Conversion of Escherichia coli into Mixotrophic CO2 Assimilation with Malate and Hydrogen Based on Recombinant Expression of 2-Oxoglutarate:Ferredoxin Oxidoreductase Using Adaptive Laboratory Evolution.

We report the mixotrophic growth of Escherichia coli based on recombinant 2-oxoglutarate:ferredoxin oxidoreductase (OGOR) to assimilate CO2 using malate as an auxiliary carbon source and hydrogen as an energy source. We employ a long-term (~184 days) two-stage adaptive evolution to convert heterotrophic E. coli into mixotrophic E. coli. In the first stage of evolution with serine, diauxic growth emerges as a prominent feature. At the end of the second stage of evolution with malate, the strain exhibits mixotrophy with CO2 as an essential substrate for growth. We expect this work will open new possibilities in the utilization of OGOR for microbial CO2 assimilation and future hydrogen-based electro-microbial conversion.

Hydrogen Bond Strength-Mediated Self-Assembly of Supramolecular Nanogels for Selective and Effective Cancer Treatment.

This study provides a significant contribution to the development of multiple hydrogen-bonded supramolecular nanocarrier systems by demonstrating that controlling the hydrogen bond strength within supramolecular polymers represents a crucial factor to tailor the drug delivery performance and enhance the effectiveness of cancer therapy. Herein, we successfully developed two kinds of poly(ethylene glycol)-based telechelic polymers Cy-PEG and UrCy-PEG having self-constituted double and quadruple hydrogen-bonding cytosine (Cy) and ureido-cytosine (UrCy) end-capped groups, respectively, which directly assemble into spherical nanogels with a number of interesting physical characteristics in aqueous solutions. The UrCy-PEG nanogels containing quadruple hydrogen-bonded UrCy dimers exhibited excellent long-term structural stability in a serum-containing biological medium, whereas the double hydrogen-bonded Cy moieties could not maintain the structural integrity of the Cy-PEG nanogels. More importantly, after the drug encapsulation process, a series of in vitro experiments clearly confirmed that drug-loaded UrCy-PEG nanogels induced selective apoptotic cell death in cancer cells without causing significant cytotoxicity to healthy cells, while drug-loaded Cy-PEG nanogels exerted nonselective cytotoxicity toward both cancer and normal cells, indicating that increasing the strength of hydrogen bonds in nanogels plays a key role in enhancing the selective cellular uptake and cytotoxicity of drugs and the subsequent induction of apoptosis in cancer cells.

Conquering the Tyranny of Number With Digital Microfluidics.

The development of large-scale integration based on soft lithography has ushered a new revolution in microfluidics. This technology, however, relies inherently on pneumatic control of micromechanical valves that require air pressure to operate, while digital microfluidics uses a purely electrical signal on an electrode for droplet manipulation. In this article, we discuss the prospect and current challenges of digital microfluidics to solve the problem of the tyranny of numbers in arbitrary fluidic manipulation. We distill the fundamental physics governing electrowetting and their implications for specifications of the control electronics. We survey existing control electronics in digital microfluidics and detail the improvements needed to realize a low-power, programmable digital microfluidic system. Such an instrument would attract wide interest in both professional and non-professional (hobbyist) communities.

A Versatile Kit Based on Digital Microfluidics Droplet Actuation for Science Education.

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported as a specific example. It also has fluorescent imaging capability and closed humidified enclosure based on an ultrasonic atomizer to prevent evaporation. The kit can be assembled within a short period of time and with minimal training in electronics and soldering. The kit allows both undergraduate/graduate students and enthusiasts to obtain hands-on experience in microfluidics in an intuitive way and be trained to gain familiarity with digital microfluidics.

Wireless bioreactor for anaerobic cultivation of bacteria.

Anaerobic cultivation methods of bacteria are indispensable in microbiology. One methodology is to cultivate the microbes in anaerobic enclosure with oxygen-adosrbing chemicals. Here, we report an electronic extension of such strategy for facultative anaerobic bacteria. The technique is based a bioreactor with entire operation including turbidity measurement, fluidic mixing, and gas delivery in an anaerobic enclosure. Wireless data transmission is employed and the anaerobic condition is achieved with gas pack. Although the technique is not meant to completely replace the anaerobic chamber for strict anaerobic bacteria, it provides a convenient way to bypass the cumbersome operation in anaerobic chamber for facultative anaerobic bacteria. Such a cultivation strategy is demonstrated with Escherichia coli with different carbon sources and hydrogen as energy source.

