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We designed a microfluidic module that generates complex and dynamic concentration profiles of multiple molecules over a large concentration range using pulse-width modulation (PWM). Our PWM module can combine up to six different inputs and select among three downstream mixing channels, as required by the application. The module can produce concentrations with a dynamic range of three decades. We created complex, temporal concentration profiles of two molecules, with each concentration independently controllable, and show that the PWM module can execute rapid concentration changes as well as long-time scale pharmacokinetic profiles. Concentration profiles were generated for molecules with molecular weights ranging from 560 Da to 150 kDa. Our PWM module produces robust and precise concentration profiles under a variety of operating conditions, making it ideal for integration with existing microfluidic devices for advanced cell and pharmacokinetic studies. [ABSTRACT FROM AUTHOR]

Rolling circle amplification (RCA) is a widely used DNA amplification method that uses circular template DNA as input and produces multimeric, linear single- or double-stranded DNA. Circle-to-circle amplification (C2CA) has further expanded this method by implementing product recircularization using restriction and ligation, leading to a higher amplification yield and enabling the generation of circular products. However, C2CA is a multistep, nonisothermal method, requiring multiple fluid manipulations and thereby compromises several advantages of RCA. Here, we improved C2CA to implement a one-pot, single step, isothermal reaction at temperatures ranging from 25 to 37 °C. Our C2CAplus method is simple, robust, and produces large quantities of product DNA that can be seen with the naked eye.

Synthetically engineered cells are powerful and potentially useful biosensors, but it remains problematic to deploy such systems due to practical difficulties and biosafety concerns. To overcome these hurdles, we developed a microfluidic device that serves as an interface between an engineered cellular system, environment, and user. We created a biodisplay consisting of 768 individually programmable biopixels and demonstrated that it can perform multiplexed, continuous sampling. The biodisplay detected 10 μg/L sodium-arsenite in tap water using a research grade fluorescent microscope, and reported arsenic contamination down to 20 μg/L with an easy to interpret "skull and crossbones" symbol detectable with a low-cost USB microscope or by eye. The biodisplay was designed to prevent release of chemical or biological material to avoid environmental contamination. The microfluidic biodisplay thus provides a practical solution for the deployment and application of engineered cellular systems.

Cell-free environments are becoming viable alternatives for implementing biological networks in synthetic biology. The reconstituted cell-free expression system (PURE) allows characterization of genetic networks under defined conditions but its applicability to native bacterial promoters and endogenous genetic networks is limited due to the poor transcription rate of Escherichia coli RNA polymerase in this minimal system. We found that addition of transcription elongation factors GreA and GreB to the PURE system increased transcription rates of E. coli RNA polymerase from sigma factor 70 promoters up to 6-fold and enhanced the performance of a genetic network. Furthermore, we reconstituted activation of natural E. coli promoters controlling flagella biosynthesis by the transcriptional activator FlhDC and sigma factor 28. Addition of GreA/GreB to the PURE system allows efficient expression from natural and synthetic E. coli promoters and characterization of their regulation in minimal and defined reaction conditions, making the PURE system more broadly applicable to study genetic networks and bottom-up synthetic biology.

Microfluidic diagnostic devices have the potential to transform the practice of medicine. We engineered a multiplexed digital-analog microfluidic platform for the rapid and highly sensitive detection of 3-4 biomarkers in quadruplicate in 16 independent and isolated microfluidic unit cells requiring only a single 5 μL sample. We comprehensively characterized the platform by performing single enzyme and digital immunoassays, achieving single molecule detection and measured as low as ∼10 fM (330 fg/mL) GFP in buffer and ∼12 fM GFP in human serum. We applied our integrated digital detection mechanism to multiplexed detection of 1pM anti-Ebola IgG in human serum and were able to differentiate three common Ebola strains. To ascertain that the device can be applied in environments beyond clinical point-of-care settings, we developed a low-cost, portable hardware system to control and read out the microfluidic device and detected anti-Ebola IgG in ultralow volume whole blood samples to levels of 100 pM in a multiplexed assay format.

Current gene synthesis methods allow the generation of long segments of dsDNA. We show that these techniques can be used to create synthetic regulatory elements and describe a method for the creation of completely defined, synthetic variants of the PHO5 promoter from the budding yeast Saccharomyces cerevisae. Overall, 128 promoters were assembled by high-temperature ligation, cloned into plasmids by isothermal assembly, maintained in E. coli, and consequently transformed into yeast by homologous recombination. Synthesis errors occurred at frequencies comparable to or lower than those achieved with current gene synthesis methods. The promoter synthesis method reported here is robust, fast, and readily accessible. Synthetically engineered promoter libraries will be useful tools for dissecting the intricacies of promoter input-output functions and may serve as tunable components for synthetic genetic networks.