Zeta potential characterization using commercial microfluidic chips.

Surface charge is a critical feature of microbes that affects their interactions with other cells and their environment. Because bacterial surface charge is difficult to measure directly, it is typically indirectly inferred through zeta potential measurements. Existing tools to perform such characterization are either costly and ill-suited for non-spherical samples or rely on microfluidic techniques requiring expensive fabrication equipment or specialized facilities. Here, we report the application of commercially available PMMA microfluidic chips and open-source data analysis workflows for facile electrokinetic characterization of particles and cells after prior zeta potential measurement with a Zetasizer for calibration. Our workflows eliminate the need for microchannel fabrication, increase measurement reproducibility, and make zeta potential measurements more accessible. This novel methodology was tested with functionalized 1 µm and 2 µm polystyrene beads as well as Escherichia coli MG1655 strain. Measured zeta potentials for these samples were in agreement with literature values obtained by conventional measurement methods. Taken together, our data demonstrate the power of this workflow to broadly enable critical measurements of particle and bacterial zeta potential for numerous applications.

A mutant fitness compendium in Bifidobacteria reveals molecular determinants of colonization and host-microbe interactions.

Bifidobacteria commonly represent a dominant constituent of human gut microbiomes during infancy, influencing nutrition, immune development, and resistance to infection. Despite interest as a probiotic therapy, predicting the nutritional requirements and health-promoting effects of Bifidobacteria is challenging due to major knowledge gaps. To overcome these deficiencies, we used large-scale genetics to create a compendium of mutant fitness in Bifidobacterium breve (Bb). We generated a high density, randomly barcoded transposon insertion pool in Bb, and used this pool to determine Bb fitness requirements during colonization of germ-free mice and chickens with multiple diets and in response to hundreds of in vitro perturbations. To enable mechanistic investigation, we constructed an ordered collection of insertion strains covering 1462 genes. We leveraged these tools to improve models of metabolic pathways, reveal unexpected host- and diet-specific requirements for colonization, and connect the production of immunomodulatory molecules to growth benefits. These resources will greatly reduce the barrier to future investigations of this important beneficial microbe.

Leveraging microfluidic dielectrophoresis to distinguish compositional variations of lipopolysaccharide in E. coli.

Lipopolysaccharide (LPS) is the unique feature that composes the outer leaflet of the Gram-negative bacterial cell envelope. Variations in LPS structures affect a number of physiological processes, including outer membrane permeability, antimicrobial resistance, recognition by the host immune system, biofilm formation, and interbacterial competition. Rapid characterization of LPS properties is crucial for studying the relationship between these LPS structural changes and bacterial physiology. However, current assessments of LPS structures require LPS extraction and purification followed by cumbersome proteomic analysis. This paper demonstrates one of the first high-throughput and non-invasive strategies to directly distinguish Escherichia coli with different LPS structures. Using a combination of three-dimensional insulator-based dielectrophoresis (3DiDEP) and cell tracking in a linear electrokinetics assay, we elucidate the effect of structural changes in E. coli LPS oligosaccharides on electrokinetic mobility and polarizability. We show that our platform is sufficiently sensitive to detect LPS structural variations at the molecular level. To correlate electrokinetic properties of LPS with the outer membrane permeability, we further examined effects of LPS structural variations on bacterial susceptibility to colistin, an antibiotic known to disrupt the outer membrane by targeting LPS. Our results suggest that microfluidic electrokinetic platforms employing 3DiDEP can be a useful tool for isolating and selecting bacteria based on their LPS glycoforms. Future iterations of these platforms could be leveraged for rapid profiling of pathogens based on their surface LPS structural identity.

Recent Advancements in Electroporation Technologies: From Bench to Clinic.

Over the past decade, the increased adoption of electroporation-based technologies has led to an expansion of clinical research initiatives. Electroporation has been utilized in molecular biology for mammalian and bacterial transfection; for food sanitation; and in therapeutic settings to increase drug uptake, for gene therapy, and to eliminate cancerous tissues. We begin this article by discussing the biophysics required for understanding the concepts behind the cell permeation phenomenon that is electroporation. We then review nano- and microscale single-cell electroporation technologies before scaling up to emerging in vivo applications.

M-TUBE enables large-volume bacterial gene delivery using a high-throughput microfluidic electroporation platform.

