SummarySynthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines, and present combinatorial methods for the production of antibody-conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Biological memory can be defined as a sustained cellular response to a transient stimulus. To understand this phenomenon, we must consider how the properties of different biological systems achieve memory of a stimulus, essentially permitting a cell to produce a lasting response. One way that cells accomplish this task is through transcriptional states, which involve populations of molecules regulating gene expression. If the transcriptional response is bistable, a chemical state becomes defined as on or off and, given certain parameters, this state can be inherited through DNA replication and cell division. In this way, a cell can produce a lasting memory of a biological response. Synthetic biologists are especially interested in transcriptional responses as a means of cellular memory because (1) much of a cell’s information processing is performed through transcription, and (2) the basic machinery for such biological behavior is well-understood. As such, transcription provides us with a set of characterized genetic units, such as promoters, activators, and repressors, that can be recombined to create novel transcriptional circuits. Furthermore, the way nature combines these biological parts to produce specific outputs, including cellular memory, has been extensively studied. Thus, we have at our fingertips the tools with which to design synthetic memory systems. The construction of synthetic memory circuits will improve our understanding of natural networks, further aiding the creation of useful, novel biological tools. For example, a device capable of remembering a biological experience might be utilized in the long-term study of particular cells within a heterogeneous population following a defined event, or applied in industry for the sustained production of desired proteins after induction by a brief stimulus. Such bio-engineered networks exemplify a primary objective of synthetic biology: to advance simple synthetic devices into expertly constructed circuits with significant applications.

A variety of biological phenomena, from disease progression to stem cell differentiation, are typified by a prolonged cellular response to a transient environmental cue. While biologically relevant, heterogeneity in these long-term responses is difficult to assess at the population level, necessitating the development of biological tools to track cell fate within subpopulations. Here we present a novel synthetic biology approach for identifying and tracking mammalian cell subpopulations. We constructed three genomically integrated circuits that use bistable autoregulatory transcriptional feedback to retain memory of exposure to brief stimuli. These ''memory devices'' are used to isolate and track the progeny of cells that responded differentially to doxycycline, hypoxia, or DNAdamaging agents. Following hypoxic or ultraviolet radiation exposure, strongly responding cells activate the memory device and exhibit changes in gene expression, growth rates, and viability for multiple generations after the initial stimulus. Taken together, these results indicate that a heritable memory of hypoxia and DNA damage exists in subpopulations that differ in long-term cell behavior.