Intracellular injection and extraction remain challenging for fungal cells that possess a cell wall. The most common methods for intracellular delivery into fungi rely on the initial degradation of the cell wall to generate protoplasts, a step that represents a major bottleneck in terms of time, efficiency, standardization, and cell viability. Here, we show that fluidic force microscopy enables the injection of solutions and cytoplasmic fluid extraction into and out of individual fungal cells, including unicellular model yeasts and multicellular filamentous fungi. The approach is strain- and cargo-independent and opens new opportunities for manipulating and analyzing fungi. We also perturb individual hyphal compartments within intact mycelial networks to study the cellular response at the single cell level.
In bottom-up neuroscience, questions on neural information processing are addressed by engineering small but reproducible biological neural networks of defined network topology in vitro. The network topology can be controlled by culturing neurons within polydimethylsiloxane (PDMS) microstructures that are combined with microelectrode arrays (MEAs) for electric access to the network. However, currently used glass MEAs are limited to 256 electrodes and pose a limitation to the spatial resolution as well as the design of more complex microstructures. The use of high density complementary metal-oxide-semiconductor (CMOS) MEAs greatly increases the spatial resolution, enabling sub-cellular readout and stimulation of neurons in defined neural networks. Unfortunately, the non-planar surface of CMOS MEAs complicates the attachment of PDMS microstructures. To overcome the problem of axons escaping the microstructures through the ridges of the CMOS MEA, we stamp-transferred a thin film of hexane-diluted PDMS onto the array such that the PDMS filled the ridges at the contact surface of the microstructures without clogging the axon guidance channels. This method resulted in 23 % of structurally fully connected but sealed networks on the CMOS MEA of which about 45 % showed spiking activity in all channels. Moreover, we provide an impedance-based method to visualize the exact location of the microstructures on the MEA and show that our method can confine axonal growth within the PDMS microstructures. Finally, the high spatial resolution of the CMOS MEA enabled us to show that action potentials follow the unidirectional topology of our circular multi-node microstructure.
Aim of this work was the establishment and validation of a novel generic functional screening approach for selection of protein variants with desired biological function in an ultra-high-throughput manner. This new process is based on the combination of a S. cerevisiae secretion host, capable of high transformation efficiencies and compatible with high-diversity protein libraries, with a mammalian-based reporter system for activity-dependent fluorescence readout of functional molecules. This strategy represents the base of a straightforward selection platform for testing a high number of different protein molecules for desired biological function. Compartmentalization of the secretor and reporter cells in distinct microreactors enables phenotype-genotype coupling while miniaturization of the test assays to volumes in the picolitre range brings the advantage of rapid biochemical reactions by fast accumulation of secreted molecules. Microfluidic-assisted encapsulation of the two cell types supports the generation of microdroplets with high speed and precision and implementation of hydrogel-forming polymers enables the recovery of uniform hydrogel microbeads surrounding the encapsulated cells. This strategy makes the system compatible to ultra-high throughput fluorescence-activated cell sorting on a commercially available device and enables the screening of high diversity protein libraries.
Gene expression control based on CRISPRi (clustered regularly interspaced short palindromic repeats interference) has emerged as a powerful tool for creating synthetic gene circuits, both in prokaryotes and in eukaryotes; yet, its lack of cooperativity has been pointed out as a potential obstacle for dynamic or multistable synthetic circuit construction. Here we use CRISPRi to build a synthetic oscillator (“CRISPRlator”), bistable network (toggle switch) and stripe pattern-forming incoherent feed-forward loop (IFFL). Our circuit designs, conceived to feature high predictability and orthogonality, as well as low metabolic burden and context-dependency, allow us to achieve robust circuit behaviors in Escherichia coli populations. Mathematical modeling suggests that unspecific binding in CRISPRi is essential to establish multistability. Our work demonstrates the wide applicability of CRISPRi in synthetic circuits and paves the way for future efforts towards engineering more complex synthetic networks, boosted by the advantages of CRISPR technology.
