Modular and orthogonal genetic logic gates are essential for building robust biologically based digital devices to customize cell signalling in synthetic biology. Here we constructed an orthogonal AND gate in Escherichia coli using a novel hetero-regulation module from Pseudomonas syringae. The device comprises two co-activating genes hrpR and hrpS controlled by separate promoter inputs, and a σ54-dependent hrpL promoter driving the output. The hrpL promoter is activated only when both genes are expressed, generating digital-like AND integration behaviour. The AND gate is demonstrated to be modular by applying new regulated promoters to the inputs, and connecting the output to a NOT gate module to produce a combinatorial NAND gate. The circuits were assembled using a parts-based engineering approach of quantitative characterization, modelling, followed by construction and testing. The results show that new genetic logic devices can be engineered predictably from novel native orthogonal biological control elements using quantitatively in-context characterized parts.

Modular cascaded amplification of cellular sensor signal. A typical cellular sensor can be abstracted as a three-stage processor comprising a sensing module that recognizes and transduces external signals into intracellular transcriptional signals, a computing module that modulates the transduced sensor signals, and an output actuating module that executes physiological responses (Fig. 1a) [1][2][3]32 . Our modular signal amplifying methodology integrates three synergistic signal amplification strategies by manipulating the first two modules step-by-step.

Cells perceive a wide variety of cellular and environmental signals, which are often processed combinatorially to generate particular phenotypic responses. Here, we employ both single and mixed cell type populations, pre-programmed with engineered modular cell signalling and sensing circuits, as processing units to detect and integrate multiple environmental signals. Based on an engineered modular genetic AND logic gate, we report the construction of a set of scalable synthetic microbe-based biosensors comprising exchangeable sensory, signal processing and actuation modules. These cellular biosensors were engineered using distinct signalling sensory modules to precisely identify various chemical signals, and combinations thereof, with a quantitative fluorescent output. The genetic logic gate used can function as a biological filter and an amplifier to enhance the sensing selectivity and sensitivity of cell-based biosensors. In particular, an Escherichia coli consortium-based biosensor has been constructed that can detect and integrate three environmental signals (arsenic, mercury and copper ion levels) via either its native two-component signal transduction pathways or synthetic signalling sensors derived from other bacteria in combination with a cell-cell communication module. We demonstrate how a modular cell-based biosensor can be engineered predictably using exchangeable synthetic gene circuit modules to sense and integrate multiple-input signals. This study illustrates some of the key practical design principles required for the future application of these biosensors in broad environmental and healthcare areas.

Recent advances in synthetic biology research have been underpinned by an exponential increase in available genomic information and a proliferation of advanced DNA assembly tools. The adoption of plasmid vector assembly standards and parts libraries has greatly enhanced the reproducibility of research and the exchange of parts between different labs and biological systems. However, a standardized modular cloning (MoClo) system is not yet available for cyanobacteria, which lag behind other prokaryotes in synthetic biology despite their huge potential regarding biotechnological applications. By building on the assembly library and syntax of the Plant Golden Gate MoClo kit, we have developed a versatile system called CyanoGate that unites cyanobacteria with plant and algal systems. Here, we describe the generation of a suite of parts and acceptor vectors for making (1) marked/unmarked knock-outs or integrations using an integrative acceptor vector, and (2) transient multigene expression and repression systems using known and previously undescribed replicative vectors. We tested and compared the CyanoGate system in the established model cyanobacterium Synechocystis sp. PCC 6803 and the more recently described fastgrowing strain Synechococcus elongatus UTEX 2973. The UTEX 2973 fast-growth phenotype was only evident under specific growth conditions; however, UTEX 2973 accumulated high levels of proteins with strong native or synthetic promoters. The system is publicly available and can be readily expanded to accommodate other standardized MoClo parts to accelerate the development of reliable synthetic biology tools for the cyanobacterial community.

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