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Living materials combine a material scaffold, that is often porous, with engineered cells that perform sensing, computing, and biosynthetic tasks. Designing such systems is difficult because little is known regarding signaling transport parameters in the material. Here, the development of a porous microplate is presented. Hydrogel barriers between wells have a porosity of 60% and a tortuosity factor of 1.6, allowing molecular diffusion between wells. The permeability of dyes, antibiotics, inducers, and quorum signals between wells were characterized. A "sentinel" strain was constructed by introducing orthogonal sensors into the genome of Escherichia coli MG1655 for IPTG, anhydrotetracycline, L‐arabinose, and four quorum signals. The strain's response to inducer diffusion through the wells was quantified up to 14 mm, and quorum and antibacterial signaling were measured over 16 h. Signaling distance is dictated by hydrogel adsorption, quantified using a linear finite element model that yields adsorption coefficients from 0 to 0.1 mol m−3. Parameters derived herein will aid the design of living materials for pathogen remediation, computation, and self‐organizing biofilms. Synopsis: Microbes interact by chemical diffusion through materials, a concept that inspires "engineered living materials". To parameterize signaling through materials, a porous culture microplate is fabricated to measure chemical signaling between isolated wells of engineered E. coli. A porous microplate is fabricated by soft lithography of the co‐polymer hydroxyethyl methacrylate‐co‐ethylene glycol dimethacrylate.Four homoserine lactone quorum sensors are evolved to minimize cross‐talk via directed evolution.Seven total orthogonal sensors are encoded into the genome of E. coli MG1655, which serves as a "sentinel strain."The biological response of the sentinel strain to inducer, quorum, and antibiotic signaling between isolated wells of the microplate are experimentally measured.A quantitative model is developed that explains the difference in transport kinetics of the various biological signals by differential absorption of the molecules to the hydrogel matrix. [ABSTRACT FROM AUTHOR]

Catalytically dead Cas9 (dCas9) is a programmable transcription factor that can be targeted to promoters through the design of small guide RNAs (sgRNAs), where it can function as an activator or repressor. Natural promoters use overlapping binding sites as a mechanism for signal integration, where the binding of one can block, displace, or augment the activity of the other. Here, we implemented this strategy in Escherichia coli using pairs of sgRNAs designed to repress and then derepress transcription through competitive binding. When designed to target a promoter, this led to 27‐fold repression and complete derepression. This system was also capable of ratiometric input comparison over two orders of magnitude. Additionally, we used this mechanism for promoter sequence‐independent control by adopting it for elongation control, achieving 8‐fold repression and 4‐fold derepression. This work demonstrates a new genetic control mechanism that could be used to build analog circuit or implement cis‐regulatory logic on CRISPRi‐targeted native genes. Synopsis: A regulatory control mechanism is developed based on the competitive binding of dCas9 to DNA by two guide RNAs. One represses a target gene (CRISPRi) and the second displaces the repressing dCas9 without interfering with the gene's transcription. Complete derepression is achieved against CRISPRi targeted to a promoter.Derepression is less efficient when CRISPRi blocks transcriptional elongation within a gene.Synthetic cis‐regulatory logic can be built using competing sgRNAs, without having to add operators to the promoter.This mechanism is used to build a ratiometric control circuit that responds to the ratio of the activities of two input promoters. [ABSTRACT FROM AUTHOR]

Aerogels are ultralight porous materials whose matrix structure can be formed by interlinking 880 nm long M13 phage particles. In theory, changing the phage properties would alter the aerogel matrix, but attempting this using the current production system leads to heterogeneous lengths. A phagemid system that yields a narrow length distribution that can be tuned in 0.3 nm increments from 50 to 2500 nm is designed and, independently, the persistence length varies from 14 to 68 nm by mutating the coat protein. A robotic workflow that automates each step from DNA construction to aerogel synthesis is used to build 1200 aerogels. This is applied to compare Ni–MnOx cathodes built using different matrixes, revealing a pareto‐optimal relationship between performance metrics. This work demonstrates the application of genetic engineering to create "tuning knobs" to sweep through material parameter space; in this case, toward creating a physically strong and high‐capacity battery. [ABSTRACT FROM AUTHOR]