Detailed profiling of carbon fixation of in silico synthetic autotrophy with reductive tricarboxylic acid cycle and Calvin-Benson-Bassham cycle in Esherichia coli using hydrogen as an energy source.

Carbon fixation is the main route of inorganic carbon in the form of CO2 into the biosphere. In nature, RuBisCO is the most abundant protein that photosynthetic organisms use to fix CO2 from the atmosphere through the Calvin-Benson-Bassham (CBB) cycle. However, the CBB cycle is limited by its low catalytic rate and low energy efficiency. In this work, we attempt to integrate the reductive tricarboxylic acid and CBB cycles in silico to further improve carbon fixation capacity. Key heterologous enzymes, mostly carboxylating enzymes, are inserted into the Esherichia coli core metabolic network to assimilate CO2 into biomass using hydrogen as energy source. Overall, such a strain shows enhanced growth yield with simultaneous running of dual carbon fixation cycles. Our key results include the following. (i) We identified two main growth states: carbon-limited and hydrogen-limited; (ii) we identified a hierarchy of carbon fixation usage when hydrogen supply is limited; and (iii) we identified the alternative sub-optimal growth mode while performing genetic perturbation. The results and modeling approach can guide bioengineering projects toward optimal production using such a strain as a microbial cell factory.

H2 Reduction Annealing Induced Phase Transition and Improvements on Redox Durability of Pt Cluster-Decorated Cu@Pd Electrocatalysts in Oxygen Reduction Reaction.

Hierarchical structures in shell with transition metal underneath is a promising design for high-performance and low-cost heterogeneous nanocatalysts (NCs). Such a design enables the optimum extent of synergetic effects in NC surface. It facilitates intermediate reaction steps and, therefore, boosts activity of NC in oxygen reduction reaction (ORR). In this study, carbon nanotube (CNT)-supported ternary metallic NC comprising Cucluster-in-Pdcluster nanocrystal and surface decoration of atomic Pt clusters (14 wt %) is synthesized by using the wet chemical reduction method with sequence and reaction time controls. By annealing in H2 environment (H2/N2 = 9:1, 10 sccm) at 600 K for 2 h, specific activity of Cu@Pd/Pt is substantially improved by ∼2.0-fold as compared to that of the pristine sample and commercial Pt catalysts. By cross-referencing results of electron microscopic, X-ray spectroscopic, and electrochemical analyses, we demonstrated that reduction annealing turns ternary NC into complex of Cu3Pt alloy and Cu x Pd1-x alloy. Such a transition preserves Pt and Pd in metallic phases, therefore improving the activity by ∼29% and the stability of NC in an accelerated degradation test (ADT) as compared to those of pristine Cu@Pd/Pt in 36 000 cycles at 0.85 V (vs RHE). This study presents robust H2 annealing for structure stabilization of NC and systematic characterizations for rationalization of the corresponding mechanisms. These results provide promising scenarios for facilitation of heterogeneous NC in ORR applications.

Conformational Effects of Pt-Shells on Nanostructures and Corresponding Oxygen Reduction Reaction Activity of Au-Cluster-Decorated NiOx@Pt Nanocatalysts.

Herein, ternary metallic nanocatalysts (NCs) consisting of Au clusters decorated with a Pt shell and a Ni oxide core underneath (called NPA) on carbon nanotube (CNT) support were synthesized by combining adsorption, precipitation, and chemical reduction methods. By a retrospective investigation of the physical structure and electrochemical results, we elucidated the effects of Pt/Ni ratios (0.4 and 1.0) and Au contents (2 and 9 wt.%) on the nanostructure and corresponding oxygen reduction reaction (ORR) activity of the NPA NCs. We found that the ORR activity of NPA NCs was mainly dominated by the Pt-shell thickness which regulated the depth and size of the surface decorated with Au clusters. In the optimal case, NPA-1004006 (with a Pt/Ni of 0.4 and Au of ~2 wt.%) showed a kinetic current (JK) of 75.02 mA cm-2 which was nearly 17-times better than that (4.37 mA cm-2) of the commercial Johnson Matthey-Pt/C (20 wt.% Pt) catalyst at 0.85 V vs. the reference hydrogen electrode. Such a high JK value resulted in substantial improvements in both the specific activity (by ~53-fold) and mass activity (by nearly 10-fold) in the same benchmark target. Those scenarios rationalize that ORR activity can be substantially improved by a syngeneic effect at heterogeneous interfaces among nanometer-sized NiOx, Pt, and Au clusters on the NC surface.

