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Digital microfluidics (DMF) has the signatures of an ideal liquid handling platform - as shown through almost two decades of automated biological and chemical assays. However, in the current state of DMF, we are still limited by the number of parallel biological or chemical assays that can be performed on DMF. Here, we report a new approach that leverages design-of-experiment and numerical methodologies to accelerate experimental optimization on DMF. The integration of the one-factor-at-a-time (OFAT) experimental technique with machine learning algorithms provides a set of recommended optimal conditions without the need to perform a large set of experiments. We applied our approach towards optimizing the radiochemistry synthesis yield given the large number of variables that affect the yield. We believe that this work is the first to combine such techniques which can be readily applied to any other assays that contain many parameters and levels on DMF.

Viral-based systems are a popular delivery method for introducing exogenous genetic material into mammalian cells. Unfortunately, the preparation of lentiviruses containing the machinery to edit the cells is labor-intensive, with steps requiring optimization and sensitive handling. To mitigate these challenges, we introduce the first microfluidic method that integrates lentiviral generation, packaging, and transduction. The new method allows the production of viral titers between 10

We present a novel "on-off", cost-effective, rapid electrochemical aptasensor combined with a microfluidics cartridge system for the detection of Δ

In this paper, we describe the design and performance of two digital microfluidics (DMF) chips capable of executing multiple ribozymatic reactions, with proper controls, in response to short single-stranded DNA inducers. Since the fluorescence output of a reaction is measurable directly from the chip, without the need for gel electrophoresis, a complete experiment involving up to eight reactions (per chip) can be carried out reliably, relatively quickly, and efficiently. The ribozymes can also be used as biosensors of the concentration of oligonucleotide inputs, with high sensitivity, low limits of quantification and of detection, and excellent signal-to-noise ratio. The presented chips are readily usable devices that can be used to automate, speed up, and reduce the costs of ribozymatic reaction experiments.

Optimization of engineered biological systems requires precise control over the rates and timing of gene expression. Optogenetics is used to dynamically control gene expression as an alternative to conventional chemical-based methods since it provides a more convenient interface between digital control software and microbial culture. Here, we describe the construction of a real-time optogenetics platform, which performs closed-loop control over the CcaR-CcaS two-plasmid system in

DNA assembly and transformation are crucial to the building process in synthetic biology. These steps are significant roadblocks when engineering increasingly complex biological systems. To address this, recent development of widespread 'biofoundry' facilities has employed automation equipment to expedite the synthetic biology workflow. Despite significant progress, there is a clear demand for lower-cost and smaller-footprint automation equipment. The field of microfluidics have emerged to provide automation capabilities to meet this demand. However, we still lack devices capable of building large multi-gene systems in a consolidated process. In response to this challenge, we have developed a digital microfluidic platform that performs "one-pot" Golden Gate DNA assembly of large plasmids and transformation of E coli. The system features a novel electrode geometry and modular design, which make these devices simple to fabricate and use, thus improving the accessibility of microfluidics. This device incorporates an impedance-based adaptive closed loop water replenishment system to compensate for droplet evaporation and maintain constant assembly reaction concentrations, which we found to be crucial to the DNA assembly efficiency. We also showcase a closed-loop temperature control system that generates precise thermodynamic profiles to optimize heat shock transformation. Moreover, we validated the system by assembling and transforming large and complex plasmids conferring a biosynthetic pathway, resulting in performance comparable to those of standard techniques. We propose that the methods described here will contribute to a new generation of accessible automation platforms aimed at speeding up the 'building' process, lowering reagent consumption and removing manual work from synthetic biology.

Synthetic biology is rapidly growing to meet the demand for inexpensive and sustainable resources. So far, the field has developed microbial strains producing biofuels, materials, drugs, as well as new tools for clinical diagnostics and gene therapy. Although rich in potential, synthetic biology still requires development – particularly in the area of automation. The price and footprint of commercially available automation equipment is restrictive to research and these tools are often not tailored to complete the entire workflow of a given project. In response to this, droplet microfluidic platforms are being developed to expedite synthetic biology. In particular, droplet microfluidic devices have been developed to assemble and transform DNA, perform high-throughput screening assays and perform directed evolution. By consolidating these capabilities and pairing them with design automation and analysis tools, droplet microfluidics will launch a new generation of synthetic biology.

