Fig. 4.

Optimized genetic elements and reliable multicycle operation of a DNA-inversion RAD module. (A) Details of an integrated DNA inversion RAD module optimized for reliable set, reset, and storage functions. Specific genetic regulatory elements controlling protein synthesis and degradation were obtained from standard biological parts collections, via computational design, or via random mutagenesis and screening, as noted (main text and SI Appendix). (B) Experimental RAD module operation over multiple duty cycles. Growing cells (doubling time ∼90 min) starting in state “1” were cycled through a “reset, hold, set, hold” input pattern, with each step lasting ∼10 generations. Cell state was assayed via multicolor cytometry. Population distribution GFP (y axis) and forward scatter (x axis) levels are shown for samples taken from the generation number following the input step given directly above each scatter plot (for example, the first scatter plot shows the population distribution at generation 10) (main text and SI Appendix). (C) Multicycle RAD module operation driven by shorter SET and RESET input pulses. As in B but with set and reset pulses lasting for ∼2.5 cell doublings (∼4 h).