Synthesizing a novel genetic sequential logic circuit: a push-on push-off switch

Abstract

Design and synthesis of basic functional circuits are the fundamental tasks of synthetic biologists. Before it is possible to engineer higher-order genetic networks that can perform complex functions, a toolkit of basic devices must be developed. Among those devices, sequential logic circuits are expected to be the foundation of the genetic information-processing systems. In this study, we report the design and construction of a genetic sequential logic circuit in Escherichia coli. It can generate different outputs in response to the same input signal on the basis of its internal state, and 'memorize' the output. The circuit is composed of two parts: (1) a bistable switch memory module and (2) a double-repressed promoter NOR gate module. The two modules were individually rationally designed, and they were coupled together by fine-tuning the interconnecting parts through directed evolution. After fine-tuning, the circuit could be repeatedly, alternatively triggered by the same input signal; it functions as a push-on push-off switch.

Figure 1

The GSLC and the related properties of its elements. (A) A simplified diagram of a sequential logic circuit. It consists of a memory module and a combinatorial gate module. (B) The effect of UV input on all repressors. The scissor represents activated RecA^*. Arrows represent UV irradiation activating RecA^* to specifically degrade CI434, CI, and LexA, but with no effect on LacI and CI_ind−. (C) Detailed schematics of the GSLC. Boxes represent the memory module and the NOR gate module. The rectangles with arrow and the full oval circles, respectively, represent genes and their translated proteins. The scissors on proteins represent those proteins that can be cleaved by RecA^*. Open circles represent terminators. Solid lines with arrow represent the regulating relations between genes. Arrows represent activation and blunt arrows represent repression. (D) The two parts interconnecting the memory and the NOR gate modules. LacI functions as an input of the NOR gate module and as an output of the memory module, whereas CI_ind− functions in the reverse manner. (E) Simulation results of the push-on push-off switch process. In simulation, the effect of UV irradiation persists for 120 min; the concentrations of repressors reach a stationary level before the next UV stimulation.

Figure 2

Construction of the bistable memory module. (A) The memory module incorporates three mechanisms: positive feedback, double-negative feedback, and the repressor binding cooperativity. (B) The arrangement of the genes and promoter region in the memory module. Rectangles with arrow represent genes. Open ovals represent terminations. Lines with arrow represent the transcriptional strength and direction of the promoters. Rectangles with colors represent the binding sites of repressors. Gray squares represent the −10 and −35 regions of the promoters. (C) Images of cells carrying the memory module. Each dot represents a colony. The image in the right panel is an enlarged view of the image in the left panel and shows the colony with mixed colors.

Figure 3

Construction of the NOR gate module. (A) The circuit for quantitative measurement of NOR gate. The promoter P_NOR is suppressed by both LacI and LexA, which can be eliminated by IPTG and UV irradiation, respectively. GFP reports the promoter's activity. (B) The truth table of the NOR gate. The output of the NOR gate is ‘ON’ only when neither LacI nor LexA exists; else the output is ‘OFF’. (C) Experimental measurement of the NOR gate by flow cytometry. values and standard deviations were determined from four parallel experiments.

Figure 4

The screening process and the switching efficiency of a functional mutant. (A) Schematics of the screening process to obtain the functional sequential logic circuits. The process is as follows: (1) The two RBS libraries for CI_ind− and LacI were transformed simultaneously into cells harboring a switch module, and the colors of mutant colonies were identified with a fluorescence stereomicroscope. (2) Green colonies were inoculated into 96-well plates, and the cultures were incubated and diluted appropriately. (3) The cultures were transferred onto two of the same type of agar plates with appropriate dilution, one of which was exposed to 25 J/m² of UV irradiation and the other was not. (4) After overnight incubation at 37°C, the colors of each mutant on the two plates were compared using the fluorescence stereomicroscope. The mutants that changed color from green to red after UV irradiation were selected for the next round of screening. The second round of selection was similar as the first one, except that colonies that changed color from red to green after UV stimulus were now selected. (B) The switching efficiency of a functional mutant from the green state (OFF) to the red state (ON). (C) The switching efficiency of the same mutant from the red state to the green state. The mark ‘before’ means samples were taken from the culture before UV irradiation; ‘30 J/m²’ and ‘0 J/m²’ represent cells exposed to 30 J/m² or unexposed to UV irradiation, respectively. Each bar represents the fraction of green cells in the population. Mean values and standard deviations were derived from eight parallel experiments.

Figure 5

A comparison of the switching process between a functional GSLC and control circuits. Indicated are colonies of cells that were exposed to 25 J/m² (labeled as UV triggered) or not exposed to UV irradiation (labeled as NULL UV triggered), respectively. Row-1 was taken from a functional GSLC 8A, whereas Row-2 to Row-4 were taken from the circuit with removed lacI gene, the circuit with removed cI_ind− gene, and the circuit with both lacI and cI_ind− genes removed, respectively.

Figure 6

Multiple sequentially switching events of the push-on push-off switch and the spontaneous switching rates of the initially green and red population. Each bar represents the fraction of green cells in the population. Mean values and standard deviations were derived from eight parallel experiments. Blue bars represent the cells that were repeatedly stimulated by 30 J/m² UV irradiation. Green bars (control-1) and red bars (control-2) represent, respectively, the initially green and red populations that were never exposed to UV irradiation. Mean values and standard deviations were derived from eight parallel experiments.