Different applications require iron oxide nanoparticles (IONPs) of varying size, shape, crystallinity, and surfaces that can be controlled through the synthesis reaction conditions. Under ambient conditions, Magnetospirillum magneticum AMB‐1 builds uniform Fe3O4 IONPs with shapes and crystal forms difficult to achieve with chemical synthesis. Genetic engineering can be used to change their properties, but there are few tools to fine‐tune expression over a wide range. To this end, ribosome binding sites, minimal constitutive promoters, and inducible systems (IPTG, aTc, and OC6) with large dynamic range are designed. These are used to control M. magneticum genes that affect IONP properties, including size (mamC), morphology (mms6), chain length (mamK), and surface coating (mamC fusions). These systems increase the fraction of IONPs that are less than 30 nm, produce rounded particles, and lead to the production of intracellular chains with 24 or more IONPs. In addition, the R5 peptide from diatoms is found to silica coat the surface of metal oxide nanoparticles (Fe, Ti, Ta, Hf) and can be genetically directed to the IONP surface. This work demonstrates the genetic control of IONP properties, but also highlights the robustness of the system, which complicates genetic engineering to produce radically different particles and structures. [ABSTRACT FROM AUTHOR]

Genetic circuits have many applications, from guiding living therapeutics to ordering process in a bioreactor, but to be useful they have to be genetically stable and not hinder the host. Encoding circuits in the genome reduces burden, but this decreases performance and can interfere with native transcription. We have designed genomic landing pads in Escherichia coli at high‐expression sites, flanked by ultrastrong double terminators. DNA payloads >8 kb are targeted to the landing pads using phage integrases. One landing pad is dedicated to carrying a sensor array, and two are used to carry genetic circuits. NOT/NOR gates based on repressors are optimized for the genome and characterized in the landing pads. These data are used, in conjunction with design automation software (Cello 2.0), to design circuits that perform quantitatively as predicted. These circuits require fourfold less RNA polymerase than when carried on a plasmid and are stable for weeks in a recA+ strain without selection. This approach enables the design of synthetic regulatory networks to guide cells in environments or for applications where plasmid use is infeasible. Synopsis: Ultra‐stable genetic circuits with customizable designs are encoded in the Escherichia coli genome by integrating genetic circuit design automation (Cello) with three highly insulated genetic landing pads on the high expression location of the genome. A library of seventeen ultra‐strong double terminators is developed to insulate three landing pads from in and outgoing transcription readthrough.Three insulated genome "landing pads" are constructed in the Escherichia coli genome where expression level is high.Six orthogonal genetic gates are optimized for the expression in the genome.A new NOR gate architecture is devised to enable sophisticated genetic circuits. [ABSTRACT FROM AUTHOR]

Diatoms produce intricately patterned silica structures under ambient conditions, a process initiated by post‐translationally modified silaffin peptides that nucleate silicic acid. Designing these peptides would enable the production of silica nanostructures with desired properties; however, the functional effects of modifications are poorly understood. Here, Escherichia coli is used to express and modify recombinant silaffin R5 peptide from the diatom Cylindrotheca fusiformis. A library of 38 enzymes is tested for R5 modifications in vitro, from which active methyltransferases, kinases, acetyltransferases, oxidases, and myristoyltransferases are identified from diatoms, humans, yeast, and bacteria. Modified R5 peptides are used for silica precipitation and the impacts on particle size, shape, porosity, and surface area are quantified. In vivo pathways are designed to coexpress R5 and a modifying enzyme and the resulting peptide is used to nucleate silica nanostructures with controlled size (100–3500 nm), porosity (20–635 m2 g−1), or embedded with melanin. It is found that phosphorylation reduces the need for inorganic phosphate during silica synthesis. The simultaneous methylation and phosphorylation of R5 leads to smaller particles requiring less inorganic phosphate. Inspired by diatoms, the use of post‐translationally modified peptides will enable the control of silica morphology under ambient conditions, with potential applications in electronics and photonics. [ABSTRACT FROM AUTHOR]