Conventional cuvette-based and microfluidics-based electroporation approaches for bacterial gene delivery have distinct advantages, but they are typically limited to relatively small sample volumes, reducing their utility for applications requiring high throughput such as the generation of mutant libraries. Here, we present a scalable, large-scale bacterial gene delivery approach enabled by a disposable, user-friendly microfluidic electroporation device requiring minimal device fabrication and straightforward operation. We demonstrate that the proposed device can outperform conventional cuvettes in a range of situations, including across Escherichia coli strains with a range of electroporation efficiencies, and we use its large-volume bacterial electroporation capability to generate a library of transposon mutants in the anaerobic gut commensal Bifidobacterium longum.

In situ continuous electrochemical quantification of bacterial adhesion to electrically polarized metallic surfaces under shear.

Biofouling creates significant human and economic losses through infections, corrosion, and drag losses on ships and in oil and food distribution pipelines. Organisms adhered to these surfaces contend with high shear rates and are actively transported to the surface. The metallic surfaces to which these organisms are adhered also conduct charge at various potentials, and the effects of surface charge on adhesion rates are little addressed in the literature. We demonstrate that mass-transport limiting current, chronoamperometry, and cyclic voltammetry can be combined to provide resulting adhesion rates similar to those in the literature. Furthermore, we demonstrate that rotating disk electrodes can be used to study adhesion of bacteria to electrically polarized metallic surfaces under shear. We study the adhesion of Escherichia coli, Bacillus subtilis, and 1µm silica microspheres over a range of shear stress from 0.15 to 37 dyncm-2 or shear rates of 14.7-3730 s-1. Unlike quartz-crystal microbalance, our methodology measures changes in the area instead of mass, simultaneously providing measurements of the protein binding. Our deposition rates agree with those found using optical systems. However, unlike optical systems, our methods apply to a wider range of materials than on-chip flow devices.

Characterizing chemical signaling between engineered "microbial sentinels" in porous microplates.

Living materials combine a material scaffold, that is often porous, with engineered cells that perform sensing, computing, and biosynthetic tasks. Designing such systems is difficult because little is known regarding signaling transport parameters in the material. Here, the development of a porous microplate is presented. Hydrogel barriers between wells have a porosity of 60% and a tortuosity factor of 1.6, allowing molecular diffusion between wells. The permeability of dyes, antibiotics, inducers, and quorum signals between wells were characterized. A "sentinel" strain was constructed by introducing orthogonal sensors into the genome of Escherichia coli MG1655 for IPTG, anhydrotetracycline, L-arabinose, and four quorum signals. The strain's response to inducer diffusion through the wells was quantified up to 14 mm, and quorum and antibacterial signaling were measured over 16 h. Signaling distance is dictated by hydrogel adsorption, quantified using a linear finite element model that yields adsorption coefficients from 0 to 0.1 mol m-3 . Parameters derived herein will aid the design of living materials for pathogen remediation, computation, and self-organizing biofilms.

Bacterial response to spatial gradients of algal-derived nutrients in a porous microplate.

Photosynthetic microalgae are responsible for 50% of the global atmospheric CO2 fixation into organic matter and hold potential as a renewable bioenergy source. Their metabolic interactions with the surrounding microbial community (the algal microbiome) play critical roles in carbon cycling, but due to methodological limitations, it has been challenging to examine how community development is influenced by spatial proximity to their algal host. Here we introduce a copolymer-based porous microplate to co-culture algae and bacteria, where metabolites are constantly exchanged between the microorganisms while maintaining physical separation. In the microplate, we found that the diatom Phaeodactylum tricornutum accumulated to cell abundances ~20 fold higher than under normal batch conditions due to constant replenishment of nutrients through the porous structure. We also demonstrate that algal-associated bacteria, both single isolates and complex communities, responded to inorganic nutrients away from their host as well as organic nutrients originating from the algae in a spatially predictable manner. These experimental findings coupled with a mathematical model suggest that host proximity and algal culture growth phase impact bacterial community development in a taxon-specific manner through organic and inorganic nutrient availability. Our novel system presents a useful tool to investigate universal metabolic interactions between microbes in aquatic ecosystems.

Anti-Black racism in academia and what you can do about it.

The experiences of Black scientists and engineers reveal that science is not a meritocracy. Here is a list of recommendations to combat anti-Black racism in academic institutions.

Microfluidic dielectrophoresis illuminates the relationship between microbial cell envelope polarizability and electrochemical activity.