The expression of a recombinant gene in a host organism through induction can be an extensively manual and labor-intensive procedure. Several methods have been developed to simplify the protocol, but none has fully replaced the traditional IPTG-based induction. To simplify this process, we describe the development of an autoinduction platform based on digital microfluidics. This system consists of a 600 nm LED and a light sensor to enable the real-time monitoring of the optical density (OD) samples coordinated with the semicontinuous mixing of a bacterial culture. A hand-held device was designed as a microbioreactor to culture cells and to measure the OD of the bacterial culture. In addition, it serves as a platform for the analysis of regulated protein expression in E. coli without the requirement of standardized well-plates or pipetting-based platforms. Here, we report for the first time, a system that offers great convenience without the user to physically monitor the culture or to manually add inducer at specific times. We characterized our system by looking at several parameters (electrode designs, gap height, and growth rates) required for an autoinducible system. As a first step, we carried out an automated induction optimization assay using a RFP reporter gene to identify conditions suitable for our system. Next, we used our system to identify active thermophilic β-glucosidase enzymes that may be suitable candidates for biomass hydrolysis. Overall, we believe that this platform may be useful for synthetic biology applications that require regulating and analyzing expression of heterologous genes for strain optimization.

Gene-editing techniques such as RNA-guided endonuclease systems are becoming increasingly popular for phenotypic screening. Such screens are normally conducted in arrayed or pooled formats. There has been considerable interest in recent years to find new technological methods for conducting these gene-editing assays. We report here the first digital microfluidic method that can automate arrayed gene-editing in mammalian cells. Specifically, this method was useful in culturing lung cancer cells for up to six days, as well as implementing automated gene transfection and knockout procedures. In addition, a standardized imaging pipeline to analyse fluorescently labelled cells was also designed and implemented during these procedures. A gene editing assay for interrogating the MAPK/ERK pathway was performed to show the utility of our platform and to determine the effects of knocking out the RAF1 gene in lung cancer cells. In addition to gene knockout, we also treated the cells with an inhibitor, Sorafenib Tosylate, to determine the effects of enzymatic inhibition. The combination of enzymatic inhibition and guide targeting on device resulted in lower drug concentrations for achieving half-inhibitory effects (IC50) compared to cells treated only with the inhibitor, confirming that lung cancer cells are being successfully edited on the device. We propose that this system will be useful for other types of gene-editing assays and applications related to personalized medicine.

The process of generating a stable knockout cell line is a complex process that can take several months to complete. In this work, we introduce a microfluidic method that is capable of isolating single cells, selecting successful edited clones, and expansion of these isoclones. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In our hybrid microfluidic device, we show that we can trap single cells and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, individual cells inside the droplet can be released from the traps or merged with other droplets by simply applying an electric potential to the electrodes that is actuated through a user interface. We use this high precision control to sort and to recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knock-out transfections.

Digital microfluidics (DMF) represents an alternative to the conventional microfluidic paradigm of transporting fluids in enclosed channels. One of the major benefits of DMF is that fluid motion and control is achieved without external pumps. The automation component of DMF have pushed the barriers of this "lab-on-chip" technology. However, integration with external components (i.e., "world-to-chip") interfaces have been a challenge. Two common "world-to-chip" challenges are (1) delivering biological samples to DMF devices and (2) accurately controlling temperatures on device. To address these challenges, this work describes two "world-to-chip" interface features that have been integrated on a DMF platform: a reagent delivery system and a thermal control apparatus. This platform enables a variety of biological or chemical experiments to be conducted on-chip while reducing manual intervention. Specifically, our platform increases reagent volumes available to device reservoirs volume by at least 50-fold eliminating the need to manually refill reservoirs while improving droplet dispensing reproducibility. In addition, we have integrated a closed-loop temperature control system that offers precise temperature control on-chip. To validate our "world-to-chip" interface, we have automated bacterial transformation and enzymatic assay protocols, showing that such a system enhances DMF performance. Overall, we propose that this system will improve biological experimentation which requires fluidic and temperature control integrated on DMF platforms.

Digital microfluidics (DMF) is a technology that provides a means of manipulating nL-μL volumes of liquids on an array of electrodes. By applying an electric potential to an electrode, these discrete droplets can be controlled in parallel which can be transported, mixed, reacted, and analyzed. Typically, an automation system is interfaced with a DMF device that uses a standard set of basic instructions written by the user to execute droplet operations. Here, we present the first feedback method for DMF that relies on imaging techniques that will allow online detection of droplets without the need to reactivate all destination electrodes. Our system consists of integrating open-source electronics with a CMOS camera and a zoom lens for acquisition of the images that will be used to detect droplets on the device. We also created an algorithm that uses a Hough transform to detect a variety of droplet sizes and to detect singular droplet dispensing and movement failures on the device. As a first test, we applied this feedback system to test droplet movement for a variety of liquids used in cell-based assays and to optimize different feedback actuation schemes to improve droplet movement fidelity. We also applied our system to a colorimetric enzymatic assay to show that our system is capable of biological analysis. Overall, we believe that using our approach of integrating imaging and feedback for DMF can provide a platform for automating biological assays with analysis.

Biological assays often require expensive reagents and tedious manipulations. These shortcomings can be overcome using digitally operated microfluidic devices that require reduced sample volumes to automate assays. One particular challenge is integrating bioassays with mass spectrometry based analysis. Towards this goal we have developed μNIMS, a highly sensitive and high throughput technique that integrates droplet microfluidics with nanostructure-initiator mass spectrometry (NIMS). Enzyme reactions are carried out in droplets that can be arrayed on discrete NIMS elements at defined time intervals for subsequent mass spectrometry analysis, enabling time resolved enzyme activity assay. We apply the μNIMS platform for kinetic characterization of a glycoside hydrolase enzyme (CelE-CMB3A), a chimeric enzyme capable of deconstructing plant hemicellulose into monosaccharides for subsequent conversion to biofuel. This study reveals NIMS nanostructures can be fabricated into arrays for microfluidic droplet deposition, NIMS is compatible with droplet and digital microfluidics, and can be used on-chip to assay glycoside hydrolase enzyme in vitro.

Acclaimed as one of the biggest scientific breakthroughs, the technology of CRISPR has brought significant improvement in the biotechnological spectrum—from editing genetic defects in diseases for gene therapy to modifying organisms for the production of biofuels. Since its inception, the CRISPR-Cas9 system has become easier and more versatile to use. Many variants have been found, giving the CRISPR toolkit a great range that includes the activation and repression of genes aside from the previously known knockout and knockin of genes. Here, in this Perspective, we describe efforts on automating the gene-editing workflow, with particular emphasis given on the use of microfluidic technology. We discuss how automation can address the limitations of gene-editing and how the marriage between microfluidics and gene-editing will expand the application space of CRISPR.

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co-planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL-sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in-house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI-H1299 lung squamous cell population resulting from loss-of-function eGFP and RAF1 gene knockout transfections.

Droplet microfluidics is a technique that has the ability to compartmentalize reactions in sub nano- (or pico-) liter volumes that can potentially enable millions of distinct biological assays to be performed on individual cells. In a typical droplet microfluidic system, droplets are manipulated by pressure-based flows. This has limited the fluidic operations that can be performed in these devices. Digital microfluidics is an alternative microfluidic paradigm with precise control and manipulation over individual droplets. Here, we implement an integrated droplet-digital microfluidic (which we call 'ID2M') system in which common fluidic operations (i.e. droplet generation, cell encapsulation, droplet merging and mixing, droplet trapping and incubation, and droplet sorting) can be performed. With the addition of electrodes, we have been able to create droplets on-demand, tune their volumes on-demand, and merge and mix several droplets to produce a dilution series. Moreover, this device can trap and incubate droplets for 24 h that can consequently be sorted and analyzed in multiple n-ary channels (as opposed to typical binary channels). The ID2M platform has been validated as a robust on-demand screening system by sorting fluorescein droplets of different concentration with an efficiency of ∼96%. The utility of the new system is further demonstrated by culturing and sorting tolerant yeast mutants and wild-type yeast cells in ionic liquid based on their growth profiles. This new platform for both droplet and digital microfluidics has the potential to be used for screening different conditions on-chip and for applications like directed evolution.

