Abstract
Cell-free gene expression systems with linear DNA expression templates (LDETs) have been widely applied in artificial cells, biochips, and high-throughput screening. However, due to the degradation caused by native nucleases in cell extracts, the transcription with linear DNA templates is weak, thereby resulting in low protein expression level, which greatly limits the development of cell-free systems using linear DNA templates. In this study, the protective sequences for stabilizing linear DNA and the transcribed mRNAs were rationally designed according to nucleases’ action mechanism, whose effectiveness was evaluated through computer simulation and cell-free gene expression. The cell-free experiment results indicated that, with the combined protection of designed sequence and GamS protein, the protein expression of LDET-based cell-free systems could reach the same level as plasmid-based cell-free systems. This study would potentially promote the development of the LDET-based cell-free gene expression system for broader applications.
Highlights
Cell-free synthetic biology (Garamella et al, 2016; Caschera, 2017; Damiati et al, 2018; Smolskaya et al, 2020) has rapidly developed as a powerful and flexible technology to overcome the inherent limitations of synthetic biology with living cells
The first principle of the protective sequence design was that the GC content affected the stability of linear DNA (Woodrow et al, 2006), so protective sequences were designed with different GC contents to explore the best GC content
An in silico linear DNA design strategy was proposed to improve the stability of linear DNA in cell-free gene expression systems
Summary
Cell-free synthetic biology (Garamella et al, 2016; Caschera, 2017; Damiati et al, 2018; Smolskaya et al, 2020) has rapidly developed as a powerful and flexible technology to overcome the inherent limitations of synthetic biology with living cells. By eliminating the constraint of sustaining life, cell-free systems provide unprecedented control over the molecular context for gene expression and metabolism (Silverman et al, 2020). Over the past 20 years, practical improvements (Dopp et al, 2019) in cell-free gene expression systems have seen its widespread adoption in basic research and industrial applications (Carlson et al, 2012; Silverman et al, 2020), such as genetic prototyping (Moore et al, 2017), artificial cells (Noireaux and Libchaber, 2004; Lai et al, 2020), high-throughput screening (Contreras-Llano and Tan, 2018), and biosensing (Verosloff et al, 2019). Compared with traditional cell-based approaches, one of the advantages of cell-free approaches is that linear DNA could be used as expression templates. There is a growing interest in studying cell-free gene expression systems with linear DNA expression templates (LDETs). LDET-based cell-free systems have been widely used in many aspects, such as biochip
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