In vitro allosteric transcription factor-based biosensing

Bacterial allosteric transcription factors (aTFs) are a new class of recognition elements for in vitro biosensing. In vitro aTF-based biosensing approaches address issues of whole-cell biosensors and open a novel route to develop tailored aTF-based biosensors. Harnessing of aTF recognition coupled with proximity effect-driven output, DNA signal output, cell-free systems, and CRISPR-Cas outputs has been developed to configure biosensors in vitro. Unique traits of aTFs endow biosensing strategies with the characteristics of high accessibility, modularity, easy fine-tuning, and low cost. Concerted efforts are required to develop a modular, systematic, and predictable workflow for aTF discovery, aTF-based biosensor configuration, and fine-tuning to readily fulfill user-defined applications. A biosensor is an analytical device that converts a biological response into a measurable output signal. Bacterial allosteric transcription factors (aTFs) have been utilized as a novel class of recognition elements for in vitro biosensing, which circumvents the limitations of aTF-based whole-cell biosensors (WCBs) and helps to meet the increasing requirement of small-molecule biosensors for diverse applications. In this review, we summarize the recent advances related to the configuration of aTF-based biosensors in vitro. Particularly, we evaluate the advantages of aTFs for in vitro biosensing and highlight their great potential for the establishment of robust and easy-to-implement biosensing strategies. We argue that key technical innovations and generalizable workflows will enhance the pipeline for facile construction of diverse aTF-based small-molecule biosensors. A biosensor is an analytical device that converts a biological response into a measurable output signal. Bacterial allosteric transcription factors (aTFs) have been utilized as a novel class of recognition elements for in vitro biosensing, which circumvents the limitations of aTF-based whole-cell biosensors (WCBs) and helps to meet the increasing requirement of small-molecule biosensors for diverse applications. In this review, we summarize the recent advances related to the configuration of aTF-based biosensors in vitro. Particularly, we evaluate the advantages of aTFs for in vitro biosensing and highlight their great potential for the establishment of robust and easy-to-implement biosensing strategies. We argue that key technical innovations and generalizable workflows will enhance the pipeline for facile construction of diverse aTF-based small-molecule biosensors. a major class of regulatory proteins that are also known as one-component signal transduction systems. On binding to small molecules, aTFs undergo a conformational change that alters their affinity to a specific DNA binding site. a bead-based assay technology used to study biomolecular interactions in a microplate format. also known as trans cleavage; nonspecific cleavage of ssDNA or RNA only when class 2 CRISPR-Cas binds to the target sequence (dsDNA or RNA). a method used in protein engineering that mimics the process of natural selection to steer proteins toward a user-defined goal. optical process of energy transfer that occurs rapidly from a donor molecule to an acceptor molecule in juxtaposition within a distance of 10 nm. an assembly of biological parts that enables cells to perform desired functions. a method that synthesizes proteins from DNA or mRNA in test tubes. It is a good platform for mimicking living cells in homogeneous buffer solutions in vitro. a nanometer-sized luminescent semiconductor crystal with unique chemical and physical properties due to its size and highly compact structure. specifically recognizes analytes. Enzymes, aptamers, antibodies, and cells are examples of recognition elements. a highly sensitive and selective isothermal amplification technique, which is currently commercialized by TwistDx. a unidirectional nucleic acid replication process. This isothermal process can produce a concatemer containing tens to hundreds of tandem repeats that are complementary to the circular template. The produced concatemer can be separated by a special endonuclease to generate many target repeats. an isothermal, in vitro nucleic acid amplification technique. It relies on a strand displacement DNA polymerase (e.g., DNA polymerase Klenow F), a DNA nicking event targeted via primer design, and a corresponding nicking endonuclease. The nicking site is regenerated with each polymerase displacement step for repeated cycles of nicking and extension, with the downstream strand displaced and free to anneal to primers in solution for amplification from the other end, resulting in exponential amplification. a module that can convert recognition events into measurable signals. using microbial cells as the recognition and transduction elements to respond to analytes and produce detectable output signals.