Cell-Free Recombinase Integrated Boolean Output System (CRIBOS)

Cell-free gene expression systems are increasingly important in fundamental research and biomanufacturing, offering a versatile platform for studying gene circuits and bio-computation. We present the Cell-Free Recombinase Integrated Boolean Output System (CRIBOS), a site-specific recombinase-based multiplex genetic circuit platform designed for cell-free environments. With CRIBOS, we build over 20 multi-input-multi-output circuits, including 2-input-1-output genetic circuits and a 2-input-4-output decoder. Combined with allosteric transcription factors (aTFs)-based sensors, the circuits demonstrate multiplex environmental sensing. Moreover, utilizing paper-based CRIBOS, which demonstrates remarkable portability and stability in a paper-based setting, we present a biological memory storage device that can preserve DNA-based biological information for over four months with minimal resources, energy costs, and maintenance requirements. Implementing CRIBOS not only expands the application of multiplex Boolean logic gates from cellular systems to the cell-free environment but also augments their overall versatility, opening new avenues for designing and applying sophisticated genetic circuits.