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

<div><p>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 <i>in vitro</i> 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.</p></div

Strategy for high-throughput evaluation of randomized T7 promoter sequences.

<p>(A) Construction of DNA fragments. DNA fragments possess a randomized T7 promoter and a random 16 bases (barcode sequence) upstream of the <i>ydaG</i> gene fragment. In addition, a universal sequence was added for NGS analysis (shown in Materials and methods). (B) NGS analysis of the DNA library. Through this analysis, the relationships between barcode sequences and T7 promoter variant sequences were clarified. In addition, the read number of each DNA sequence was counted. (C) Construction of a RNA library. RNA fragments were obtained by <i>in vitro</i> transcription of the DNA library. Transcribed RNA included barcode sequences. (D) NGS analysis of the RNA library. Through this analysis, the read count of each barcode was determined by NGS. (E) Integration of NGS data. By dividing RNA-derived barcode counts by the corresponding DNA counts, the transcriptional activity of each T7 promoter variant was calculated.</p

Strategy for construction of DNA and RNA samples.

<p>(A) Strategy for construction of DNA samples. DNA samples were constructed by two PCRs. The first PCR was performed to add universal sequence, a randomized T7 promoter, and a barcode sequence to the template. A second PCR was performed to amplify the DNA samples over 30 cycles. (B) Theoretical distribution of the number of base substitution for each randomization ratio. Blue line shows the retention rate at 90%. Orange line shows the retention rate at 70%. Black line shows the retention rate at 50%.</p

The nucleotide bias at each position of the T7 promoter library.

<p>The nucleotide bias at each position of the T7 promoter library.</p

Coverage rate (%) of each base substitution group.

<p>Coverage rate (%) of each base substitution group.</p

Distribution of the transcriptional activity of each T7 promoter variant sequence.

<p>(A) Distribution of all T7 promoter variants. The vertical axis means the transcriptional activity. Transcriptional activity was defined as the number of RNA reads/DNA reads. All T7 variants (from #1 to #7847) were listed in order of transcriptional activity. Blue circle plots show transcriptional activity of each randomized T7 promoter sequence. Five red triangle plots show transcriptional activity of original T7 promoter sequence with different barcodes. Four Black square plots show T7 promoter sequences used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196905#pone.0196905.g005" target="_blank">Fig 5</a> experiment. There is some overlap between plots. (B) Distribution of T7 variants from #7604 to #7847.</p

Distribution of the number of base substitution.

<p>(A) Distribution of number of substituted bases in the high group (transcriptional activity over 1% compared with original sequence). (B) Distribution of number of substituted bases in the low group (transcriptional activity under 1% compared with original sequence). Horizontal axis denotes the number of base substitutions. Vertical axis denotes the read counts.</p

Correlation between the transcriptional activity and translational activity of <i>LacZ</i> gene with T7 promoter variant sequences.

<p>The plot illustrates the correlation between the transcriptional activity versus translational activity of LacZ production for four T7 promoter variants. Their relative transcriptional activities are 0.5, 0.2, 0.1 and 0.01. We used original T7 promoter sequence as a standard. In addition, we carried out an experiment with no DNA fragment as a negative control, and the transcriptional activity was set as zero. The translational activity was measured as the hydrolysis rate of 5-chloromethylfluorescein di-β-d-galactopyranoside (CMFDG). Each plot was averaged across three independent experiments. The error bars indicate standard errors of the means. RFU means relative fluorescence units.</p