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.Boolean logic gates integrate multiple digital inputs into a digital output. Electronic integrated circuits consist of many layered gates. In cells, regulatory networks encode logic operations that integrate environmental and cellular signals [6][7][8] . Synthetic genetic logic gates have been constructed, including those that perform AND, OR and NOT functions [9][10][11][12] , and have been used in pharmaceutical and biotechnological applications 13,14 . Multiple gates can be layered to build more complex programs [15][16][17] , but it remains difficult to predict how a combination of circuits will behave on the basis of the functions of the individuals 11,18 . Here we have compartmentalized a simple logic gate into separate E. coli strains and use quorum signalling to allow communication between the strains 5 . Compartmentalizing the circuit produces more reliable computation by population-averaging the response. In addition, a program can be built from a smaller number of orthogonal parts (for example transcription factors) by re-using them in multiple cells. NOR and NAND gates are unique because they are functionally complete. That is, any computational operation can be implemented by layering either of these gates alone 19 . Of these, the NOR gate is the simplest to implement using existing genetic parts. A NOR gate is 'on' only when both inputs are 'off' (Fig. 1a). We designed a simple NOR gate by adding a second input promoter to a NOT gate 20 . Tandem promoters with the same orientation drive the expression of a transcriptional repressor (Fig. 1b). Tandem promoters are common in prokaryotic genomes 21 . This is expected to produce an OR function; however, interference between the promoters can occur (Supplementary Figure 3)....

Genetic programs function to integrate environmental sensors, implement signal processing algorithms and control expression dynamics1. These programs consist of integrated genetic circuits that individually implement operations ranging from digital logic to dynamic circuits2–6, 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 circuits1,7. 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.

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 do not interact with other promoters. Each repressor:promoter pair was converted to a NOT gate and characterized. Used as a set of 16 NOR gates, there are >1054 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.

Synthetic genetic programs promise to enable novel applications in industrial processes. For such applications, the genetic circuits that compose programs will require fidelity in varying and complex environments. In this work, we report the performance of two synthetic circuits in Escherichia coli under industrially relevant conditions, including the selection of media, strain, and growth rate. We test and compare two transcriptional circuits: an AND and a NOR gate. In E. coli DH10B, the AND gate is inactive in minimal media; activity can be rescued by supplementing the media and transferring the gate into the industrial strain E. coli DS68637 where normal function is observed in minimal media. In contrast, the NOR gate is robust to media composition and functions similarly in both strains. The AND gate is evaluated at three stages of early scale-up: 100 ml shake-flask experiments, a 1 ml MTP microreactor, and a 10 L bioreactor. A reference plasmid that constitutively produces a GFP reporter is used to make comparisons of circuit performance across conditions. The AND gate function is quantitatively different at each scale. The output deteriorates late in fermentation after the shift from exponential to constant feed rates, which induces rapid resource depletion and changes in growth rate. In addition, one of the output states of the AND gate failed in the bioreactor, effectively making it only responsive to a single input. Finally, cells carrying the AND gate show considerably less accumulation of biomass. Overall, these results highlight challenges and suggest modified strategies for developing and characterizing genetic circuits that function reliably during fermentation.

Many applications require cells to switch between discrete phenotypic states. Here, we harness the FimBE inversion switch to flip a promoter, allowing expression to be toggled between two genes oriented in opposite directions. The response characteristics of the switch are characterized using two-color cytometry. This switch is used to toggle between orthogonal chemosensory pathways by controlling the expression of CheW and CheW*, which interact with the Tar (aspartate) and Tsr* (serine) chemoreceptors, respectively. CheW* and Tsr* each contain a mutation at their proteinprotein interface such that they interact with each other. The complete genetic program containing an arabinose-inducible FimE controlling CheW/CheW* (and constitutively-expressed tar/tsr*) is transformed into an E. coli strain lacking all native chemoreceptors. This program enables bacteria to swim towards serine or aspartate in the absence or presence of arabinose, respectively. Thus, the program functions as a multiplexer with arabinose as the selector. This demonstrates the ability of synthetic genetic circuits to connect to a natural signaling network to switch between phenotypes.