Logic and memory are essential functions of circuits that generate complex, state-dependent responses. Here we describe a strategy for efficiently assembling synthetic genetic circuits that use recombinases to implement Boolean logic functions with stable DNA-encoded memory of events. Application of this strategy allowed us to create all 16 two-input Boolean logic functions in living Escherichia coli cells without requiring cascades comprising multiple logic gates. We demonstrate long-term maintenance of memory for at least 90 cell generations and the ability to interrogate the states of these synthetic devices with fluorescent reporters and PCR. Using this approach we created two-bit digital-to-analog converters, which should be useful in biotechnology applications for encoding multiple stable gene expression outputs using transient inputs of inducers. We envision that this integrated logic and memory system will enable the implementation of complex cellular state machines, behaviors and pathways for therapeutic, diagnostic and basic science applications.

The activity of four native FDHs and four engineered FDH variants on 93 low molecular weight arenes was used to generate FDH substrate activity profiles. These profiles provided insights into how substrate class, functional group substitution, electronic activation, and binding impact FDH activity and selectivity. The enzymes studied could halogenate a far greater range of substrates than previously recognized, but significant differences in their substrate specificity and selectivity were observed. Trends between the electronic activation of each site on a substrate and halogenation conversion at that site were established, and these data, combined with docking simulations, suggest that substrate binding can override electronic activation even on compounds differing appreciably from native substrates. These findings provide a useful framework for understanding and exploiting FDH reactivity for organic synthesis.

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The synthetic manufacture of functional proteins enables a bottom-up understanding of the workings of biological systems and opens new opportunities for the treatment of disease. Cell-free protein synthesis is a practical approach for enabling such manufacturing, however, it is typically carried out in fairly large volumes, when compared to a natural cell, leading to increases in cost and loss of efficiency. Here we demonstrate continuous cell free protein synthesis in arrays of cellular scale containers that continuously exchange energy and materials with their environment. A multiscale fabrication process allows the monolithic integration of nanoporous silicon containers within an addressable microfluidic network. Synthesis of enhanced green fluorescent protein (eGFP) in the containers continues beyond 24 h and yields more than twice the amount of protein, on a per volume basis, than conventional scale batch reactions. By mimicking the physical volume and controlled flux of a natural cell, the resulting "cell mimic" devices can enable fundamental studies of biological systems as well as serve applications related to the functional screening of proteins and the on-demand production of biologics.

Multi-scale lithography and cryogenic deep reactive ion etching techniques were used to create ensembles of nanoporous, picoliter volume, reaction vessels within a microfluidic system. The fabrication of these vessels is described and how this process can be used to tailor vessel porosity by controlling the width of slits that constitute the vessel pores is demonstrated. Control of pore size allows the containment of nucleic acids and enzymes that are the foundation of biochemical reaction systems, while allowing smaller reaction constituents to traverse the container membrane and continuously supply the reaction. In this work, a 5.4kB DNA plasmid was retained within the reaction vessels and labeled under microfluidic control with ethidium bromide as an initial proof-of-principle. Subsequently, a coupled enzyme reaction, in which glucose oxidase and horseradish peroxidase were contained and fed with a substrate solution of glucose and Amplex Red™ to produce fluorescent Resorufin, was carried out under microfluidic control and monitored using fluorescent microscopy. The fabrication techniques presented are broadly applicable and can be adapted to produce devices in which a variety of high aspect ratio, nanoporous silicon structures can be integrated within a microfluidic network. The devices shown here are amenable to being scaled in number and organized to implement more complex reaction systems for applications in sensing and actuation as well as fundamental studies of biological reaction systems.