Cell-Free Protein Synthesis as a Prototyping Platform for Mammalian Synthetic Biology

Abstract

The field of mammalian synthetic biology is expanding quickly, and technologies for engineering large synthetic gene circuits are increasingly accessible. However, for mammalian cell engineering, traditional tissue culture methods are slow and cumbersome, and are not suited for high-throughput characterization measurements. Here we have utilized mammalian cell-free protein synthesis (CFPS) assays using HeLa cell extracts and liquid handling automation as an alternative to tissue culture and flow cytometry-based measurements. Our CFPS assays take a few hours, and we have established optimized protocols for small-volume reactions using automated acoustic liquid handling technology. As a proof-of-concept, we characterized diverse types of genetic regulation in CFPS, including T7 constitutive promoter variants, internal ribosomal entry sites (IRES) constitutive translation-initiation sequence variants, CRISPR/dCas9-mediated transcription repression, and L7Ae-mediated translation repression. Our data shows simple regulatory elements for use in mammalian cells can be quickly prototyped in a CFPS model system.

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

“…The traditionally cumbersome requirements for in vitro tissue culture maintenance slow down efforts to explore mammalian tissue engineering. However, the optimization of various cell-free protein synthesis reactions run in microfluidic chambers or microchips may encourage high-throughput prototyping in a mammalian cell-free protein synthesis system [172,173].…”

Biological Materials: The Next Frontier for Cell-Free Synthetic Biology

“…Cell-free workflows are also amenable to automation. Indeed, several studies have successfully utilized acoustic liquid handling robots to rapidly setup large-scale, low-volume (≤10 µl) prototyping cell-free reactions in a 384 well plate format (Moore et al, 2018;Kopniczky et al, 2020). Microfluidic (Swank et al, 2019), droplet array or multiplex (Yim et al, 2019) strategies have also been used to enable high-throughput cell-free experiments.…”

Intelligent Biotech

“…To help bypass the complexities of eukaryotic and particularly mammalian cells, their recent paper demonstrates mammalian cell-free protein synthesis (CFPS) assays based on HeLa cell extracts and liquid handling automation as an alternative to conventional cell-based methods such as tissue culture and flow cytometry. 5 As a proof of concept study, the authors used this cell-free system to optimise regulatory gene networks for use in mammalian cell engineering applications.…”