Probing the operability regime of an engineered ribocomputing unit in terms of dynamic range maintenance with extracellular changes and time

Roser Montagud-Martínez,Arantxa Rosado,Jordi Ventura,Rafael Ballesteros-Garrido,Guillermo Rodrigo

doi:10.1186/s13036-020-00234-5

Abstract

Synthetic biology aims at engineering gene regulatory circuits to end with cells (re)programmed on purpose to implement novel functions or discover natural behaviors. However, one overlooked question is whether the resulting circuits perform as intended in variety of environments or with time. Here, we considered a recently engineered genetic system that allows programming the cell to work as a minimal computer (arithmetic logic unit) in order to analyze its operability regime. This system involves transcriptional and post-transcriptional regulations. In particular, we studied the analog behavior of the system, the effect of physicochemical changes in the environment, the impact on cell growth rate of the heterologous expression, and the ability to maintain the arithmetic functioning over time. Conclusively, our results suggest 1) that there are wide input concentration ranges that the system can correctly process, the resulting outputs being predictable with a simple mathematical model, 2) that the engineered circuitry is quite sensitive to temperature effects, 3) that the expression of heterologous small RNAs is costly for the cell, not only of heterologous proteins, and 4) that a proper genetic reorganization of the system to reduce the amount of heterologous DNA in the cell can improve its evolutionary stability.

Highlights

The design and subsequent implementation of synthetic gene circuits provide valuable information about the corresponding counterparts found in nature [1]
We present experimental results that served us to critically analyze the operability regime of this synthetic circuit, which is of importance on the light of the considerable growth of RNA synthetic biology in recent years [17]
We monitored the expression of the monomeric red fluorescent protein and the superfolder green fluorescent protein for each induction condition (Fig. 2)

Summary

Introduction

The design and subsequent implementation of synthetic gene circuits provide valuable information about the corresponding counterparts found in nature [1]. To what extent the design principles exploited to engineer synthetic gene circuits [1] are not influenced by more complex processes (linked to the physicochemical properties of the different biological species and the intricate regulatory circuitry of the cell) is not entirely known [6], there are ongoing efforts in this direction [7]. Intra- or intermolecular RNA interactions are quite sensitive to changes in temperature, pH, osmolarity, or metal ions [8, 9].

Methods

Results

Conclusion