Abstract
Tuned gene expression is crucial to the proper growth and response to the environmental changes of an organism. To enable tunable gene expression as designed is desirable in both scientific research and industrial application. Here, we introduce a novel promoter switching method based on the DDI2 promoter (PDDI2) that can fine tune the expression of target genes. We constructed a recyclable cassette (PDDI2-URA3-PDDI2) and integrated it upstream of yeast target genes to replace the native promoters by DDI2 promoter without introducing any junk sequence. We found that the presence or absence of cyanamide as an inducer could turn on or off the expression of target genes. In addition, we showed that PDDI2 could act as a gene switch to linearly regulate the expression levels of target genes in vivo. We switched the original promoters of RAD18, TUP1, and CDC6 with PDDI2 as a proof-of-concept.
Highlights
For each cell, the transcriptional programs are modified to maintain specific intracellular conditions to ensure optimal growth and function[1,2]
We demonstrate that PDDI2 is a strong inducible promoter
Our study suggests the potential of PDDI2 for linear control of gene expression and as a tight on-off switch
Summary
The transcriptional programs are modified to maintain specific intracellular conditions to ensure optimal growth and function[1,2]. Yeast cells prefer glucose and fructose as carbon sources, when PGAL1 is used to induce target gene expression, the original carbon source must be replaced by galactose. The tetracycline regulatory system (Tet-on and Tet-off), which is originally from bacteria, has been widely used to regulate gene expression in eukaryotes[19] In eukaryotic cells, this system has been applied to control the RNA polymerase III-driven transcription of eukaryotic tRNA genes[20,21]. We propose to utilize the DDI2 promoter to develop a novel gene expression regulating system induced by cyanamide. We used the superfolder green fluorescent protein gene (sfGFP) as a reporter gene to determine the strength and regulation of PDDI2 compared with three other widely-used promoters (PADH1, PCUP1 and PGAL1)[32]. We demonstrated a linear gene expression control on the target gene correlated with the inducing strength, which further facilitates the precise regulation of gene expression in budding yeast
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