We have developed a new hybrid droplet-to-digital microfluidic platform (D2D) that integrates droplet-in-channel microfluidics with digital microfluidics (DMF) for performing multi-step assays. This D2D platform combines the strengths of the two formats-droplets-in-channel for facile generation of droplets containing single cells, and DMF for on-demand manipulation of droplets including control of different droplet volumes (pL-μL), creation of a dilution series of ionic liquid (IL), and parallel single cell culturing and analysis for IL toxicity screening. This D2D device also allows for automated analysis that includes a feedback-controlled system for merging and splitting of droplets to add reagents, an integrated Peltier element for parallel cell culture at optimum temperature, and an impedance sensing mechanism to control the flow rate for droplet generation and preventing droplet evaporation. Droplet-in-channel is well-suited for encapsulation of single cells as it allows the careful manipulation of flow rates of aqueous phase containing cells and oil to optimize encapsulation. Once single cell containing droplets are generated, they are transferred to a DMF chip via a capillary where they are merged with droplets containing IL and cultured at 30 °C. The DMF chip, in addition to permitting cell culture and reagent (ionic liquid/salt) addition, also allows recovery of individual droplets for off-chip analysis such as further culturing and measurement of ethanol production. The D2D chip was used to evaluate the effect of IL/salt type (four types: NaOAc, NaCl, [C2mim] [OAc], [C2mim] [Cl]) and concentration (four concentrations: 0, 37.5, 75, 150 mM) on the growth kinetics and ethanol production of yeast and as expected, increasing IL concentration led to lower biomass and ethanol production. Specifically, [C2mim] [OAc] had inhibitory effects on yeast growth at concentrations 75 and 150 mM and significantly reduced their ethanol production compared to cells grown in other ILs/salts. The growth curve trends obtained by D2D matched conventional yeast culturing in microtiter wells, validating the D2D platform. We believe that our approach represents a generic platform for multi-step biochemical assays such as drug screening, digital PCR, enzyme assays, immunoassays and cell-based assays.

Sialyl-LewisX and LewisX are cell-surface glycans that influence cell-cell adhesion behaviors. These glycans are assembled by α(1,3)-fucosyltransferase enzymes. Their increased expression plays a role in inflammatory disease, viral and microbial infections, and cancer. Efficient screens for specific glycan modifications such as those catalyzed by fucosyltransferases are tended toward costly materials and large instrumentation. We demonstrate for the first time a fucosylation inhibition assay on a digital microfluidic system with the integration of image-based techniques. Specifically, we report a novel lab-on-a-chip approach to perform a fluorescence-based inhibition assay for the fucosylation of a labeled synthetic disaccharide, 4-methylumbelliferyl β-N-acetyllactosaminide. As a proof-of-concept, guanosine 5′-diphosphate has been used to inhibit Helicobacter pylori α(1,3)-fucosyltransferase. An electrode shape (termed “skewed wave”) is designed to minimize electrode density and improve droplet movement compared to conventional square-based electrodes. The device is used to generate a 10 000-fold serial dilution of the inhibitor and to perform fucosylation reactions in aqueous droplets surrounded by an oil shell. Using an image-based method of calculating dilutions, referred to as “pixel count,” inhibition curves along with IC(50) values are obtained on-device. We propose the combination of integrating image analysis and digital microfluidics is suitable for automating a wide range of enzymatic assays.

Background: Dried blood spot (DBS) sampling is emerging as a valuable technique in a variety of fields, including clinical and preclinical testing of pharmaceuticals. Despite this popularity, current DBS sampling and analysis processes remain laborious and time consuming. Digital microfluidics, a microscale liquid-handling technique, characterized by the manipulation of discrete droplets on open electrode arrays, offers a potential solution to these problems. Results: We report a new digital microfluidic method for multiplexed extraction and analysis of pharmaceuticals in DBS samples. In the new method, four DBS samples are extracted in microliter-sized droplets containing internal standard, and the extract is delivered to dedicated nanoelectrospray ionization emitters for direct analysis by tandem mass spectometry and selected reaction monitoring. Conclusion: The new method allows for an order of magnitude reduction in processing time and approximately three-times reduction in extraction solvent relative to conventional techniques, while maintaining acceptable analytical performance for most drugs tested.

Synthetic biology follows the traditional engineering paradigm of designing, building, testing and learning to create new biological systems. While such approaches have enormous potential, major challenges still exist in this field including increasing the speed at which this workflow can be performed. Here, we present recently developed microfluidic tools that can be used to automate the synthetic biology workflow with the goal of advancing the likelihood of producing desired functionalities. With the potential for programmability, automation, and robustness, the integration of microfluidics and synthetic biology has the potential to accelerate advances in areas such as bioenergy, health, and biomaterials.

