• Overview
• Background
• Design
• Protocol
• Summary and Result

1. Overview

Cells sense the environment, process information, and make response to stimuli. To make cells work well in complex natural environments, lots of processes have to be preset to react to various signals. However, when well-characterized modules are combined to construct higher order systems, unpredictable behaviors often occur because of the interplay between modules. Another significant problem is that complex integrated systems composed of numerous parts may cause cell overload.

We proposed an elegant method to design higher order systems. Instead of merely combining different functional modules, we constructed one integrated processing module with fewer parts by utilizing the common structures between modules. The circuit we designed is a rewirable one and the topological structure of the processing module can be altered to adapt to environmental change. The basic idea is to rewire the connections between parts and devices to implement multiple functions with the help of the site-specific recombination systems.

Based on the design principle we put forward, we built two circuits to verify our idea. Each circuit has three modules including an input module, a processing module, and an output module. The input module receives environmental signal and triggers the rewiring of the processing module. The output module monitors real-time processes using fluorescence intensity.

Our design approach may lead to a revolutionary step towards system integration in synthetic biology. Potential fields of application include organism development, living therapeutics and environment improvement.

2. Background

Since its inception more than a decade ago, synthetic biology has undergone considerable development and has attained significant achievements with the help of the engineering slant. However, there are still obstacles to build a cell. Engineers try to abstract the DNA sequences into some standard functional parts and assemble them using some principles in electrical engineering. So far, the limited understanding of biological system prevents us to combine parts and modules to create larger scale systems. The complexity of synthetic systems didn’t increase rapidly as the Moore’s law (Purnick and Weiss, 2009).

Challenges

There are some common problems that make the circuits we designed not work as our expected. Many failure modes have been collated by Brophy and Voigy in their review (Brophy and Voigt, 2014). In our project, we mainly focus on two modes, crosstalk and host overload, that emerge especially when we create more sophisticated systems. More specifically, regulators may interact with each other’s targets leading to errors in the desired operation, and the synthetic circuits may compete with natural parts that maintain the normal cellular processes for limited resources.

Solution

We designed a time-sharing system that can process information according to the input signal. Cells rewire its synthetic circuit to alter the topological structure of regulatory pathway when they receive the corresponding stimuli. In this way, we reuse the existing synthetic module rather than add a new one to implement another function, which reduces the resource cost in running unnecessary function and prevents the interplay between parallel modules. After overcoming these two big problems, our engineered cells are more versatile and flexible in information processing.