Our understanding of translation underpins our capacity to engineer living systems. The canonical start codon (AUG) and a few near-cognates (GUG, UUG) are considered as the ‘start codons’ for translation initiation in Escherichia coli. Translation is typically not thought to initiate from the 61 remaining codons. Here, we quantified translation initiation of green fluorescent protein and nanoluciferase in E. coli from all 64 triplet codons and across a range of DNA copy number. We detected initiation of protein synthesis above measurement background for 47 codons. Translation from non-canonical start codons ranged from 0.007 to 3% relative to translation from AUG. Translation from 17 non-AUG codons exceeded the highest reported rates of non-cognate codon recognition. Translation initiation from non-canonical start codons may contribute to the synthesis of peptides in both natural and synthetic biological systems.

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Combinatorial chemical evolution is used to select oil-water droplet interfaces that drive inorganic nanoparticle synthesis.

The way nature evolves and sculpts materials using proteins inspires new approaches to materials engineering but is still not completely understood. Here, we present a cell-free synthetic biological platform to advance studies of biologically synthesized solid-state materials. This platform is capable of simultaneously exerting many of the hierarchical levels of control found in natural biomineralization, including genetic, chemical, spatial, structural, and morphological control, while supporting the evolutionary selection of new mineralizing proteins and the corresponding genetically encoded materials that they produce. DNA-directed protein expression and enzymatic mineralization occur on polystyrene microbeads in waterin-oil emulsions, yielding synthetic surrogates of biomineralizing cells that are then screened by flow sorting, with light-scattering signals used to sort the resulting mineralized composites differentially. We demonstrate the utility of this platform by evolutionarily selecting newly identified silicateins, biomineralizing enzymes previously identified from the silica skeleton of a marine sponge, for enzyme variants capable of synthesizing silicon dioxide (silica) or titanium dioxide (titania) composites. Mineral composites of intermediate strength are preferentially selected to remain intact for identification during cell sorting, and then to collapse postsorting to expose the encoding genes for enzymatic DNA amplification. Some of the newly selected silicatein variants catalyze the formation of crystalline silicates, whereas the parent silicateins lack this ability. The demonstrated bioengineered route to previously undescribed materials introduces in vitro enzyme selection as a viable strategy for mimicking genetic evolution of materials as it occurs in nature. directed evolution | in vitro compartmentalization | DNA shuffling | metal oxide nanoparticles | self-assembly

DNA-based information systems drive the combinatorial optimization processes of natural evolution, including the evolution of biominerals. Advances in high-throughput DNA sequencing expand the power of DNA as a potential information platform for combinatorial engineering, but many applications remain to be developed due in part to the challenge of handling large amounts of sequence data. Here we employ high-throughput sequencing and a recently developed clustering method (AutoSOME) to identify single-stranded DNA sequence families that bind specifically to ZnO semiconductor mineral surfaces. These sequences were enriched from a diverse DNA library after a single round of screening, whereas previous screening approaches typically require 5-15 rounds of enrichment for effective sequence identification. The consensus sequence of the largest cluster was poly d(T)30. This consensus sequence exhibited clear aptamer behavior and was shown to promote the synthesis of crystalline ZnO from aqueous solution at near-neutral pH. This activity is significant, as the crystalline form of this wide-bandgap semiconductor is not typically amenable to solution synthesis in this pH range. High-resolution TEM revealed that this DNA synthesis route yields ZnO nanoparticles with an amorphous-crystalline core-shell structure, suggesting that the mechanism of mineralization involves nanoscale coacervation around the DNA template. We thus demonstrate that our new method, termed Single round Enrichment of Ligands by deep Sequencing (SEL-Seq), can facilitate biomimetic synthesis of technological nanomaterials by accelerating combinatorial selection of biomolecular-mineral interactions. Moreover, by enabling direct characterization of sequence family demographics, we anticipate that SEL-Seq will enhance aptamer discovery in applications employing additional rounds of screening.

