Multiplexed Gene Expression Tuning with Orthogonal Synthetic Gene Circuits

Abstract

Understanding the individual and joint contribution of multiple protein levels toward a phenotype requires precise and tunable multigene expression control. Here we introduce a pair of mammalian synthetic gene circuits that linearly and orthogonally control the expression of two reporter genes in mammalian cells with low variability in response to chemical inducers introduced into the growth medium. These gene expression systems can be used to simultaneously probe the individual and joint effects of two gene product concentrations on a cellular phenotype in basic research or biomedical applications.

Context-aware synthetic biology by controller design: Engineering the mammalian cell

“…Input-output linearization has been successful for two inducible systems (TetR/Dox and PhlF/DAPG) in mammalian cells (Szenk et al, 2020), both of which involve a small molecule inhibiting negative autoregulation by the TF. Transcriptional repressors feeding back on their own production are classically modeled as proportional feedback modules.…”

“…Biosensors and the genetic/cellular responses that they generate are the basic building blocks of a cell therapy that can sense and respond to cues in the body. Controllers can be applied when developing such sensors in order to linearize their I/O responses (Nevozhay et al, 2013;Nunns and Goentoro, 2018;Sturm et al, 2010;Szenk et al, 2020), decrease noise (Dublanche et al, 2006;Jones et al, 2021), and ensure that the sensor functions are robust to perturbations (Jones et al, 2021;M€ uller et al, 2019;Steel et al, 2019). Thus, controllers can help both in the development of devices with ideal properties, as well as in ultimately yielding systems that will be more likely to work in varying contexts, such as when moved from cell lines (e.g., Jurkat T cells) to primary cells, or when in vitro tests with those engineered primary cells are moved in vivo.…”

An endoribonuclease-based feedforward controller for decoupling resource-limited genetic modules in mammalian cells

“…There are many sources of context dependence, including unexpected off-target interactions between regulators and their targets 6 – 8 , transcription factor (TF) loading by DNA targets 9 , 10 , gene orientation 11 , and resource loading by expressed genes 12 , 13 . To date, much effort has gone into identifying and engineering gene regulators with unique binding specificity, e.g., between TFs and their DNA-binding sites, with the goal of finding gene regulators that work orthogonally 7 , 14 – 18 . Nevertheless, even if subsystems are entirely composed of putatively orthogonal regulators, their gene expression levels can still become coupled to each other via competition for shared cellular resources 2 , 12 , 13 , 19 , 20 .…”

Development of Host‐Orthogonal Genetic Systems for Synthetic Biology

“…In addition, a plentiful of mutually orthogonal genetic elements that are both orthogonal to the host cell and each other have been mined and developed in various organisms, which made it possible for constructing more complex and user‐defined multigene expression programs in various hosts. [ 164,171 ] Currently, the large‐scale applications of host‐orthogonal systems are still facing a number of major limitations. First, the host strain is difficult to fully accommodate integrated orthogonal elements.…”