Genomic mining of prokaryotic repressors for orthogonal logic gates

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[superscript 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.United States. Air Force Office of Scientific Research (Award FA9550-11-C-0028)American Society for Engineering Education. National Defense Science and Engineering Graduate Fellowship (32 CFR 168a)United States. Defense Advanced Research Projects Agency. Chronical of Lineage Indicative of Origins (N66001-12-C-4016)United States. Office of Naval Research (N00014-13-1-0074)National Institutes of Health (U.S.) (GM095765)National Science Foundation (U.S.). Synthetic Biology Engineering Research Center (SA5284-11210

Synthetic Logic Circuits in Escherichia coli

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

Genetic Programs Constructed from Layered Logic Gates in Single Cells

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.United States. Defense Advanced Research Projects Agency (Chronicle of Lineage Indicative of Origins N66001-12-C-4018)United States. Office of Naval Research (N00014-10-1-0245)National Science Foundation (U.S.) (CCF-0943385)National Institutes of Health (U.S.) (AI067699)National Science Foundation (U.S.) (Synthetic Biology Engineering Research Center SA5284-11210