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Characterization and mitigation of gene expression burden in mammalian cells.

ABSTRACT: Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematical model, 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 use of endogenous miRNAs as elementary 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.

SUBMITTER: Frei T

PROVIDER: S-EPMC7492461 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Characterization and mitigation of gene expression burden in mammalian cells.

Frei Timothy T Cella Federica F Tedeschi Fabiana F Gutiérrez Joaquín J Stan Guy-Bart GB Khammash Mustafa M Siciliano Velia V

Nature communications 20200915 1

Despite recent advances in circuit engineering, the design of genetic networks in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here, we demonstrate that transiently expressed genes in mammalian cells compete for limited transcriptional and translational resources. This competition results in the coupling of otherwise independent exogenous and endogenous genes, creating a divergence between intended and actual function. Guided by a resource-aware mathematica ...[more]

PMID: 32934213

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Project description:The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2?26. eGFP-Kir6.2?26 was expressed in HEK293?cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2?26 retains native single channel conductance (64?±?3.3?pS), mean open times (?1?=?0.72?ms, ?2?=?15.3?ms) and ATP affinity (IC50?=?115?±?25??M) when expressed in HEK293?cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2?26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-?-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5?mol ratio. Reconstituted eGFP-Kir6.2?26 displayed similar single channel conductance (61.8?±?0.54?pS) compared to eGFP-Kir6.2?26 expressed in HEK293 membranes; however, channel mean open times increased (?1?=?7.9?ms, ?2?=?61.9?ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2?26 reconstituted into BLMs (IC50?=?3.14?±?0.4?mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

Dataset Information

Characterization and mitigation of gene expression burden in mammalian cells.

Publications

Characterization and mitigation of gene expression burden in mammalian cells.

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