Screening and selection of artificial riboswitches

• Published in: Methods (San Diego, Calif.) Calif.), 2018-07, Vol.143, p.77-89
• Main Authors: Harbaugh, Svetlana V., Martin, Jennifer A., Weinstein, Jenna, Ingram, Grant, Kelley-Loughnane, Nancy
• Format: Article
• Language: English
• Subjects: Aptamer; Aptamers, Nucleotide - chemical synthesis; Aptamers, Nucleotide - genetics; DNA, Complementary - genetics; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Gene Expression Regulation, Bacterial; Gene Library; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; High-Throughput Screening Assays - instrumentation; High-Throughput Screening Assays - methods; In vivo selection; Ligands; Luminescent Proteins - chemistry; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Promoter Regions, Genetic - genetics; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Red Fluorescent Protein; Riboswitch; Riboswitch - genetics
• ISSN: 1046-2023; 1095-9130
• DOI: 10.1016/j.ymeth.2018.05.012

In vivo screening for functional riboswitches. Colonies showing green fluorescence are transferred in liquid media in 96-well plates and grown overnight. Obtained cell cultures are used for screening. Screening is performed in cell cultures without and in the presence of the analyte (TNT). New cell cultures are grown overnight, aliquots are transferred in black plates, fluorescence intensities of GFPa1 and mKate2 are measured and riboswitch activation ratios are calculated. [Display omitted] Synthetic riboswitches are engineered to regulate gene expression in response to a variety of non-endogenous small molecules, and a challenge to select this engineered response requires robust screening tools. A new synthetic riboswitch can be created by linking an in vitro-selected aptamer library with a randomized expression platform followed by in vivo selection and screening. In order to determine response to analyte, we developed a dual-color reporter comprising elements of the E. coli fimbriae phase variation system: recombinase FimE controlled by a synthetic riboswitch and an invertible DNA segment (fimS) containing a constitutively active promoter placed between two fluorescent protein genes. Without an analyte, the fluorescent reporter constitutively expressed green fluorescent protein (GFPa1). Addition of the analyte initiated translation of fimE causing unidirectional inversion of the fimS segment and constitutive expression of red fluorescent protein (mKate2). The dual color reporter system can be used to select and to optimize artificial riboswitches in E. coli cells. In this work, the enriched library of aptamers incorporated into the riboswitch architecture reduces the sequence search space by offering a higher percentage of potential ligand binders. The study was designed to produce structure switching aptamers, a necessary feature for riboswitch function and efficiently quantify this function using the dual color reporter system.