Exploration Of Biosensing Based On Cell-free Systems

In order to bypass the effects of cell membrane and complex signaling pathways in cells on genetic circuit design,cell-free synthetic biology has gradually become one of the commonly used method for researchers.Cell-free synthesis system does not need to maintain cell viability as well as life activity and has no complex signaling pathways compared to cellular systems.It has a simple and open environment,which provides a platform for researchers doing flexible engineering design.This paper introduces the applications of cell-free systems and then illustrates the current circumstance of cell-free biosensing in details.After that,we used the cell-free biosynthesis system as a platform and tried to establish a series of cell-free biosensing systems,including cell-free inorganic ion biosensing systems,cell-free organic molecular biosensing systems,cell-free blue-light sensing systems,and cell-free temperature sensing system.Five cell-free inorganic ion and organic molecular biosensing systems with better response performance were obtained by screening cell extracts types and Mg²⁺concentration.Inorganic ion and organic molecular biosensing systems were explored from three aspects:sensitivity,response time and selectivity.The sensitivity of cell-free biosensing systems could reach nanomolar level.Fluorescence could be seen in 3 h and the response time was less than that of cell systems.The selectivity experiments showed that cell-free biosensing of inorganic ions had no signal interference and good selectivity.There has been a lack of a convenient control switch on protein synthesis in cell-free systems.Optogenetic tool is an ideal control switch for its quick response time,non-toxicity,and good spatial temporal conversion.A blue light regulated two-component system YF1/Fix J was introduced into cell-free systems to control protein synthesis.After a series of exploration,the photosensitive protein YF1 and Fix J were induced into cell extract,and the cell-free system was able to realize the blue light regulated sensing function.Moreover,the mass ratio of two photosensitive proteins YF1 and Fix J was optimized and finally determined to be 0.25:1 to enable an ideal blue light sensing in cell-free systems.Cell-free system achieved five-fold dynamic expression of protein by blue light repressing and three-fold dynamic expression by blue light activating.To demonstrate the universality of cell-free blue light sensing systems,using the optimized system,blue light controlled imaging and antibody proteins synthesis could be successfully performed.Temperature is a special switch that can be specifically activated in living organism.The establishment of a thermal sensing circuit in cell-free system can be used for site-specific synthesizing drugs in vivo.The experiment combined purified temperature-sensitive protein Tlp A36 with its corresponding promoter in vitro,which demonstrating that temperature-sensitive protein cannot achieve thermal sensing in cell-free system.In this study,inorganic ions biological detection,organic molecules biological detection,and blue light controlled protein synthesis were successfully achieved by establishing genetic circuits in cell-free system.These cell-free biosensing systems not only provide design methods for cell-free gene circuits,increase the control of cell-free systems,but also expand the application of cell-free systems,from diagnostics,education to medication,and promote the development of cell-free synthetic biology.