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Circuit training for bioengineers For the creativity of synthetic biologists to be unleashed, basic circuits must become truly interchangeable — that is, modular and scalable. Two papers in this week's Nature take steps towards that goal — one from the Escherichia coli camp and the other using yeast. Tamsir et al . harness bacterial 'quorum sensing' in E. coli and Regot et al . exploit yeast pheromone communication to achieve complex computation through communication between individual cells performing simple logic functions. Such extracellular 'chemical wiring' is one promising way to get around the difficulty of insulating different genetic circuits when these operate within a single cell. For synthetic biologists' creativity to be unleashed, basic circuits must become truly interchangeable, that is, modular and scalable. This study, one of two linked papers, has harnessed bacterial 'quorum sensing' to achieve complex computation through communication between individual cells performing simple logic functions. Such extracellular 'chemical wiring' is one promising way to get around intracellular noise when building more complex genetic circuitry. Computation underlies the organization of cells into higher-order structures, for example during development or the spatial association of bacteria in a biofilm 1 , 2 , 3 . Each cell performs a simple computational operation, but when combined with cell–cell communication, intricate patterns emerge. Here we study this process by combining a simple genetic circuit with quorum sensing to produce more complex computations in space. We construct a simple NOR logic gate in Escherichia coli by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum-sensing ‘sender’ and ‘receiver’ devices 4 , 5 . The quorum molecules form the wires between gates. By arranging the colonies in different spatial configurations, all possible two-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the ‘on’ and ‘off’ states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between cells.

The creation of orthogonal ‘AND’ logic gates by combining DNA-binding proteins into complex, layered circuits opens the way to the design of programmable integrated circuits in synthetic biology. New levels of complexity for synthetic gene circuits Synthetic genetic circuits tend to interfere with one another, a complication that restricts the number of circuits that can be used to program a cell. Chris Voigt and colleagues have mined a collection of DNA-binding proteins that depend on specific 'chaperone' proteins to activate the transcription of their target genes, and combined them into complex, layered circuits of orthogonal 'AND' logic gates. Using this system, the authors constructed one of the largest genetic programs built so far, consisting of seven integrated sensors/circuits and eleven regulatory proteins. This work opens the way for the design of programmable integrated circuits in synthetic biology. Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics 1 . These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits 2 , 3 , 4 , 5 , 6 , and they have been used in various cellular engineering applications, including the implementation of process control in metabolic networks and the coordination of spatial differentiation in artificial tissues. A key limitation is that the circuits are based on biochemical interactions occurring in the confined volume of the cell, so the size of programs has been limited to a few circuits 1 , 7 . Here we apply part mining and directed evolution to build a set of transcriptional AND gates in Escherichia coli . Each AND gate integrates two promoter inputs and controls one promoter output. This allows the gates to be layered by having the output promoter of an upstream circuit serve as the input promoter for a downstream circuit. Each gate consists of a transcription factor that requires a second chaperone protein to activate the output promoter. Multiple activator–chaperone pairs are identified from type III secretion pathways in different strains of bacteria. Directed evolution is applied to increase the dynamic range and orthogonality of the circuits. These gates are connected in different permutations to form programs, the largest of which is a 4-input AND gate that consists of 3 circuits that integrate 4 inducible systems, thus requiring 11 regulatory proteins. Measuring the performance of individual gates is sufficient to capture the behaviour of the complete program. Errors in the output due to delays (faults), a common problem for layered circuits, are not observed. This work demonstrates the successful layering of orthogonal logic gates, a design strategy that could enable the construction of large, integrated circuits in single cells.

In synthetic biology designs, circuit components can generally move within the cell, meaning that functional cross-talk can cause faulty wiring. Genome mining, synthetic promoter construction and cross-reactivity screening now identify 20 orthogonal TetR repressor-promoter pairs for use in complex applications. Genetic circuits perform computational operations based on interactions between freely diffusing molecules within a cell. When transcription factors are combined to build a circuit, unintended interactions can disrupt its function. Here, we apply 'part mining' to build a library of 73 TetR-family repressors gleaned from prokaryotic genomes. The operators of a subset were determined using an in vitro method, and this information was used to build synthetic promoters. The promoters and repressors were screened for cross-reactions. Of these, 16 were identified that both strongly repress their cognate promoter (5- to 207-fold) and exhibit minimal interactions with other promoters. Each repressor-promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOT/NOR gates, there are >10 54 circuits that could be built by changing the pattern of input and output promoters. This represents a large set of compatible gates that can be used to construct user-defined circuits.

Increasing biological nitrogen (N) fixation (BNF) in maize production could reduce the environmental impacts of N fertilizer use, but reactive N in the rhizosphere of maize limits the BNF process. Using non-transgenic methods, we developed gene-edited strains of Klebsiella variicola ( Kv 137-2253) and Kosakonia sacchari ( Ks 6-5687) bacteria optimized for root-associated BNF and ammonium excretion in N-rich conditions. The aim of this research was to elucidate the mechanism of action of these strains. We present evidence from in vitro, in planta and field experiments that confirms that our genetic remodeling strategy derepresses BNF activity in N-rich systems and increases ammonium excretion by orders of magnitude above the respective wildtype strains. BNF is demonstrated in controlled environments by the transfer of labeled 15 N 2 gas from the rhizosphere to the chlorophyll of inoculated maize plants. This was corroborated in several 15 N isotope tracer field experiments where inoculation with the formulated, commercial-grade product derived from the gene-edited strains (PIVOT BIO PROVEN® 40) provided on average 21 kg N ha -1 to the plant by the VT-R1 growth stages. Data from small-plot and on-farm trials suggest that this technology can improve crop N status pre-flowering and has potential to mitigate the risk of yield loss associated with a reduction in synthetic N fertilizer inputs.

Computation underlies the organization of cells into higher-order structures; for example, during development or the spatial association of bacteria in a biofilm. Each cell performs a simple computational operation, but when combined with cell-cell communication, intricate patterns emerge. Here, we study this process by combining a simple genetic circuit with quorum sensing in order to produce more complex computations in space. A simple NOR gate is constructed by arranging two tandem promoters that function as inputs to drive the transcription of a repressor. The repressor inactivates a promoter that serves as the output. Individual colonies of E. coli carry the same NOR gate, but the inputs and outputs are wired to different orthogonal quorum sensing “sender” and “receiver” devices. The quorum molecules form the wires between gates. By arranging the colonies in different spatial configurations, all possible 2-input gates are produced, including the difficult XOR and EQUALS functions. The response is strong and robust, with 5- to >300-fold changes between the ON and OFF states. This work helps elucidate the design rules by which simple logic can be harnessed to produce diverse and complex calculations by rewiring communication between cells.