Electrons can be transported from microbes to external insoluble electron acceptors (e.g., metal oxides or electrodes in an electrochemical cell). This process is known as extracellular electron transfer (EET) and has received considerable attention due to its applications in environmental remediation and energy conversion. However, the paucity of rapid and noninvasive phenotyping techniques hinders a detailed understanding of microbial EET mechanisms. Most EET phenotyping techniques assess microorganisms based on their metabolism and growth in various conditions and/or performance in electrochemical systems, which requires large sample volumes and cumbersome experimentation. Here, we use microfluidic dielectrophoresis to show a strong correlation between bacterial EET and surface polarizability. We analyzed surface polarizabilities for wild-type strains and cytochrome-deletion mutants of two model EET microbes, Geobacter sulfurreducens and Shewanella oneidensis, and for Escherichia coli strains heterologously expressing S. oneidensis EET pathways in various growth conditions. Dielectrophoretic phenotyping is achieved with small cell culture volumes (~100 µl) in a short amount of time (1 to 2 min per strain). Our work demonstrates that cell polarizability is diminished in response to deletions of crucial outer-membrane cytochromes and enhanced due to additions of EET pathways. Results of this work hold exciting promise for rapid screening of direct EET or other cell envelope phenotypes using cell polarizability as a proxy, especially for microbes difficult to cultivate in laboratory conditions.

Continuous shear stress alters metabolism, mass-transport, and growth in electroactive biofilms independent of surface substrate transport.

Electroactive bacteria such as Geobacter sulfurreducens and Shewanella onedensis produce electrical current during their respiration; this has been exploited in bioelectrochemical systems. These bacteria form thicker biofilms and stay more active than soluble-respiring bacteria biofilms because their electron acceptor is always accessible. In bioelectrochemical systems such as microbial fuel cells, corrosion-resistant metals uptake current from the bacteria, producing power. While beneficial for engineering applications, collecting current using corrosion resistant metals induces pH stress in the biofilm, unlike the naturally occurring process where a reduced metal combines with protons released during respiration. To reduce pH stress, some bioelectrochemical systems use forced convection to enhance mass transport of both nutrients and byproducts; however, biofilms' small pore size limits convective transport, thus, reducing pH stress in these systems remains a challenge. Understanding how convection is necessary but not sufficient for maintaining biofilm health requires decoupling mass transport from momentum transport (i.e. fluidic shear stress). In this study we use a rotating disc electrode to emulate a practical bioelectrochemical system, while decoupling mass transport from shear stress. This is the first study to isolate the metabolic and structural changes in electroactive biofilms due to shear stress. We find that increased shear stress reduces biofilm development time while increasing its metabolic rate. Furthermore, we find biofilm health is negatively affected by higher metabolic rates over long-term growth due to the biofilm's memory of the fluid flow conditions during the initial biofilm development phases. These results not only provide guidelines for improving performance of bioelectrochemical systems, but also reveal features of biofilm behavior. Results of this study suggest that optimized reactors may initiate operation at high shear to decrease development time before decreasing shear for steady-state operation. Furthermore, this biofilm memory discovered will help explain the presence of channels within biofilms observed in other studies.

Numerical study of the effect of soft layer properties on bacterial electroporation.

We present a numerical model of electroporation in a gram-positive bacterium, which accounts for the presence of a negatively charged soft polyelectrolyte layer (which may include a periplasmic space, peptidoglycan layer, cilia, flagella, and other surface appendages) surrounding its plasma membrane. We model the ion transport within and outside the soft layer using the soft layer electrokinetics-based Poisson-Nernst-Planck formalism. Additionally, we model the electroporation dynamics on the plasma membrane using the pore nucleation-based electroporation formalism developed by Krassowska and Filev. We find that ion transport within the soft layer (surface conduction), which depends on the relative importance of the soft layer charged group concentration compared to the buffer concentration, significantly alters the transmembrane voltage across the plasma membrane and hence the pore characteristics. Our numerical simulations suggest that surface conduction significantly lowers the pore number in the plasma membrane. This observation is consistent with experimental studies that show that gram-positive bacteria, in general, have lower transformation efficiencies compared to gram-negative bacteria. Our studies highlight a strong dependence of bacterial electroporation on cell envelope properties and buffer conditions, which need to be taken into consideration when designing electroporation protocols.

