The functioning of synthetic gene circuits depends on their local chemical context defined by the types and concentrations of biomolecules in the surrounding milieu that influences gene transcription and translation. This chemical-context dependence of synthetic gene circuits arises from significant yet unknown cross talk between engineered components, host cells, and environmental factors and has been a persistent challenge for synthetic biology. Here, we show that the sensitivity of synthetic gene networks to their extracellular chemical contexts can be minimized, and their designed functions rendered robust using artificial cells, which are synthetic biomolecular compartments engineered from the bottom-up using liposomes that encapsulate the gene networks. Our artificial cells detect, interact with, and kill bacteria in simulated external environments with different chemical complexity. Our work enables the engineering of synthetic gene networks with minimal dependency on their extracellular chemical context and creates a new frontier in controlling robustness of synthetic biological systems using bioinspired mechanisms.

Assembly of recombinant multiprotein systems requires multiple culturing and purification steps that scale linearly with the number of constituent proteins. This problem is particularly pronounced in the preparation of the 34 proteins involved in transcription and translation systems, which are fundamental biochemistry tools for reconstitution of cellular pathways ex vivo. Here, we engineer synthetic microbial consortia consisting of between 15 and 34 Escherichia coli strains to assemble the 34 proteins in a single culturing, lysis, and purification procedure. The expression of these proteins is controlled by synthetic genetic modules to produce the proteins at the correct ratios. We show that the pure multiprotein system is functional and reproducible, and has low protein contaminants. We also demonstrate its application in the screening of synthetic promoters and protease inhibitors. Our work establishes a novel strategy for producing pure translation machinery, which may be extended to the production of other multiprotein systems.

The incorporation of cell-free transcription and translation systems into high-throughput screening applications enables the in situ and on-demand expression of peptides and proteins. Coupled with modern microfluidic technology, the cell-free methods allow the screening, directed evolution and selection of desired biomolecules in minimal volumes within a short timescale. Cell-free high-throughput screening applications are classified broadly into in vitro display and on-chip technologies. In this review, we outline the development of cell-free high-throughput screening methods. We further discuss operating principles and representative applications of each screening method. The cell-free high-throughput screening methods may be advanced by the future development of new cell-free systems, miniaturization approaches, and automation technologies.

The biosynthesis of antioxidant pigments, namely, betalains, was believed to be restricted to Caryophyllales plants. This paper changes this paradigm, and enzyme mining from bacterial hosts promoted the discovery of bacterial cultures producing betalains. The spectrum of possible sources of betalain pigments in nature is broadened by our description of the first betalain-forming bacterium, Gluconacetobacter diazotrophicus. The enzyme-specific step is the extradiol cleavage of the precursor amino acid l-dihydroxyphenylalanine (l-DOPA) to form the structural unit betalamic acid. Molecular and functional work conducted led to the characterization of a novel dioxygenase, a polypeptide of 17.8 kDa with a Km of 1.36 mM, with higher activity and affinity than those of its plant counterparts. Its superior activity allowed the first experimental characterization of the early steps in the biosynthesis of betalains by fully characterizing the presence and time evolution of 2,3- and 4,5-seco-DOPA intermediates. Furthermore, spontaneous chemical reactions are characterized and incorporated into a comprehensive enzymatic-chemical mechanism that yields the final pigments. IMPORTANCE Several studies have demonstrated the health-promoting effects of betalains due to their high antioxidant capacity and their positive effect on the dose-dependent inhibition of cancer cells and their proliferation. To date, betalains were restricted to plants of the order Caryophyllales and some species of fungi, but the present study reveals the first betalain-producing bacterium, as well as the first steps in the formation of pigments. This finding demonstrates that betalain biosynthesis can be expanded to prokaryotes.