Engineered modular and orthogonal genetic logic gates for robust digital-like synthetic biology had proof of concept built inside E Coli

Nature Communications – Engineering modular and orthogonal genetic logic gates for robust digital-like synthetic biology

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.

A modular and orthogonal genetic AND gate design. The AND gate is designed on the basis of the σ54-dependent hrpR/hrpS hetero-regulation module. Two environment-responsive promoters, P1 and P2, act as the inputs to drive the transcriptions of hrpR and hrpS, and respond to the small molecules I1 and I2, respectively. The transcription of the output hrpL promoter is turned on only when both proteins HrpR and HrpS are present and bind the upstream activator sequence to remodel the closed σ54-RNAP-hrpL transcription complex to an open one through ATP hydrolysis. The output shown is a gfp reporter. The RBS is used for tuning the dynamic range of the device inputs or output. The regulatory promoter inputs and gfp output are both interchangeable. The AND gate is orthogonal to the E. coli genetic background and is independent of its normally used σ70-dependent transcriptional pathway.

A modular and orthogonal genetic AND gate was constructed in E. coli using a new strictly regulated orthogonal system from P. syringae. The hrpR/hrpS hetero-regulation of the σ54-dependent transcription allowed the AND gate to be tightly controlled with a robust near-digital logic behaviour. The device integrated two individual environmental signals through the two transcriptional inputs to generate one output response, accordingly, in the logic AND manner. The AND gate was shown to be modular by exchanging the inputs and output of the circuit while preserving the logic AND function. A modular combinatorial logic NAND gate was generated by directly connecting the AND gate output to a NOT gate module. Because of modularity, the AND and NAND gates can be reconnected to different sensor inputs to integrate multiple environmental and cellular signals and thus be applied to identify a specific combinatorial condition, like the cancer microenvironment. The transcription-based AND gate was shown to be robust with functionality in a range of different tested contexts. Moreover, the signal response surfaces of the AND and NAND gates show the sigmoid response property for both inputs, largely due to the high cooperativity of the two transcriptional activator proteins. The sigmoidal response filtering property can suppress the analogue noise in biomolecular logic systems and increase signal robustness enhancing the error tolerance capability and the scalability of the logic gates. Having a similar role as their electronic counterparts, the engineered AND and NAND gates are the fundamental components in designing biologically based digital systems to control cell signalling and can be easily applied in different contexts, owing to their modularity and orthogonality, to implement human-designed intra- and extra-cellular control functions.

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