Synthetic genetic circuits offer the potential to wield computational control over biology, but their complexity is limited by the accuracy of mathematical models. Here, we present advances that enable the complete encoding of an electronic chip in the DNA carried by Escherichia coli (E. coli). The chip is a binary‐coded digit (BCD) to 7‐segment decoder, associated with clocks and calculators, to turn on segments to visualize 0–9. Design automation is used to build seven strains, each of which contains a circuit with up to 12 repressors and two activators (totaling 63 regulators and 76,000 bp DNA). The inputs to each circuit represent the digit to be displayed (encoded in binary by four molecules), and output is the segment state, reported as fluorescence. Implementation requires an advanced gate model that captures dynamics, promoter interference, and a measure of total power usage (RNAP flux). This project is an exemplar of design automation pushing engineering beyond that achievable "by hand", essential for realizing the potential of biology. Synopsis: A complete electronic chip is encoded in Escherichia coli DNA by combining genetic circuit design automation (Cello) with more accurate mathematical models and gates with reduced impact on the host cell. The circuits encode a function used by calculators to turn on segments and display the digits 0–9. A new gate model captures the impact of placing repressible promoters in series and this is used to more accurately predict how to connect NOR gates.A simple mathematical model is introduced that captures the dynamics of gates using only two parameters that describe the characteristic on and off times for the gate.The dynamic model is able to predict the circuit response over 4 days when cells are transitioned between states.When resource utilization crosses a threshold, the circuits are rapidly lost from the cells. [ABSTRACT FROM AUTHOR]

Natural melanin polymers, biosynthesized from free tyrosine, assemble into microstructures that interact with light, producing a wide range of photonic properties. These structures are formed by mechanisms that are difficult to control to create new optical materials. Here, simple pentapeptides YXXXY is presented, where the XXX controls self‐assembly into microstructures, after which the Y's are polymerized by tyrosinase into melanin. It sought to computationally predict peptides that will assemble into two‐layer films that exhibit structural color and infrared (IR) scattering. Three peptides (YPIIY, YLPIY, YVPAY) are synthesized and found to form a two‐layer film exhibiting structural color. YLPIY‐melanin also forms a stable coating with scattering peaks in the near‐ and mid‐IR. This coating reduces the signature of a hotplate (37 °C) as seen by a thermal camera. This synthetic peptidic material is the first to exhibit IR‐scattering, opening the possibility of incorporating this function into bio‐compatible fibers, textiles, and plastics. [ABSTRACT FROM AUTHOR]

Genetic circuits implement computational operations within a cell. Debugging them is difficult because their function is defined by multiple states (e.g., combinations of inputs) that vary in time. Here, we develop RNA-seq methods that enable the simultaneous measurement of: (i) the states of internal gates, (ii) part performance (promoters, insulators, terminators), and (iii) impact on host gene expression. This is applied to a three-input one-output circuit consisting of three sensors, five NOR/NOT gates, and 46 genetic parts. Transcription profiles are obtained for all eight combinations of inputs, from which biophysical models can extract part activities and the response functions of sensors and gates. Various unexpected failure modes are identified, including cryptic antisense promoters, terminator failure, and a sensor malfunction due to media-induced changes in host gene expression. This can guide the selection of new parts to fix these problems, which we demonstrate by using a bidirectional terminator to disrupt observed antisense transcription. This work introduces RNA-seq as a powerful method for circuit characterization and debugging that overcomes the limitations of fluorescent reporters and scales to large systems composed of many parts. [ABSTRACT FROM AUTHOR]

A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit ( NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering. [ABSTRACT FROM AUTHOR]

Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA: DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on-target repression (56- to 440-fold) and negligible off-target interactions (< 1.3-fold). These gates were connected to build larger circuits, including the Boolean-complete NOR gate and a 3-gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor ( malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance). [ABSTRACT FROM AUTHOR]

Synthetic genetic systems share resources with the host, including machinery for transcription and translation. Phage RNA polymerases ( RNAPs) decouple transcription from the host and generate high expression. However, they can exhibit toxicity and lack accessory proteins (σ factors and activators) that enable switching between different promoters and modulation of activity. Here, we show that T7 RNAP (883 amino acids) can be divided into four fragments that have to be co-expressed to function. The DNA-binding loop is encoded in a C-terminal 285-aa 'σ fragment', and fragments with different specificity can direct the remaining 601-aa 'core fragment' to different promoters. Using these parts, we have built a resource allocator that sets the core fragment concentration, which is then shared by multiple σ fragments. Adjusting the concentration of the core fragment sets the maximum transcriptional capacity available to a synthetic system. Further, positive and negative regulation is implemented using a 67-aa N-terminal 'α fragment' and a null (inactivated) σ fragment, respectively. The α fragment can be fused to recombinant proteins to make promoters responsive to their levels. These parts provide a toolbox to allocate transcriptional resources via different schemes, which we demonstrate by building a system which adjusts promoter activity to compensate for the difference in copy number of two plasmids. [ABSTRACT FROM AUTHOR]

