Abstract
Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Here we address this issue at the level of prokaryotic transcription by insulating minimal promoters and operators to prevent their interaction and enable the biophysical modeling of synthetic transcription without free parameters. This approach allows genetic circuit design with extraordinary precision and diversity, and consequently simplifies the design-build-test-learn cycle of circuit engineering to a mix-and-match workflow. As a demonstration, combinatorial promoters encoding NOT-gate functions were designed from scratch with mean errors of <1.5-fold and a success rate of >96% using our insulated transcription elements. Furthermore, four-node transcriptional networks with incoherent feed-forward loops that execute stripe-forming functions were obtained without any trial-and-error work. This insulation-based engineering strategy improves the resolution of genetic circuit technology and provides a simple approach for designing genetic circuits for systems and synthetic biology.
Highlights
Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels
Synthetic biology seeks to address this issue by developing foundational theories and technologies at the systems level[4,5,6] using various approaches, such as developing design strategies for synthetic genetic circuits based on principles of mature engineering disciplines[7, 8], reverse engineering of natural genetic circuits to create their synthetic counterparts[9,10,11], and exploration of design constraints for dealing with the complex interactions between genetic circuits and their biochemical, host, and environmental factors[12,13,14]
Developments have occurred at the biological parts and modules level and include composable parts that regulate gene expression with high orthogonality and have minimal interferences with the host cells[15,16,17,18], biological insulators that eliminate or buffer against unexpected interferences at the functional[19] and physical[20,21,22,23] interfaces of parts/modules, and a corresponding programming environment that supports automated, highthroughput composing of parts/modules for non-experts[24]
Summary
Rational engineering of biological systems is often complicated by the complex but unwanted interactions between cellular components at multiple levels. Developments have occurred at the biological parts and modules level and include composable parts that regulate gene expression with high orthogonality and have minimal interferences with the host cells[15,16,17,18], biological insulators that eliminate or buffer against unexpected interferences at the functional[19] and physical[20,21,22,23] interfaces of parts/modules, and a corresponding programming environment that supports automated, highthroughput composing of parts/modules for non-experts[24] Despite these abovementioned developments that enable composing biological parts to build circuits by managing or reducing the systems complexity, methods that enable the rational design of individual basic parts, such as promoters, a crucial need for understanding and manipulating basic processes in gene expression, remain elusive. Despite the intensive efforts focused on interpreting and dissecting these modes of interplays in order to develop a rational basis for promoter engineering, either computationally or experimentally[29, 31,32,33], the bottom-up design of prokaryotic promoters using sub-promoter elements is still largely an ad hoc exercise, with prokaryotic promoters are generally regarded as functionally “indivisible” parts in genetic circuit engineering
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