Biocatalysis provides a potentially sustainable means of chemical manufacturing. However, the tailoring of enzymes to industrial processes is often laborious and time consuming, which limits the broad implementation of this approach. High-throughput screening methods can expedite the search for suitable catalysts, but are often constrained by the need for labelled substrates. The generalization of such techniques would therefore significantly expand their impact. Here we have established a versatile ultrahigh-throughput microfluidic assay that enables isolation of functional oxidases from libraries that contain up to 107 members. The increased throughput over prevalent methods led to complete active-site remodelling of cyclohexylamine oxidase in one round of directed evolution. A 960-fold increase in catalytic efficiency afforded an enzyme with wild-type levels of activity for a non-natural substrate, allowing biocatalytic synthesis of a sterically demanding pharmaceutical intermediate with complete stereocontrol. The coupled enzyme assay is label free and can be easily adapted to re-engineer any oxidase.
MicroRNA (miRNA) is a class of short RNA that is emerging as an ideal biomarker, as its expression level has been found to correlate with different types of diseases including diabetes and cancer. The detection of miRNA is highly beneficial for early diagnostics and disease monitoring. However, miRNA sensing remains difficult because of its small size and low expression levels. Common techniques such as quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization and Northern blotting have been developed to quantify miRNA in a given sample. Nevertheless, these methods face common challenges in point-of-care practice as they either require complicated sample handling and expensive equipment, or suffer from low sensitivity. Here we present a new tool based on dark-field microwells to overcome these challenges in miRNA sensing. This miniaturized device enables the readout of a gold nanoparticle assay without the need of a dark-field microscope. We demonstrate the feasibility of the dark-field microwells to detect miRNA in both buffer solution and cell lysate. The dark-field microwells allow affordable miRNA sensing at a high throughput which make them a promising tool for point-of-care diagnostics.
Horizontal transfer of plasmids among bacterial populations is critical to genetic exchange and adaptation to environmental stresses. Studies have examined how bacterial cell density, donor-recipient ratio and nutrient availability affect plasmid transfer rates in homogeneous cultures. The insights are useful, however, they often neglect the ubiquitous physical heterogeneity of natural microbial habitats such as soil environments. Our hypothesis is that unsaturated and dynamic water conditions prevailing in most soils lead to fragmentation of the aquatic bacterial habitat, which could enhance plasmid spread through local (microscale) higher cell densities with reduced competition. However, the biophysical processes at play eschew quantitative observations at the cell level owing to soil complexity and opacity. We report and new microfluidic chip comprised of connected microhabitats that permit external control of aqueous fragmentation and observations at the single-cell level. These elements were used to quantify conjugation events in the microchip at various scales as function of fragmentation dynamics. We used the soil bacterium Pseudomonas putida as donor and recipient of a conjugative plasmid carrying a tetracycline resistance gene, while a tagging system with autofluorescent proteins allowed us to distinguish donors and transconjugants with epifluorescence microscopy. We successfully generated various patterns of aqueous fragmentation in the microchips, hence resulting in disconnected aqueous habitats for the bacteria. Plasmid transfer rate in individual microhabitats was linked to local cell densities as well as aqueous and gas phase distribution. This study highlights the importance of microhydrological processes that affect the ecology and evolution of bacteria in natural soil habitats.