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σs as well as the specificity of their interaction with anti-σs. DNA synthesis was used to build 86 ECF σs (two from every subgroup), their promoters, and 62 anti-σs identified from the genomes of diverse bacteria. A subset of 20 σs and promoters were found to be highly orthogonal to each other. This set can be increased by combining the -35 and -10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal σs, anti-σs, and promoters were used to build synthetic genetic switches in Escherichia coli. This represents a genome-scale resource of the properties of ECF σs and a resource for synthetic biology, where this set of well-characterized regulatory parts will enable the construction of sophisticated gene expression programs. [ABSTRACT FROM AUTHOR]

The article presents a study on the characterization of combinatorial patterns generated by multiple two-component sensors in Escherichia coli (E.coli) that respond to many stimuli. It examines the responses of three two-component systems in E. coli which have been reported to respond to many environmental stimuli. Results show that the data demonstrate one mechanism by which bacteria are able to use a limited set of sensors to identify a diverse set of compounds and environmental conditions.

The computational algorithm SCHEMA was developed to estimate the disruption caused when amino acid residues that interact in the three-dimensional structure of a protein are inherited from different parents upon recombination. To evaluate how well SCHEMA predicts disruption, we have shuffled the distantly-related β-lactamases PSE-4 and TEM-1 at 13 sites to create a library of 214 (16,384) chimeras and examined which ones retain lactamase function. Sequencing the genes from ampicillin-selected clones revealed that the percentage of functional clones decreased exponentially with increasing calculated disruption ( E = the number of residue-residue contacts that are broken upon recombination). We also found that chimeras with low E have a higher probability of maintaining lactamase function than chimeras with the same effective level of mutation but chosen at random from the library. Thus, the simple distance metric used by SCHEMA to identify interactions and compute E allows one to predict which chimera sequences are most likely to retain their function. This approach can be used to evaluate crossover sites for recombination and to create highly mosaic, folded chimeras. [ABSTRACT FROM AUTHOR]

Significant efforts exist to develop living/non‐living composite materials—known as biohybrids—that can support and control the functionality of biological agents. To enable the production of broadly applicable biohybrid materials, new tools are required to improve replicability, scalability, and control. Here, the Hybrid Living Material (HLM) fabrication platform is presented, which integrates computational design, additive manufacturing, and synthetic biology to achieve replicable fabrication and control of biohybrids. The approach involves modification of multimaterial 3D‐printer descriptions to control the distribution of chemical signals within printed objects, and subsequent addition of hydrogel to object surfaces to immobilize engineered Escherichia coli and facilitate material‐driven chemical signaling. As a result, the platform demonstrates predictable, repeatable spatial control of protein expression across the surfaces of 3D‐printed objects. Custom‐developed orthogonal signaling resins and gene circuits enable multiplexed expression patterns. The platform also demonstrates a computational model of interaction between digitally controlled material distribution and genetic regulatory responses across 3D surfaces, providing a digital tool for HLM design and validation. Thus, the HLM approach produces biohybrid materials of wearable‐scale, self‐supporting 3D structure, and programmable biological surfaces that are replicable and customizable, thereby unlocking paths to apply industrial modeling and fabrication methods toward the design of living materials. [ABSTRACT FROM AUTHOR]

Living cells can impart materials with advanced functions, such as sense‐and‐respond, chemical production, toxin remediation, energy generation and storage, self‐destruction, and self‐healing. Here, an approach is presented to use light to pattern Escherichia coli onto diverse materials by controlling the expression of curli fibers that anchor the formation of a biofilm. Different colors of light are used to express variants of the structural protein CsgA fused to different peptide tags. By projecting color images onto the material containing bacteria, this system can be used to pattern the growth of composite materials, including layers of protein and gold nanoparticles. This is used to pattern cells onto materials used for 3D printing, plastics (polystyrene), and textiles (cotton). Further, the adhered cells are demonstrated to respond to sensory information, including small molecules (IPTG and DAPG) and light from light‐emitting diodes. This work advances the capacity to engineer responsive living materials in which cells provide diverse functionality. [ABSTRACT FROM AUTHOR]