This article presents a simple and highly reliable method for preparing PDMS microfluidic double emulsion devices that employs a single-step oxidative plasma treatment to both pattern the wettability of microchannels and to bond the chips. As a key component of our strategy we use epoxy glue to define the required hydrophobic zones and then remove this after plasma treatment, but prior to bonding. This novel approach achieves surface modification and device sealing in a single process, which reduces chip preparation times to minutes and eliminates the need for unreliable coating processes. The second key element of our procedure is the maintenance of the patterned surfaces, where we demonstrate that immediate filling of the prepared device with water or the use of solventextracted PDMS vastly extends the operational lifetimes of the chips. The reliability of this technique is confirmed by generating water-in-oil-in-water (W/O/W) double emulsion droplets with controlled core/shell structures and volumes, while its versatility is demonstrated by simply using a different placement of the epoxy glue on the same chip design to create oil-in-water-in-oil (O/W/O) double emulsion droplets. Both W/O/W and O/W/O double emulsion droplets can therefore be created from the same soft-lithography mould. This simple method overcomes one of the key problems limiting the wider use of double emulsions lack of reliability while its speed and simplicity will facilitate the high-throughput production of monodisperse double emulsions. Our method is demonstrated to produce double emulsion down to 55 µm in diameter and could be readily extended to produce microfluidic chips with more complex hydrophilic and hydrophobic patterns.

This new approach enables alignment to within ±10 µm and allows greater flexibility in choosing the dimensions of the capillary, which contributes to the size and stability of formation of the double emulsion. Importantly, it also allows the user to compensate for the deviations from ideal shape that occur in pulled glass capillaries, which has been a source of failure with previous methods. A detailed description of the critical design and operational parameters that affect double emulsion generation in these capillary microfluidic devices is provided.

these techniques are limited by the requirement for complex enzymatic reactions. As an alternative strategy that bypasses the need for genetic engineering, combinatorial methods can be employed. These can be used to explore tens to hundreds of reaction conditions, where the most promising or "lead" conditions may be selected based on the structures or properties of the resultant material. [ 17,18 ] Lead conditions can then be used to narrow the reaction landscape in successive screening rounds. Surprisingly, although combinatorial methods are often used in solid-state chemistry to explore, for example, different reagent compositions, [ 18 ] we are not aware of their use in identifying soluble additives capable of directing mineralization.In this article we demonstrate how combinatorial methods can be used in conjunction with effi cient screening processes to rapidly identify combinations of small organic molecules that are capable of directing the formation of photoluminescent quantum dot minerals in aqueous solution and at room temperature. Indeed, a key feature of biomineralization processes is that control over mineral formation is achieved using many soluble additives that operate in concert. That this feature has seldom been addressed in bioinspired methods is almost certainly due to the vast number of potential variables, which renders a full, systematic exploration intractable. As a solution to this challenge, we here utilize a genetic algorithm as a bioinspired heuristic that mimics natural evolution. Genetic algorithms use selection, recombination, and mutation strategies to rapidly identify and optimize the combination of conditions (here, soluble additives), which gives rise to materials with target properties. [ 19 ] Using a pipetting robot to prepare reaction sets and a UV-light table to rapidly assess the reactions for the formation of photoluminescent minerals, we are able to rapidly identify the key additives that promote the formation of quantum dot superstructures from one-pot aqueous reactions.Our initial library of organic mediators included 23 components, of which 17 were amino acids and 6 were surfactants. Stock solutions of the amino acids were prepared to initial concentrations of 100 × 10 −3 M , and explored at concentrations ranging from 0.01 to 50 × 10 −3 M , while surfactants were prepared to near their solubility limits in water. Surfactants were included as potential structure-directing agents to drive hierarchical assembly in aqueous solution. The overall screening approach used to identify the key additive set is summarized in Figure 1 . First, library amino acids and surfactants were randomly mixed in 48 wells of a multi-well plate, such that each well contained between 1-6 amino acids and 1-3 surfactants (Figure 1 A). Cadmium chloride and thioacetic acid (as a sulfur source) [ 20 ] were then added to a concentration of 1 × 10 −3 M in all wells, as precursors for CdS. After 3 d the plate was viewed under UV illumination (Figure 1 A) and with a fl uorimetric Biomineralization, whi...