Aims and Objectives. Transfer of conjugative plasmids among soil bacteria is an important evolutionary driver for fostering adaptation to environmental stresses such as antibiotic or xenobiotic contamination. However, plasmids impose a metabolic burden on host cells and the reason for their persistence over evolutionary times remains unclear. Here, we hypothesize that soil environments favor plasmid maintenance due to habitat fragmentation and nutrient spatial heterogeneity that promote plasmid transfer rates and reduce competition in microniches. We aim to test that hypothesis and disentangle the contributions of transmission and selection. Materials and Methods. The soil bacterium Pseudomonas putida was used as donor and recipient of the conjugative plasmid pIPO2tet (conferring resistance to tetracycline). A tagging system with fluorescent proteins allowed us to visually discriminate recipients, donors and transconjugants using microscope image analysis and plating on selective media. Bacteria were grown in controlled systems of varying complexity, from agar surfaces to micromodels, and in presence or absence of tetracycline as a selective agent. Results. Experiments on homogeneous agar surfaces showed that transfer rate and the final size of the plasmid-carrying population increased with cell density, while competition (as measured by selection coefficient) tended to decrease. The presence of antibiotics at sub-inhibitory concentrations also affected plasmid transfer rate and selection. To address the role of spatial isolation of microhabitats, we have designed micromodels that allow us to observe local variations in the spread of bacterial plasmids, with results still pending. Conclusions. Local (microscale) conditions such as cell density and spatial confinement can enhance or suppress plasmid transfer among bacterial populations, as well as affect the selective effects of carrying a plasmid. The study offers new insights linking soil microhabitats to ecological and evolutionary adaptations of soil bacteria.
This protocol provides a detailed description of how to fabricate and use the dual-flowRootChip (dfRootChip), a novel microfluidic platform for investigating root nutrition, root-microbe interactions and signaling and development in controlled asymmetric conditions. The dfRootChip was developed primarily to investigate how plants roots interact with their environment by simulating environmental heterogeneity. The goal of this protocol is to provide a detailed resource for researchers in the biological sciences wishing to employ the dfRootChip in particular, or microfluidic devices in general, in their laboratory.
Theoretical and in vivo neuroscience research suggests that functional information transfer within neuronal networks is influenced by circuit architecture. Due to the dynamic complexities of the brain, it remains a challenge to test the correlation between structure and function of a defined network. Engineering controlled neuronal networks in vitro offers a way to test structural motifs; however, no method has achieved small, multi-node networks with stable, unidirectional connections. Here, we screened ten different microchannel architectures within polydimethylsiloxane (PDMS) devices to test their potential for axonal guidance. The most successful design had a 92% probability of achieving strictly unidirectional connections between nodes. Networks built from this design were cultured on multielectrode arrays and recorded on days in vitro 9, 12, 15 and 18 to investigate spontaneous and evoked bursting activity. Transfer entropy between subsequent nodes showed up to 100 times more directional flow of information compared to the control. Additionally, directed networks produced a greater amount of information flow, reinforcing the importance of directional connections in the brain being critical for reliable communication. By controlling the parameters of network formation, we minimized response variability and achieved functional, directional networks. The technique provides us with a tool to probe the spatio-temporal effects of different network motifs.
High-throughput screening of a DNA library expressed in a bacterial population for identifying potentially rare members displaying a property of interest is a crucial step for success in many experiments such as directed evolution of proteins and synthetic circuits and deep mutational scanning to identify gain- or loss-of-function mutants. Here, I describe a protocol for high-throughput screening of bacterial (E. coli) microcolonies in gel beads. Single cells are encapsulated into monodisperse water-in-oil emulsion droplets produced with a microfluidic device. The aqueous solution also contains agarose that gelates upon cooling on ice, so that solid gel beads form inside the droplets. During incubation of the emulsion, the cells grow into monoclonal microcolonies inside the beads. After isolation of the gel beads from the emulsion and their sorting by fluorescence activated cell sorting (FACS), the bacteria are recovered from the gel beads and are then ready for a further round of sorting, mutagenesis or analysis. In order to sort by FACS, this protocol requires a fluorescent readout, such as the expression of a fluorescent reporter protein. Measuring the average fluorescent signals of microcolonies reduces the influence of high phenotypic cell-to-cell variability and increases the sensitivity compared to the sorting of single cells. We applied this method to sort a pBAD promoter library at ON and OFF states (Duarte et al., 2017).
Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g. being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclona bacterial microcolonies (e.g. expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.