Cellular checkpoint control using programmable sequential logic

INTRODUCTION Modern computing is based on sequential logic, in which the state of a circuit depends both on the present inputs as well as the input history (memory). Implementing sequential logic inside a living cell would enable it to be programmed to progress through discrete states. For example, cells could be designed to differentiate into a multicellular structure or order the multistep construction of a material. A key challenge is that sequential logic requires the implementation of regulatory feedback, which has proven difficult to design and scale. RATIONALE We present a quantitative method to design regulatory circuits that encode sequential logic. Our approach uses NOT gates as the core unit of regulation, in which an input promoter drives the expression of a repressor protein that turns off an output promoter. Each gate is characterized by measuring its response function, in other words, how changing the input affects the output at steady state. Mathematically, the response functions are treated as nullclines, and tools from nonlinear dynamics (phase plane and bifurcation analyses) are applied to predict how combining gates leads to multiple steady states and dynamics. The circuits can be connected to genetic sensors that respond to environmental information. This is used to implement checkpoint control, in which the cell waits for the right signals before continuing to the next state. Circuits are built that instruct Escherichia coli to proceed through a linear or cyclical sequence of states. RESULTS First, pairs of repressors are combined to build the simplest unit of sequential logic: a set-reset (SR) latch, which records a digital bit of information. The SR latches can be easily connected to each other and to sensors because they are designed such that the inputs and outputs are both promoters. Each latch requires two repressors that inhibit each other’s expression. A total of 11 SR latches were designed by using a phase plane analysis. The computation accurately predicts the existence of multiple steady states by using only the empirical NOT gate response functions. A set of 43 circuits was constructed that connects these latches to different combinations of sensors that respond to small molecules in the media. These circuits are shown to reliably hold their state for >48 hours over many cell divisions, only switching states in response to the sensors that connect to the set and reset inputs of the latch. Larger circuits are constructed by combining multiple SR latches and additional feedback loops. A gated data (D) latch, common in electronic integrated circuits, is constructed where one input sets the state of the circuit and the second input locks this state. Up to three SR latches (based on six repressors) are combined in a single cell, thus allowing three bits to be reversibly stored. The performances of these circuits closely match those predicted by the responses of the component gates and a bifurcation analysis. Circuits are designed to implement checkpoint control, in which cells wait indefinitely in a state until the correct signals are received to progress to the next state. The progression can be designed to be cyclical, analogous to cell cycle phases, during which cells progress through a series of states until returning to the starting state. The length of time in each state is indefinite, which is confirmed by demonstrating stability for days when the checkpoint conditions are not met. CONCLUSION This work demonstrates the implementation of sequential logic circuits in cells by combining reliable units of regulation according to simple rules. This approach is conducive to design automation software, which can use these rules to combine gates to build larger circuits. This provides a designable path to building regulatory networks with feedback loops, critical to many cellular functions and ubiquitous in natural networks. This represents a critical step toward performing advanced computing inside of cells.