Construction Of A Modular Cell-free Protein Expression System Based On Digital Microfluidics

With the development of microfluidic technology,the progress of Lab-on-a-chip for biochemistry has developed into a trend.In current microfluidic systems,digital microfluidics(DMF)technology,which drives droplets through the dielectric wetting effect(EWOD),can manipulate droplets by controlling the movement,mixing,separation and other actions of individual droplets with high performance.The advantages of flexibility,high automation and high throughput have received extensive attention in the field of cell-free biology.Cell-free biology has gained rapid development in vitro in recent years due to the openness and control ability of its reaction environment.However,complex liquid handling protocols are cumbersome,especially for large scale,simultaneous execution of multiple different reactions.Digital microfluidic technology is a new micro droplet manipulation technology based on specific electrode array and using the dielectric wetting phenomenon of droplets on hydrophobic surface to realize the orderly,accurate and controllable discrete droplets.We can design and develop an automatic portable synthesis platform suitable for a variety of scenes by changing the form of electrode array.In this paper,a digital microfluidic platform for an integrated cell-free protein expression system suitable for a variety of scenarios was developed,which realizes the DNA assembly and chip synthesis of several common proteins.In this paper,a digital microfluidic platform for an integrated cell-free protein expression system suitable for various scenarios was developed,and several common proteins were synthesized on a chip.Firstly,from the perspective of application,two common digital microfluidic chips were made in this article:printed circuit(PCB)chip and glass based gold electrode array chip.It has the advantage of developing a set of programs based on the graphical control board and python,which can be developed for the whole desktop control system at the same time.Using this platform,we have successfully realized the DNA assembly and amplification of the closed digital microfluidic platform.Compared with traditional methods,digital microfluidic technology can make DNA assembly consume less reagent and time,but the open reaction environment and droplet movement on the chip surface require the following additional conditions:appropriate amplification and assembly conditions;appropriate amount of nonionic surfactant;additional functional factors.After the completion of DNA assembly,we integrated the synthesis system of cell-free protein with the DNA assembly and amplification system,built a set of modular cell-free protein expression system based on digital microfluidic,and successfully synthesized GFP,mCherry,mOrange and other proteins from encoded DNA.The result can reach 80-90%of the yield of conventional methods,and its yield is even higher than that of commercial kits.Compared with traditional methods,the platform is simple to operate,low-cost,saves the time of gene cloning,transformation screening and other operations,and has a good application prospect in the fields of clinical diagnosis,real-time detection and so on.