Biology subjects

Siciliano, V.

Publications and source records attributed to Siciliano, V..

2 recordsLinked to original sources

bioRxiv2019

An endoribonuclease-based incoherent feedforward loop for decoupling resource-limited genetic modules

Synthetic biology has the potential to bring forth advanced genetic devices for applications in healthcare and biotechnology. However, accurately predicting the behavior of engineered genetic devices remains difficult due to lack of modularity, wherein a devices output does not depend only on its intended inputs but also on its context. One contributor to lack of modularity is competition among genes for shared cellular resources, such as those required for transcription and translation, which can induce coupling among otherwise independently-regulated genes. Here, we quantify the effects of resource sharing on engineered genetic systems in mammalian cells and develop an endoribonuclease-based incoherent feedforward loop (iFFL) to make gene expression levels robust to changes in resource availability. Our iFFL accurately controls gene expression levels in various cell lines and in the presence of significant resource sequestration by transcriptional activators. In addition to mitigating resource sharing, our iFFL also adapts gene expression to multiple log decades of DNA copy number variation, substantially improving upon previously-described miRNA-based iFFLs. Ultimately, our iFFL device will enable predictable, robust, and context-independent control of gene expression in mammalian cells.

synthetic biology↗

bioRxiv2019

Characterization, modelling and mitigation of gene expression burden in mammalian cells

Despite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

synthetic biology↗