"Do It Yourself" Microbial Cultivation Techniques for Synthetic and Systems Biology: Cheap, Fun, and Flexible.

With the emergence of inexpensive 3D printing technology, open-source platforms for electronic prototyping and single-board computers, "Do it Yourself" (DIY) approaches to the cultivation of microbial cultures are becoming more feasible, user-friendly, and thus wider spread. In this perspective, we survey some of these approaches, as well as add-on solutions to commercial instruments for synthetic and system biology applications. We discuss different cultivation designs, including capabilities and limitations. Our intention is to encourage the reader to consider the DIY solutions. Overall, custom cultivation devices offer controlled growth environments with in-line monitoring of, for example, optical density, fluorescence, pH, and dissolved oxygen, all at affordable prices. Moreover, they offer a great degree of flexibility for different applications and requirements and are fun to design and construct. We include several illustrative examples, such as gaining optogenetic control and adaptive laboratory evolution experiments.

Programming ORR Activity of Ni/NiO x @Pd Electrocatalysts via Controlling Depth of Surface-Decorated Atomic Pt Clusters.

Carbon nanotube supported ternary metallic nanocatalysts (NCs) comprising Nicore-Pdshell structure and Pt atomic scale clusters in shell (namely, Ni@Pd/Pt) are synthesized by using wet chemical reduction method with reaction time control. Effects of Pt4+ adsorption time and Pt/Pd composition ratios on atomic structure with respect to electrochemical performances of experimental NCs are systematically investigated. By cross-referencing results of high-resolution transmission electron microscopy, X-ray diffraction, X-ray absorption, density functional theoretical calculations, and electrochemical analysis, we demonstrate that oxygen reduction reaction (ORR) activity is dominated by depth and distribution of Pt clusters in a Ni@Pd/Pt NC. For the optimum case (Pt4+ adsorption time = 2 h), specific activity of Ni@Pd/Pt is 0.732 mA cm-2 in ORR. Such a value is 2.8-fold higher as compared to that of commercial J.M.-Pt/C at 0.85 V (vs reversible hydrogen electrode). Such improvement is attributed to the protection of defect sites from oxide reaction in the presence of Pt clusters in NC surface. When adsorption time is 10 s, Pt clusters tends to adsorb in the Ni@Pd surface. A substantially increased galvanic replacement between Pt4+ ion and Pd/Ni metal is found to result in the formation of Ni@Pd shell with Pt cluster in the interface when adsorption time is 24 h. Both structures increase the surface defect density and delocalize charge density around Pt clusters, thereby suppressing the ORR activity of Ni@Pd/Pt NCs.

Trapping of Micro Particles in Nanoplasmonic Optical Lattice.

The plasmonic optical tweezer has been developed to overcome the diffraction limits of the conventional far field optical tweezer. Plasmonic optical lattice consists of an array of nanostructures, which exhibit a variety of trapping and transport behaviors. We report the experimental procedures to trap micro-particles in a simple square nanoplasmonic optical lattice. We also describe the optical setup and the nanofabrication of a nanoplasmonic array. The optical potential is created by illuminating an array of gold nanodiscs with a Gaussian beam of 980 nm wavelength, and exciting plasmon resonance. The motion of particles is monitored by fluorescence imaging. A scheme to suppress photothermal convection is also described to increase usable optical power for optimal trapping. Suppression of convection is achieved by cooling the sample to a low temperature, and utilizing the near-zero thermal expansion coefficient of a water medium. Both single particle transport and multiple particle trapping are reported here.

Mini Photobioreactors for in Vivo Real-Time Characterization and Evolutionary Tuning of Bacterial Optogenetic Circuit.

The current standard protocols for characterizing the optogenetic circuit of bacterial cells using flow cytometry in light tubes and light exposure of culture plates are tedious, labor-intensive, and cumbersome. In this work, we engineer a bioreactor with working volume of ∼10 mL for in vivo real-time optogenetic characterization of E. coli with a CcaS-CcaR light-sensing system. In the bioreactor, optical density measurements, reporter protein fluorescence detection, and light input stimuli are provided by four light-emitting diode sources and two photodetectors. Once calibrated, the device can cultivate microbial cells and record their growth and gene expression without human intervention. We measure gene expression during cell growth with different organic substrates (glucose, succinate, acetate, pyruvate) as carbon sources in minimal medium and demonstrate evolutionary tuning of the optogenetic circuit by serial dilution passages.