We present a water-in-oil droplet microfluidic platform for transformation, culture and expression of recombinant proteins in multiple host organisms including bacteria, yeast and fungi. The platform consists of a hybrid digital microfluidic/channel-based droplet chip with integrated temperature control to allow complete automation and integration of plasmid addition, heat-shock transformation, addition of selection medium, culture, and protein expression. The microfluidic format permitted significant reduction in consumption (100-fold) of expensive reagents such as DNA and enzymes compared to the benchtop method. The chip contains a channel to continuously replenish oil to the culture chamber to provide a fresh supply of oxygen to the cells for long-term (∼5 days) cell culture. The flow channel also replenished oil lost to evaporation and increased the number of droplets that could be processed and cultured. The platform was validated by transforming several plasmids into Escherichia coli including plasmids containing genes for fluorescent proteins GFP, BFP and RFP; plasmids with selectable markers for ampicillin or kanamycin resistance; and a Golden Gate DNA assembly reaction. We also demonstrate the applicability of this platform for transformation in widely used eukaryotic organisms such as Saccharomyces cerevisiae and Aspergillus niger. Duration and temperatures of the microfluidic heat-shock procedures were optimized to yield transformation efficiencies comparable to those obtained by benchtop methods with a throughput up to 6 droplets/min. The proposed platform offers potential for automation of molecular biology experiments significantly reducing cost, time and variability while improving throughput.

© 2015 American Chemical Society. New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

We report the first digital microfluidic (DMF) system capable of impedance sensing of mammalian cells. The new system was validated in three assays: calibration, proliferation, and serum sensing. In the first assay, three cell lines (HeLa, CHO-K1, and NIH-3T3) were seeded at different densities to determine the relationship between impedance and cell number, which was found to be linear for each type of cell. In the proliferation assay, cells were grown for four days and their proliferation rates were determined by regular impedance measurements. In the serum sensing assay, a dilution series of cell media containing different concentrations of serum was evaluated using impedance measurements to determine the optimum conditions for proliferation. The DMF impedance system is label-free, does not require imaging, and is compatible with long-term cell culture. We propose that this system will be useful for the growing number of scientists who are seeking methods other than fluorescence or cell sorting to analyze adherent cells in situ.

Dried blood spot (DBS) samples on filter paper are surging in popularity as a sampling and storage vehicle for a wide range of clinical and pharmaceutical applications. For example, a DBS sample is collected from every baby born in the province of Ontario, Canada, for quantification of approximately one hundred analytes that are used to screen for 28 conditions, including succinylacetone (SA), a marker for hepatorenal tyrosinemia. Unfortunately, the conventional methods used to evaluate DBS samples for newborn screening and other applications are tedious and slow, with limited options for automated analysis. In response to this challenge, we have developed a method to couple digital microfluidics (DMF) to nanoelectrospray ionization mass spectrometry (nESI-MS) for SA quantification in DBS samples. The new system is formed by sandwiching a pulled glass capillary emitter between the two DMF substrates such that the capillary emitter is immobilized without external seals or gaskets. Moreover, we introduce a new feedback control system that enables high-fidelity droplet manipulation across DBS samples without manual intervention. The system was validated by application to on-chip extraction, derivatization, and analysis of SA and other analytes from DBS samples, with comparable performance to gold-standard methods. We propose that the new methods described here can potentially contribute to a new generation of analytical techniques for quantifying analytes in DBS samples for a wide range of applications.

We introduce a micro-scale bioreactor for automated culture and density analysis of microorganisms. The microbioreactor is powered by digital microfluidics (DMF) and because it is used with bacteria, algae and yeast, we call it the BAY microbioreactor. Previous miniaturized bioreactors have relied on microchannels which often require valves, mixers and complex optical systems. In contrast, the BAY microbioreactor is capable of culturing microorganisms in distinct droplets on a format compatible with conventional bench-top analyzers without the use of valves, mixers or pumps. Bacteria, algae and yeast were grown for up to 5 days with automated semi-continuous mixing and temperature control. Cell densities were determined by measuring absorbances through transparent regions of the devices, and growth profiles were shown to be comparable to those generated in conventional, macro-scale systems. Cell growth and density measurements were integrated in the microbioreactor with a fluorescent viability assay and transformation of bacteria with a fluorescent reporter gene. These results suggest that DMF may be a useful new tool in automated culture and analysis of microorganisms for a wide range of applications.

