Research Article

Cellular checkpoint control using programmable sequential logic

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Science  21 Sep 2018:
Vol. 361, Issue 6408, eaap8987
DOI: 10.1126/science.aap8987

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Building smarter synthetic biological circuits

Synthetic genetic and biological regulatory circuits can enable logic functions to form the basis of biological computing; synthetic biology can also be used to control cell behaviors (see the Perspective by Glass and Alon). Andrews et al. used mathematical models and computer algorithms to combine standardized components and build programmable genetic sequential logic circuits. Such circuits can perform regulatory functions much like the biological checkpoint circuits of living cells. Circuits composed of interacting proteins could be used to bypass gene regulation, interfacing directly with cellular pathways without genome modification. Gao et al. engineered proteases that regulate one another, respond to diverse inputs that include oncogene activation, process signals, and conditionally activate responses such as those leading to cell death. This platform should facilitate development of “smart” therapeutic circuits for future biomedical applications.

Science, this issue p. eaap8987, p. 1252; see also p. 1199

Structured Abstract

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.

Quantitative design of sequential logic in living cells.

Cells can be genetically programmed to respond to temporal stimuli by using complex sequential logic circuits. (Left) Checkpoint control is one such example in which the circuit state (s0 and s1) transitions when the specified input signals are presented. (Middle) Sequential logic circuits can be designed from simple steady-state response functions measured in relative promoter units by using principles of nonlinear dynamics. Bistable latches are used as rewritable memory. The colored symbols represent gates. (Right) The circuit output (Y) was measured for cells that were grown in inputs that were varied over time. The square waveforms indicate the presence or absence of the input signals. Over multiple days, the cells can be cycled through the circuit states or held waiting for the next checkpoint.

Abstract

Biological processes that require orderly progression, such as growth and differentiation, proceed via regulatory checkpoints where the cell waits for signals before continuing to the next state. Implementing such control would allow genetic engineers to divide complex tasks into stages. We present genetic circuits that encode sequential logic to instruct Escherichia coli to proceed through a linear or cyclical sequence of states. These are built with 11 set-reset latches, designed with repressor-based NOR gates, which can connect to each other and sensors. The performance of circuits with up to three latches and four sensors, including a gated D latch, closely match predictions made by using nonlinear dynamics. Checkpoint control is demonstrated by switching cells between multiple circuit states in response to external signals over days.

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