Heterogeneous Cu-Pd binary interface boosts stability and mass activity of atomic Pt clusters in the oxygen reduction reaction.

A ternary metallic CuPdPt nanocatalyst (NC) is synthesized using a wet chemical reduction method, which is sequentially designed, in the presence of acid treated carbon nanotubes. This NC is a nanocrystal with a configuration of a Cu@Pd core and atomic Pt clusters (∼9 wt%) on the top (Cu@Pd/Pt). A residual current of 92.6%, 5.2 times higher than that of commercial Pt catalysts (at 0.85 V vs. RHE), is retained after 40 000 cycles of an accelerated degradation test (ADT). Atomic and electronic structure analyses show that such exclusive stability mainly results from electron localization at Pt clusters in heterogeneous interfaces of the Cu-Pd core. Most importantly, we develop a robust ternary NC, which shows outstanding MA, superior chemical durability, and ∼90 wt% lower Pt loading than commercial Pt NCs in the oxygen reduction reaction.

Design and Use of a Low Cost, Automated Morbidostat for Adaptive Evolution of Bacteria Under Antibiotic Drug Selection.

We describe a low cost, configurable morbidostat for characterizing the evolutionary pathway of antibiotic resistance. The morbidostat is a bacterial culture device that continuously monitors bacterial growth and dynamically adjusts the drug concentration to constantly challenge the bacteria as they evolve to acquire drug resistance. The device features a working volume of ~10 ml and is fully automated and equipped with optical density measurement and micro-pumps for medium and drug delivery. To validate the platform, we measured the stepwise acquisition of trimethoprim resistance in Escherichia coli MG 1655, and integrated the device with a multiplexed microfluidic platform to investigate cell morphology and antibiotic susceptibility. The approach can be up-scaled to laboratory studies of antibiotic drug resistance, and is extendible to adaptive evolution for strain improvements in metabolic engineering and other bacterial culture experiments.

Characterization of the near-field and convectional transport behavior of micro and nanoparticles in nanoscale plasmonic optical lattices.

Here, we report the characterization of the transport of micro- and nanospheres in a simple two-dimensional square nanoscale plasmonic optical lattice. The optical potential was created by exciting plasmon resonance by way of illuminating an array of gold nanodiscs with a loosely focused Gaussian beam. This optical potential produced both in-lattice particle transport behavior, which was due to near-field optical gradient forces, and high-velocity (∼µm/s) out-of-lattice particle transport. As a comparison, the natural convection velocity field from a delocalized temperature profile produced by the photothermal heating of the nanoplasmonic array was computed in numerical simulations. This work elucidates the role of photothermal effects on micro- and nanoparticle transport in plasmonic optical lattices.

Theory of a microfluidic serial dilution bioreactor for growth of planktonic and biofilm populations.

We present the theory of a microfluidic bioreactor with a two-compartment growth chamber and periodic serial dilution. In the model, coexisting planktonic and biofilm populations exchange by adsorption and detachment. The criteria for coexistence and global extinction are determined by stability analysis of the global extinction state. Stability analysis yields the operating diagram in terms of the dilution and removal ratios, constrained by the plumbing action of the bioreactor. The special case of equal uptake function and logistic growth is analytically solved and explicit growth curves are plotted. The presented theory is applicable to generic microfluidic bioreactors with discrete growth chambers and periodic dilution at discrete time points. Therefore, the theory is expected to assist the design of microfluidic devices for investigating microbial competition and microbial biofilm growth under serial dilution conditions.

Review of Microfluidic Photobioreactor Technology for Metabolic Engineering and Synthetic Biology of Cyanobacteria and Microalgae.

One goal of metabolic engineering and synthetic biology for cyanobacteria and microalgae is to engineer strains that can optimally produce biofuels and commodity chemicals. However, the current workflow is slow and labor intensive with respect to assembly of genetic parts and characterization of production yields because of the slow growth rates of these organisms. Here, we review recent progress in the microfluidic photobioreactors and identify opportunities and unmet needs in metabolic engineering and synthetic biology. Because of the unprecedented experimental resolution down to the single cell level, long-term real-time monitoring capability, and high throughput with low cost, microfluidic photobioreactor technology will be an indispensible tool to speed up the development process, advance fundamental knowledge, and realize the full potential of metabolic engineering and synthetic biology for cyanobacteria and microalgae.