Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Similar to other engineering disciplines, synthetic biology aims to standardize protocols, allowing tools with highly reproducible functions to be utilized for exploring questions relevant to biological systems^1,2. One domain in synthetic biology where many control systems have been built is the area of gene expression regulation^3,4. Gene expression control can target both protein levels and variability (noise or coefficient of variation, CV = σ/µ, measured as the standard deviation over the mean), which are crucial cellular characteristics d....

1. Gene circuit design

1. Select genetic components to combine into a single gene circuit/plasmid (e.g., mammalian DNA integration sequence motifs³², light-responsive elements³³, or functional genes³⁴).
2. Using any genetic engineering and/or molecular cloning software, store the DNA sequences for later use and reference, annotate each sequence, and examine all the necessary features (e.g., START codons, regulatory, or translated sequ.......

Gene circuit assembly and stable cell line generation within this article were based on commercial, modified HEK-293 cells containing a transcriptionally active, single stable FRT site (Figure 1). The gene circuits were constructed into vectors that had FRT sites within the plasmid, allowing for the Flp-FRT integration into the HEK-293 cell genome. This approach is not limited to Flp-In cells, as FRT sites can be added to any cell line of interest anywhere in the genome using DNA editing tec.......

Readers of this article can gain insight into the steps vital for characterizing optogenetic gene circuits (as well as other gene expression systems), including 1) gene circuit design, construction, and validation; 2) cell engineering for introducing gene circuits into stable cell lines (e.g., Flp-FRT recombination); 3) induction of the engineered cells with a light-based platform such as the LPA; 4) initial characterization of light induction assays via fluorescence microscopy; and 5) final gene expression characterizat.......

We would like to thank Balázsi lab for comments and suggestions, Dr. Karl P. Gerhardt and Dr. Jeffrey J. Tabor for helping us construct the first LPA, and Dr. Wilfried Weber for sharing the LOV2-degron plasmids. This work was supported by the National Institutes of Health [R35 GM122561 and T32 GM008444]; The Laufer Center for Physical and Quantitative Biology; and a National Defense Science and Engineering Graduate (NDSEG) Fellowship. Funding for open access charge: NIH [R35 GM122561].

Author contributions: M.T.G. and G.B. conceived the project. M.T.G., D.C., and L.G., performed the experiments. M.T.G., D.C., L.G., and G.B. analyzed th....