ArticlePloS one2018
High-throughput evaluation of T7 promoter variants using biased randomization and DNA barcoding.
Article in PloS one, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 21 papers.
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Who cites it
21 citing papers in PubMed, 56 citations in OpenAlex.
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- Synthetic Biology of Plants and Microbes for Agriculture, Environment, and Future Applications.Chemical reviews · 2026Review
- Reconstituting alternative life using the test-bed of cell-free systems.Philosophical transactions of the Royal Society of London. Series B, Biological sciences · 2025Review
- Simultaneous in vitro expression of minimal 21 transfer RNAs by tRNA array method.Nature communications · 2025Article
- Self-splicing RNA circularization facilitated by intact group I and II introns.Nature communications · 2025Article
- Article
- Regulatory Components for Bacterial Cell-Free Systems Engineering.ACS synthetic biology · 2024Review
- