Robust multicellular computing using genetically encoded NOR gates and chemical ‘wires’

Abstract

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)....

Specification and Simulation of Synthetic Multicelled Behaviors

“…The mean μ and variance σ 2 of the division size are also parameters that can be set in gro. By default, μ = 3.14 fL and σ 2 = 0.005 fL 2 . gro simulates a simple N step counting process tuned to result in the desired division size statistics.…”

Spatial dispersal of bacterial colonies induces a dynamical transition from local to global quorum sensing

“…However, the quantitative agreement between the analytical theory and numerical simulations is not as strong -in particular, the mean-field theory appears to overestimate the LQS boundary by a factor of two, with the resulting GQS boundaries being similarly distorted. The origin of this discrepancy can be attributed to the simplifying approximation used in equation (25), namely, treating a finite-radius cell as a localized Dirac Delta function point source. In Figure 7, we compare the actual numerical results for the steadystate concentration due to a single cell with the analytical prediction of the point-source Green's function.…”

“…In this work, we restrict ourselves to a regime where the timescale of sigmoidal quorum sensing activation is taken to be much faster than the timescale for signal production and diffusion, which implies that in the limit of coarse-grained, long-time integrations, the activation can be approximated by an instantaneous all-or-none step function (for a specific recent study analyzing the range of validity of this approximation in quorum sensing systems, we refer the reader to [30], and for further validation data com- a Taken directly from [8]. b Selected as a lower limit from the reported range of 5-fold to 300-fold change in [25], for numerical convenience.…”

Genetic Circuit for the Early Warning of Lung Cancer using iBioSim

“…Anderson designed an AND logic gate based on the transcription of a gene, for which translation is dependent on the co-transcription of an engineered tRNA [12]. Tam sir and Moon finished comprehensive engineering of robust transcriptional logic including all 16 elementary logic gates, as well as the engineering of a multiple input logic network using a multitier transcriptional cascade [13][14]. Chau used protein signalling circuits to produce spatial polarization in yeast by engineering circuits from components that can be self-organized into localized distributions [15].…”