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

Cis-regulatory elements (CREs) are one of the important factors to control gene expression. The elucidation of their roles has been attracting great interest. We developed an improved method for analyzing a large variety of mutant CRE sequences in a simple and high-throughput manner. In our method, mutant CREs with unique barcode sequences were obtained by biased-randomization in a single PCR reaction. The original T7 promoter sequence was randomized by biased randomization, and the target number of base substitutions was set to be within a range of 0 to 5. The DNA library and transcribed RNA library were sequenced by NGS to quantify transcriptional activity of each mutant. As a result, we succeeded in producing randomized T7 promoter library with high saturation ratio. In a single NGS analysis, we quantified transcriptional activity of 7847 T7 promoter variants. We confirmed that the bases from -9 to -7 played an important role in the transcriptional activity of T7 promoter. Furthermore, using an in vitro transcription/translation system, we found that transcriptional activities of these T7 variants were in nearly direct proportion with resultant protein amount. We could demonstrate that our method enabled simple and high-throughput analysis of the effects of various CRE mutations on transcriptional regulation.