Synthesis of an Event Detector in a Cell-Free Expression System

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Jongmin Kim, Ishan Khetarpal, Arjun Ravikumar, Enoch Yeung, Richard M. Murray

California Institute of Technology, United States

In biological organisms, efficient processing of the temporal sequence of inputs represents an important task for development and survival. Over the last decade, engineering approaches in synthetic biology community demonstrated several logic gates and complex circuits in biological organisms. Recently, a recombinase-based cascade of memory units was successfully utilized for synthetic circuits that respond to a sequence of inducer inputs, indicating its potential for general implementation of temporal logic in cells.
Synthetic biology approaches in cell-free expression system offers an attractive alternative to cellular engineering for rapid prototyping and engineering of controller for production of natural and unnatural, even cytotoxic products. Here, we focus on the construction and characterization of an event detector in a cell-free expression system. The design of an event detector utilizes a previously characterized synthetic transcriptional memory module that responds to two distinct DNA inputs, ‘A’ and ‘B’, resulting in two stable steady-states depending on which input becomes available first. Utilizing modular and programmable switch motifs, it is straightforward to implement an event detector that distinguishes the temporal sequence of inputs.
The functionality of event detector is first demonstrated in a cell-free transcription system. When the event detector is configured to recognize ‘input A then input B’ case, the output signal, an RNA aptamer for fluorescent dye, is produced only upon the introduction of input ‘A’ followed by the input ‘B’, whereas no output signal is detected upon the introduction of any single input or the two inputs in reverse order. To further develop the general applicability of such circuits, we also tested the event detector in a cell-free transcription/translation system (PURExpressTM), where the transcription switch modules support efficient translational control of target proteins by generating programmable antisense signals. Together, the modular transcriptional circuit architecture in cell-free expression system offers a powerful platform for temporal logic control.