Abstract
The robust and precise on and off switching of one or more genes of interest, followed by expression or repression is essential for many biological circuits as well as for industrial applications. However, many regulated systems published to date influence the viability of the host cell, show high basal expression or enable only the overexpression of the target gene without the possibility of fine regulation. Herein, we describe an AND gate designed to overcome these limitations by combining the advantages of three well established systems, namely the scaffold RNA CRISPR/dCas9 platform that is controlled by Gal10 as a natural and by LexA-ER-AD as heterologous transcription factor. We hence developed a predictable and modular, versatile expression control system. The selection of a reporter gene set up combining a gene of interest (GOI) with a fluorophore by the ribosomal skipping T2A sequence allows to adapt the system to any gene of interest without losing reporter function. In order to obtain a better understanding of the underlying principles and the functioning of our system, we backed our experimental findings with the development of a mathematical model and single-cell analysis.
Highlights
Synthetic biology aims at designing modular genetic circuits that combine and interconnect different logic gates
Instead of a single-guide RNA (sgRNA), we used a scaffold RNA (scRNA), which was extended by an additional loop containing the viral sequence MS2 that binds to a tetO promoter region [31] (Precise scRNA construction in Supplementary Figure S2)
This arrangement was shown by Zalatan et al to allow expression and repression of reporter genes in versatile ways in S. cerevisiae as well as in mammalian cells and offers the possibility to work with different components on DNA and RNA level [10]
Summary
Synthetic biology aims at designing modular genetic circuits that combine and interconnect different logic gates. Natural transcription factors influence cell growth and depend on host cell genes for correct performance [2,3,4]. Another approach relies on the use of synthetic transcription factors such as e.g. zinc fingers. Though these transcription factors do not considerably influence cell growth, each one has to be designed individually to target a specific locus [5]. Recent research efforts rely on the design of individually inducible genetic switches that show low basal activity, high levels of transcription activation and preferentially no toxicity [6,7]
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