Controlling gene expression during a bioprocess enables real‐time metabolic control, coordinated cellular responses, and staging order‐of‐operations. Achieving this with small molecule inducers is impractical at scale and dynamic circuits are difficult to design. Here, we show that the same set of sensors can be integrated by different combinatorial logic circuits to vary when genes are turned on and off during growth. Three Escherichia coli sensors that respond to the consumption of feedstock (glucose), dissolved oxygen, and by‐product accumulation (acetate) are constructed and optimized. By integrating these sensors, logic circuits implement temporal control over an 18‐h period. The circuit outputs are used to regulate endogenous enzymes at the transcriptional and post‐translational level using CRISPRi and targeted proteolysis, respectively. As a demonstration, two circuits are designed to control acetate production by matching their dynamics to when endogenous genes are expressed (pta or poxB) and respond by turning off the corresponding gene. This work demonstrates how simple circuits can be implemented to enable customizable dynamic gene regulation. Synopsis: Genetically encoded sensors responding to feedstock (glucose), growth conditions (oxygen), and byproduct accumulation (acetate) are designed and their response is measured during growth. Sensor integration by combinatorial logic circuits generates different temporal responses and can be used to control metabolism. Genetically encoded sensors for glucose and oxygen are built based on synthetic Escherichia coli promoters that respond to native regulators and optimized using oligo synthesis and high‐throughput screening.As shown computationally, combinatorial logic circuits that implement different truth tables can integrate the three sensors to produce different dynamic responses during a growth experiment.The circuit output can be used to quickly and strongly repress a target gene by combining CRISPRi (transcriptional control) and a targeted protease (post‐translational control).Genetic circuits with CRISPRi/protease outputs were able to dynamically repress native target genes and regulate acetate metabolism. Genetically encoded sensors responding to feedstock (glucose), growth conditions (oxygen), and byproduct accumulation (acetate) are designed and their response is measured during growth. Sensor integration by combinatorial logic circuits generates different temporal responses and can be used to control metabolism. [ABSTRACT FROM AUTHOR]

The type III secretion system (T3SS) exports proteins from the cytoplasm, through both the inner and outer membranes, to the external environment. Here, a system is constructed to harness the T3SS encoded within Salmonella Pathogeneity Island 1 to export proteins of biotechnological interest. The system is composed of an operon containing the target protein fused to an N-terminal secretion tag and its cognate chaperone. Transcription is controlled by a genetic circuit that only turns on when the cell is actively secreting protein. The system is refined using a small human protein (DH domain) and demonstrated by exporting three silk monomers (ADF-1, -2, and -3), representative of different types of spider silk. Synthetic genes encoding silk monomers were designed to enhance genetic stability and codon usage, constructed by automated DNA synthesis, and cloned into the secretion control system. Secretion rates up to 1.8 mg l−1 h−1 are demonstrated with up to 14% of expressed protein secreted. This work introduces new parts to control protein secretion in Gram-negative bacteria, which will be broadly applicable to problems in biotechnology. [ABSTRACT FROM AUTHOR]

Microorganisms use genetic circuits to integrate environmental information. We have constructed a synthetic AND gate in the bacterium Escherichia coli that integrates information from two promoters as inputs and activates a promoter output only when both input promoters are transcriptionally active. The integration occurs via an interaction between an mRNA and tRNA. The first promoter controls the transcription of a T7 RNA polymerase gene with two internal amber stop codons blocking translation. The second promoter controls the amber suppressor tRNA supD. When both components are transcribed, T7 RNA polymerase is synthesized and this in turn activates a T7 promoter. Because inputs and outputs are promoters, the design is modular; that is, it can be reconnected to integrate different input signals and the output can be used to drive different cellular responses. We demonstrate this modularity by wiring the gate to integrate natural promoters (responding to Mg2+ and AI-1) and using it to implement a phenotypic output (invasion of mammalian cells). A mathematical model of the transfer function is derived and parameterized using experimental data. [ABSTRACT FROM AUTHOR]