Nonlinear electrokinetic effects in insulator-based dielectrophoretic systems.

Insulator-based dielectrophoresis (iDEP) has emerged as a powerful tool for multiple biomicrofluidic operations, such as cell separation and concentration. The key feature for iDEP systems is the alteration of insulating microchannel geometries to create strong electric field gradients. Under AC electric fields, this strong electric field gradient can affect fluid flow by (at least) two nonlinear electrokinetic phenomena; (a) electrothermal flow due to Joule heating and (b) induced charge electroosmosis (ICEO) near the microchannel constrictions of small (but finite) permittivity and conductivity. This paper presents an experimental and theoretical study on the interplay of electrothermal and ICEO flows near microchannel constrictions with various geometries and fluid ionic strengths, which are crucial design factors for iDEP systems. Temperature rise and fluid velocities in 2D Gaussian-shaped constrictions were studied experimentally with supporting analytical estimations and numerical simulations. Additionally, we show qualitatively distinct recirculating flow patterns in 2D and 3D microchannel constrictions used for iDEP systems. Approximate analytical expressions for electrothermal and ICEO velocity scales are provided as a function of constriction geometry, bulk electrolyte concentration, and the applied electric field. Insights from this study will be useful in designing microfluidic systems for electrokinetic particle manipulation.

Toward establishing model organisms for marine protists: Successful transfection protocols for Parabodo caudatus (Kinetoplastida: Excavata).

We developed protocols for, and demonstrated successful transfection of, the free-living kinetoplastid flagellate Parabodo caudatus with three plasmids carrying a fluorescence reporter gene (pEF-GFP with the EF1 alpha promoter, pUB-GFP with Ubiquitin C promoter, and pEYFP-Mitotrap with CMV promoter). We evaluated three electroporation approaches: (1) a square-wave electroporator designed for eukaryotes, (2) a novel microfluidic transfection system employing hydrodynamically-controlled electric field waveforms, and (3) a traditional exponential decay electroporator. We found the microfluidic device provides a simple and efficient platform to quickly test a wide range of electric field parameters to find the optimal set of conditions for electroporation of target species. It also allows for processing large sample volumes (>10 ml) within minutes, increasing throughput 100 times over cuvettes. Fluorescence signal from the reporter gene was detected a few hours after transfection and persisted for 3 days in cells transfected by pEF-GFP and pUB-GFP plasmids and for at least 5 days post-transfection for cells transfected with pEYFP-Mitotrap. Expression of the reporter genes (GFP and YFP) was also confirmed using reverse transcription-PCR (RT-PCR). This work opens the door for further efforts with this taxon and close relatives toward establishing model systems for genome editing.

Bioaerosol generation by raindrops on soil.

Aerosolized microorganisms may play an important role in climate change, disease transmission, water and soil contaminants, and geographic migration of microbes. While it is known that bioaerosols are generated when bubbles break on the surface of water containing microbes, it is largely unclear how viable soil-based microbes are transferred to the atmosphere. Here we report a previously unknown mechanism by which rain disperses soil bacteria into the air. Bubbles, tens of micrometres in size, formed inside the raindrops disperse micro-droplets containing soil bacteria during raindrop impingement. A single raindrop can transfer 0.01% of bacteria on the soil surface and the bacteria can survive more than one hour after the aerosol generation process. This work further reveals that bacteria transfer by rain is highly dependent on the regional soil profile and climate conditions.

High efficiency hydrodynamic bacterial electrotransformation.

Synthetic biology holds great potential for addressing pressing challenges for mankind and our planet. One technical challenge in tapping into the full potential of synthetic biology is the low efficiency and low throughput of genetic transformation for many types of cells. In this paper, we discuss a novel microfluidic system for improving bacterial electrotransformation efficiency and throughput. Our microfluidic system is comprised of non-uniform constrictions in microchannels to facilitate high electric fields with relatively small applied voltages to induce electroporation. Additionally, the microfluidic device has regions of low electric field to assist in electrophoretic transport of nucleic acids into the cells. The device features hydrodynamically controlled electric fields that allow cells to experience a time dependent electric field that is otherwise difficult to achieve using standard electronics. Results suggest that transformation efficiency can be increased by ∼4×, while throughput can increase by 100-1000× compared to traditional electroporation cuvettes. This work will enable high-throughput and high efficiency genetic transformation of microbes, facilitating accelerated development of genetically engineered organisms.