Clinical proteomics has emerged as an important new discipline, promising the discovery of biomarkers that will be useful for early diagnosis and prognosis of disease. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from extremely heterogeneous fluids (i.e. serum, whole blood, etc.) and (ii) extensive biochemical processing prior to analysis. Here, we report a new digital microfluidics (DMF) based method integrating several processing steps used in clinical proteomics. This includes protein extraction, resolubilization, reduction, alkylation and enzymatic digestion. Digital microfluidics is a microscale fluid-handling technique in which nanoliter-microliter sized droplets are manipulated on an open surface. Droplets are positioned on top of an array of electrodes that are coated by a dielectric layer - when an electrical potential is applied to the droplet, charges accumulate on either side of the dielectric. The charges serve as electrostatic handles that can be used to control droplet position, and by biasing a sequence of electrodes in series, droplets can be made to dispense, move, merge, mix, and split on the surface. Therefore, DMF is a natural fit for carrying rapid, sequential, multistep, miniaturized automated biochemical assays. This represents a significant advance over conventional methods (relying on manual pipetting or robots), and has the potential to be a useful new tool in clinical proteomics. Mais J. Jebrail, Vivienne N. Luk, and Steve C. C. Shih contributed equally to this work. Sergio L. S. Freire's current address is at the University of the Sciences in Philadelphia located at 600 South 43rd Street, Philadelphia, PA 19104.

Polytopic alpha-helical membrane proteins present one of the final frontiers for protein structural biology, with significant challenges causing severe under-representation in the protein structure databank. However, with the advent of hardware and methodology geared to the study of large molecular weight complexes, solution NMR is being increasingly considered as a tool for structural studies of these types of membrane proteins. One method that has the potential to facilitate these studies utilizes uniformly deuterated samples with protons reintroduced at one or two methyl groups of leucine, valine and isoleucine. In this work we demonstrate that in spite of the increased proportion of these amino acids in membrane proteins, the quality of structures that can be obtained from this strategy is similar to that obtained for all alpha-helical water soluble proteins. This is partly attributed to the observation that NOEs between residues within the transmembrane helix did not have an impact on structure quality. Instead the most important factors controlling structure accuracy were the strength of dihedral angle restraints imposed and the number of unique inter-helical pairs of residues constrained by NOEs. Overall these results suggest that the most accurate structures will arise from accurate identification of helical segments and utilization of inter-helical distance restraints from various sources to maximize the distribution of long-range restraints.

The transmembrane (TM) segment of the major coat protein from Ff bacteriophage has been extensively studied as an example of dimerization in detergent and lipid bilayer systems. However, almost all the information regarding this interaction has been gained through mutagenesis studies, with little direct structural information being available. To this end solution NMR has the potential to provide new insights into structure of the dimer. In order to evaluate the utility of this approach we have studied a selectively 15N-labeled peptide containing the TM segment of MCP (MCPTM) by solution NMR. This peptide was found to give rise to detergent concentration-dependent spectra that were assigned to monomeric and dimeric forms. The standard free energy of this interaction in SDS was estimated from these spectra and found to be consistent with weak but specific dimerization. In addition, similar spectra could be obtained in β-octyl glucoside with intermolecular paramagnetic relaxation experiments demonstrating a parallel arrangement of TM helices in the dimer. In both detergents backbone chemical shift differences between monomeric and dimeric forms of MCPTM showed that the largest changes occur around its GXXXG motif. The resulting structural model is consistent with observations made for MCP mutants previously characterized in biological membranes, opening the door to detailed structural characterization of this form of MCP. These results also have general implications for the study of weakly interacting TM segments by solution NMR since the use of similar sample conditions should allow structural data to be accessed for oligomeric states from a wide range systems that undergo biologically relevant but weak associations in the membrane.

We report a digital microfluidic system designed for droplet-based long-term culture and analysis of algae. The system includes unique innovations relative to standard devices including an active reservoir structure to maintain homogeneous cell density, a customized device layout capable of controlling a wide range of different droplet volumes, vertical interconnects to collect spent reagents, detection zones compatible with parallel-scale optical measurements using a standard multiwell plate reader, and optimized features for droplet dispensing in parallel. The method allows for automated, multiplexed analysis with significant reductions in human intervention, representing a decrease from 600 pipette steps (for a conventional screen in multiwell plates) to fewer than 20 (for the new microfluidic technique). The system was applied to screen conditions favourable for lipid generation in the widely used algal model for biofuel production, Cyclotella cryptica. A dependence on illumination wavelength was observed, with the best conditions (representing a four-fold increase relative to control) comprising an alternation between yellow (∼580 nm) and blue (∼450 nm) illumination wavelengths. These effects were observed for both micro- and macro-scale cultures, and are consistent with a putative mechanism involving photooxidative stress. We propose that the microfluidic system described here is an attractive new screening tool with potential advantages for applications in renewable energy, biotechnology, materials science, and beyond.