Baojun Wang

Synthetic biology aims to customize biological systems by programming genetic circuits. However, the scale and complexity of genetic circuits are limited by a shortage of composable, programmable, and orthogonal regulatory parts. Recently, split-intron-enabled trans-splicing riboregulators (SENTRs) have emerged as a promising tool for regulating gene expression at the post-transcriptional level, featuring low leakage expression, wide dynamic range (> 1000-fold), high predictability using machine learning, and orthogonality across multiple intronic components. SENTR’s composability with other regulatory modalities, such as split inteins, enables the establishment of a novel split-biomolecule-enabled circuit design paradigm, unleashing the information-processing capacity of a single regulator gene for complex cellular logic computation with up to six inputs. Here, we describe the methods and strategies to implement SENTRs for efficient gene regulation, including computational design, experimental assessment, and statistical analysis of SENTR-based genetic circuits. Validated on ten genes encoding fluorescent proteins, transcription factors, and ncRNAs, this method can be readily utilized to develop SENTRs for diverse applications, such as stringent gene regulation, complex logic computation, and biosensing for intracellular RNAs or physiological biomarkers.

Synthetic biology employs engineering principles to construct genetic circuits with customized functionality, empowering unprecedented control over biological systems. By harnessing this capability to precisely manipulate biological systems, synthetic biosensors are being developed as promising biosensing platforms for on‐site, sustainable, affordable, and easy‐to‐use detection across diverse scenarios, such as environmental monitoring, disease diagnosis, food safety control, and bioproduction optimization. However, the field deployment and real‐world application of synthetic biosensors face considerable challenges in biosensing sensitivity, specificity, speed, stability, and biosafety. This review summarizes recent advancements of genetic circuit‐enabled synthetic biosensors, focusing on their sensory mechanisms, designs, and applications. Moreover, the design principles, enabling tools, and engineering strategies for creating a high‐performing synthetic biosensor are analyzed. In particular, methods for tuning various characteristics of the dose‐response curve, including detection limit, detection threshold, operating range, dynamic range, and leakiness, are thoroughly examined. Finally, this review discusses the functional extension of biosensors by customizing signal‐processing and output modules, and outlines future directions to expedite the transition of synthetic biosensors from laboratory settings to field applications. Genetic circuit‐enabled synthetic biosensors, in collaboration with materials science, electronic engineering, and artificial intelligence, will tremendously expand the application space of synthetic biology.

Current gene circuits designed to time gene expression depend on the intricate interactions among various regulators and their targets, which confines them to a limited range of temporal tunability. Here, we report a programmable timer switch of gene expression termed BioFuse, which allows the reaction time ranging from hours to days. BioFuse comprises a series of fuse-like tandem DNA cassettes that can be sequentially edited by the adenine base editors (ABEs), resulting in either the activation or deactivation of a downstream gene once the editing is complete. Adjusting the number of DNA cassettes incorporated allows precise programming of BioFuse’s reaction time. Applying BioFuse to control carotenoid biosynthesis genes decouples lycopene production from growth in E. coli and increases lycopene yield without external inducers. Using BioFuse in a bacterial autolysis system enables timely and efficient protein release. BioFuse offers a versatile tool for precise, wide-range timing of gene expression and metabolic activities in bacteria, with potential applications in industry and biomedicine.

Biosensors exploit the capabilities of biological systems to acquire a huge variety of chemical or physical information and convert molecular signals into actionable data. Here we took a bottom-up synthetic biology approach to combine the versatility and programmability of whole-cell bacterial biosensors with the sensitivity of electrochemical sensing devices. We built genetic modules to produce different phenazines and wired these to various sensing and information processing modules. A whole-cell bioelectronic sensor with a T7 RNAP-based signal amplifier was first constructed that detected mercury contaminants below the level of WHO safe limit for drinking water. We demonstrated the modularity and programmability of the sensor design by incorporating Boolean logic computation into a dual-input sensor. We subsequently engineered a sensor strain that can produce two phenazine types, giving a two-channel electrochemical output signal based on the detection of differentiated midpoint potentials. Our modular bioelectronic sensor therefore can be readily adapted for different applications and forms the basis for development of low-cost, field-deployable sensing devices.