Comparison between Effects of Retroactivity and Resource Competition upon Change in Downstream Reporter Genes of Synthetic Genetic Circuits

Abstract

Reporter genes have contributed to advancements in molecular biology. Binding of an upstream regulatory protein to a downstream reporter promoter allows quantification of the activity of the upstream protein produced from the corresponding gene. In studies of synthetic biology, analyses of reporter gene activities ensure control of the cell with synthetic genetic circuits, as achieved using a combination of in silico and in vivo experiments. However, unexpected effects of downstream reporter genes on upstream regulatory genes may interfere with in vivo observations. This phenomenon is termed as retroactivity. Using in silico and in vivo experiments, we found that a different copy number of regulatory protein-binding sites in a downstream gene altered the upstream dynamics, suggesting retroactivity of reporters in this synthetic genetic oscillator. Furthermore, by separating the two sources of retroactivity (titration of the component and competition for degradation), we showed that, in the dual-feedback oscillator, the level of the fluorescent protein reporter competing for degradation with the circuits’ components is important for the stability of the oscillations. Altogether, our results indicate that the selection of reporter promoters using a combination of in silico and in vivo experiments is essential for the advanced design of genetic circuits.

From Specification to Implementation: Assume-Guarantee Contracts for Synthetic Biology

“…At the same time, due to the resources being used by a subsystem, loading or retroactivity effects are commonly observed experimentally. Various system analysis approaches in the literature have analyzed retroactivity using mathematical models [26]- [28]. The contract-based design framework that we proposed in this paper can be easily scaled to describe such environmental assumptions as well.…”

Designing Biological Circuits: From Principles to Applications

“…53 A combination of in silico and in vivo methods have been used to study retroactivity in a dualfeedback oscillator circuit. 54 Retroactivity in an in vitro system consisting of an oscillator driving two types of loads: (a) a pair of DNA tweezers, and (b) an RNA aptamer for malachite green, was studied by Franco et al 55 In the case of the DNA tweezer, the oscillator times the closing of the tweezer hands, while for the RNA aptamer, the oscillator periodically switches on/off the production of the RNA. Two types of coupling of the oscillator and the load can occur depending on whether the oscillator species driving the load is consumed or not.…”

“…This effect is known as retroactivity and is an important context-dependent effect that must be addressed for the reliable design of genetic circuits . A combination of in silico and in vivo methods have been used to study retroactivity in a dual-feedback oscillator circuit . Retroactivity in an in vitro system consisting of an oscillator driving two types of loads: (a) a pair of DNA tweezers, and (b) an RNA aptamer for malachite green, was studied by Franco et al In the case of the DNA tweezer, the oscillator times the closing of the tweezer hands, while for the RNA aptamer, the oscillator periodically switches on/off the production of the RNA.…”

Designing biological circuits: from principles to applications

“…This effect is known as retroactivity and is an important context-dependent effect that must be addressed for the reliable design of genetic circuits [44]. A combination of in silico and in vivo methods have been used to study retroactivity in a dual-feedback oscillator circuit [27].…”