High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding

• Published in: PloS one Vol. 13; no. 5; p. e0196905
• Main Authors: Komura, Ryo, Aoki, Wataru, Motone, Keisuke, Satomura, Atsushi, Ueda, Mitsuyoshi
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 07.05.2018; Public Library of Science (PLoS)
• Subjects: Bacteriophage T7 - genetics; Biochemistry; Biology and life sciences; Cell cycle; Computer and information sciences; Deoxyribonucleic acid; DNA; DNA barcoding; DNA Barcoding, Taxonomic - methods; DNA-Directed RNA Polymerases - genetics; E coli; Escherichia coli; Gene expression; Gene Expression Regulation - genetics; Gene mutation; Gene regulation; Gene sequencing; Genetic research; High-Throughput Nucleotide Sequencing - methods; Life sciences; Methods; Mutagenesis; Mutation; Nucleotide sequence; Promoter Regions, Genetic; Promoters (Genetics); Proteins; Randomization; Regulatory sequences; Regulatory Sequences, Nucleic Acid - genetics; Research and analysis methods; Ribonucleic acid; RNA; RNA polymerase; Transcription; Transcription (Genetics); Transcription, Genetic; Japan
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0196905

Cis-regulatory elements (CREs) are one of the important factors in controlling gene expression and elucidation of their roles has been attracting great interest. We have developed an improved method for analyzing a large variety of mutant CRE sequences in a simple and high-throughput manner. In our approach, mutant CREs with unique barcode sequences were obtained by biased randomization in a single PCR amplification. The original T7 promoter sequence was randomized by biased randomization, and the target number of base substitutions was set to be within the range of 0 to 5. The DNA library and subsequent transcribed RNA library were sequenced by next generation sequencers (NGS) to quantify transcriptional activity of each mutant. We succeeded in producing a randomized T7 promoter library with high coverage rate at each target number of base substitutions. In a single NGS analysis, we quantified the transcriptional activity of 7847 T7 promoter variants. We confirmed that the bases from -9 to -7 play an important role in the transcriptional activity of the T7 promoter. This information coincides with the previous researches and demonstrated the validity of our methodology. Furthermore, using an in vitro transcription/translation system, we found that transcriptional activities of these T7 variants were well correlated with the resultant protein abundance. We demonstrate that our method enables simple and high-throughput analysis of the effects of various CRE mutations on transcriptional regulation.