Abstract
Precise control of gene expression is critical for biological research and biotechnology. However, transient plasmid transfections in mammalian cells produce a wide distribution of copy numbers per cell, and consequently, high expression heterogeneity. Here, we report plasmid-based synthetic circuits – Equalizers – that buffer copy-number variation at the single-cell level. Equalizers couple a transcriptional negative feedback loop with post-transcriptional incoherent feedforward control. Computational modeling suggests that the combination of these two topologies enables Equalizers to operate over a wide range of plasmid copy numbers. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. We also show that episome-encoded Equalizers enable the rapid generation of extrachromosomal cell lines with stable and uniform expression. Overall, Equalizers are simple and versatile devices for homogeneous gene expression and can facilitate the engineering of synthetic circuits that function reliably in every cell.
Highlights
Precise control of gene expression is critical for biological research and biotechnology
Modeling suggests that negative feedback (NF) and incoherent feedforward (IFF) circuits can function synergistically for dosage compensation
To guide the development of a more effective gene dosage compensation system, we modeled different control topologies and quantified how their circuit output varied as a function of plasmid copy number
Summary
Precise control of gene expression is critical for biological research and biotechnology. We report plasmid-based synthetic circuits – Equalizers – that buffer copy-number variation at the single-cell level. We demonstrate experimentally that Equalizers outperform other gene dosage compensation topologies and produce as low cell-to-cell variation as chromosomally integrated genes. In an ideal compensation circuit, the per-plasmid expression rate is inversely proportional to the copy number; the total protein expression remains constant (Fig. 1) These circuits promise to combine the versatility and convenience of plasmids with the lower cell-to-cell variability of chromosomal expression. Existing mammalian circuits buffer gene dosage variation across a limited range of plasmid copy numbers. Their ability to reduce cell-to-cell expression variability within a transfected population has not been demonstrated or has been incompletely quantified. We describe computational models that guided circuit design a No gene dosage compensation b Perfect gene dosage compensation
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