Dynamic allocation of orthogonal ribosomes facilitates uncoupling of co-expressed genes

Abstract

Introduction of synthetic circuits into microbes creates competition between circuit and host genes for shared cellular resources, such as ribosomes. This can lead to the emergence of unwanted coupling between the expression of different circuit genes, complicating the design process and potentially leading to circuit failure. By expressing a synthetic 16S rRNA with altered specificity, we can partition the ribosome pool into host-specific and circuit-specific activities. We show mathematically and experimentally that the effects of resource competition can be alleviated by targeting genes to different ribosomal pools. This division of labour can be used to increase flux through a metabolic pathway. We develop a model of cell physiology which is able to capture these observations and use it to design a dynamic resource allocation controller. When implemented, this controller acts to decouple genes by increasing orthogonal ribosome production as the demand for translational resources by a synthetic circuit increases.

Effects of spatial heterogeneity on bacterial genetic circuits

“…In this case, as a result of translation being localized at the gene location, the effective ribosome binding site strength will also depend on gene location through the BCF. We may also consider the role of spatial heterogeneity on orthogonal translational machinery [39]. From our models, we predict that one can tune the rate at which orthogonal ribosomes are formed by creating larger synthetic 16S rRNA.…”

“…From our models, we predict that one can tune the rate at which orthogonal ribosomes are formed by creating larger synthetic 16S rRNA. Furthermore, once the production of orthogonal ribosomes is placed in a feedback form to decouple genetic circuits [39], our framework suggests that the feedback efficiency may depend on the spatial location of the synthetic 16S rRNA gene. The value of the parameter r * , whose squared value is inversely proportional to the average chromosome density (2), is critical in determining the extent of spatial effects.…”

Design trade-offs and robust architectures for combined transcriptional and translational resource allocation controllers

“…In [17], we developed a translational controller which dynamically allocates the distribution between host and orthogonal ribosomes by placing the o-rRNA gene, g r under the control of a protein, p f , which itself is translated by the orthogonal ribosome pool. The controller protein, p f , sequesters free rRNA promoters to an inactive complex, k r :…”

“…To alleviate translational resource competition, we have previously designed and implemented a translational resource allocation controller in E. coli [17,18]. The ribosome is a large ribonucleoprotein complex encoded by multiple rRNA and r-protein genes; therefore, unlike in the case of transcription, there does not exist a truly orthogonal ribosome which can be co-opted from another species.…”

“…The translational controller works by regulating the production of the synthetic 16S rRNA. This negative feedback controller takes the form of a repressor protein (LacI was used in [17]) which inhibits 16S rRNA production and itself is translated by the o-ribosome pool. As demand for orthogonal translation increases, the level of the repressor falls due to resource competition and so o-rRNA, and hence orthogonal ribosome, production increases (matching demand).…”