Microfluidic Screening of Electric Fields for Electroporation.

Electroporation is commonly used to deliver molecules such as drugs, proteins, and/or DNA into cells, but the mechanism remains poorly understood. In this work a rapid microfluidic assay was developed to determine the critical electric field threshold required for inducing bacterial electroporation. The microfluidic device was designed to have a bilaterally converging channel to amplify the electric field to magnitudes sufficient to induce electroporation. The bacterial cells are introduced into the channel in the presence of SYTOX(®), which fluorescently labels cells with compromised membranes. Upon delivery of an electric pulse, the cells fluoresce due to transmembrane influx of SYTOX(®) after disruption of the cell membranes. We calculate the critical electric field by capturing the location within the channel of the increase in fluorescence intensity after electroporation. Bacterial strains with industrial and therapeutic relevance such as Escherichia coli BL21 (3.65 ± 0.09 kV/cm), Corynebacterium glutamicum (5.20 ± 0.20 kV/cm), and Mycobacterium smegmatis (5.56 ± 0.08 kV/cm) have been successfully characterized. Determining the critical electric field for electroporation facilitates the development of electroporation protocols that minimize Joule heating and maximize cell viability. This assay will ultimately enable the genetic transformation of bacteria and archaea considered intractable and difficult-to-transfect, while facilitating fundamental genetic studies on numerous diverse microbes.

Nanoporous microscale microbial incubators.

Reconstruction of phylogenetic trees based on 16S rRNA gene sequencing reveals abundant microbial diversity that has not been cultured in the laboratory. Many attribute this so-called 'great plate count anomaly' to traditional microbial cultivation techniques, which largely facilitate the growth of a single species. Yet, it is widely recognized that bacteria in nature exist in complex communities. One technique to increase the pool of cultivated bacterial species is to co-culture multiple species in a simulated natural environment. Here, we present nanoporous microscale microbial incubators (NMMI) that enable high-throughput screening and real-time observation of multi-species co-culture. The key innovation in NMMI is that they facilitate inter-species communication while maintaining physical isolation between species, which is ideal for genomic analysis. Co-culture of a quorum sensing pair demonstrates that the NMMI can be used to culture multiple species in chemical communication while monitoring the growth dynamics of individual species.

Antiwetting Fabric Produced by a Combination of Layer-by-Layer Assembly and Electrophoretic Deposition of Hydrophobic Nanoparticles.

This work describes a nanoparticle coating method to produce durable antiwetting polyester fabric. Electrophoretic deposition is used for fast modification of polyester fabric with silica nanoparticles embedded in polymeric networks for high durability coatings. Typically, electrophoretic deposition (EPD) is utilized on electrically conductive substrates due to its dependence on an applied electrical field. EPD on nonconductive materials has been attempted but are limited by weak adhesion, cracks, and other irregularities. To resolve these issues, we coat polyester fabric with thin polymer layers using electrostatic self-assembly (layer-by-layer self-assembly). Next, silica nanoparticles are uniformly dispersed on the polymer layers. Finally, polymerically stabilized silica nanoparticles are deposited by EPD on the fabric, followed by heat treatment. The modified fabric shows high static contact angle and low contact angle hysteresis, while keeping its original color, flexibility, and air permeability. During a skin fiction resistance test, the hydrophobicity of the coating layer was maintained over 500 h. Furthermore, we also show that this approach facilitates patterned regions of wettability by modifying the electric field in EPD.

Antimicrobial behavior of novel surfaces generated by electrophoretic deposition and breakdown anodization.

Biofilms have devastating impacts on many industries such as increased fuel consumption and damage to surfaces in maritime industries. Ideal biofouling management is inhibition of initial bacterial attachment. The attachment of a model marine bacterium (Halomonas pacfica g) was investigated to evaluate the potential of these new novel surfaces to resist initial bacterial adhesion. Novel engineered surfaces were generated via breakdown anodization or electrophoretic deposition, to modify three parameters: hydrophobicity, surface chemistry, and roughness. Mass transfer rates were determined using a parallel plate flow chamber under relevant solution chemistries. The greatest deposition was observed on the superhydrophilic surface, which had micro- and nano-scale hierarchical structures composed of titanium oxide deposited on a titanium plate. Conversely, one of the hydrophobic surfaces with micro-porous films overlaid with polydimethylsiloxane appeared to be most resistant to cell attachment.