Abstract
Bioreporter systems based on detectable enzyme activity, such as that of beta-galactosidase or luciferase, are key in novel bacterial promoter discovery and study. While these systems permit quantification of gene expression, their use is limited by the toxicity of the expressed reporter enzymes in a given host. Indeed, the most potent promoters may be overlooked if their activity causes a lethal overproduction of the reporter genes when screening for transcriptional activity of potential promoter sequences with the luxCDABE cassette. To overcome this limitation, a variation of the mini-CTX-lux plasmid has been designed which allows reduction of promoter activity via the addition of an adjacent fluoride riboswitch. The riboswitch adds a layer of regulation between the promoter and the reporter gene, allowing cloning of stronger promoters by weakening expression, while giving the potential to induce with fluoride to provide a good signal for weaker promoters, thus circumventing limitations associated with reporter toxicity. We noticed the riboswitch potential portability issues between species, suggesting caution when using riboswitches non-native to the species where it is being used. This study introduces a new molecular biology tool which will allow for the identification of previously unverifiable or uncharacterized potent promoters and also provides a cloning vector for translational fusion with luciferase in a plasmid compatible with many species such as from the genera Burkholderia and Pseudomonas.
Highlights
Reporter genes encoding for proteins which are detectable through sensitive and simple means are key elements to numerous gene expression studies and critical to decipher regulatory elements, including the discovery of new promoters and their characterization in terms of strength and dynamics
We identified the fluoride riboswitch (F RS) as a potential candidate for mitigating the potency of two promoters to be used in a reporter gene system: the constitutive S7 ribosomal protein gene promoter (PS7) from Paraburkholderia xenovorans strain LB400 and the P1 integron promoter originally from R388, a trimethoprim-resistance broad-host-range plasmid (Zolg and Hanggi, 1981; DeShazer and Woods, 1996; Massey et al, 2011)
In this study we designed a plasmid which allows for straightforward swapping of promoters and 5′ UnTranslated Regions (UTRs) translationally fused sequences directly from PCR amplified inserts using Gibson assembly-based homologous cloning
Summary
Reporter genes encoding for proteins which are detectable through sensitive and simple means (colorimetry, fluorescence, luminescence) are key elements to numerous gene expression studies and critical to decipher regulatory elements, including the discovery of new promoters and their characterization in terms of strength and dynamics. As a rule of thumb, when gene regulatory elements are cloned upstream of a reporter gene, a high reporter protein signal indicates a strong promoter, while a low signal is attributed to a weak promoter. Strategies have been developed to allow for weak promoter detection and characterization via reporter gene assays (Guo et al, 2019), Riboswitch-Mediated Expression Dampening to our knowledge, no strategy for the detection and study of circumstantially lethal potent promoters, have been suggested. Classical gene reporter assays may have biases against the most potent promoters. The toxicity caused by overexpression of reporter proteins could inhibit the growth of potential clones causing an important gap in new promoter discovery
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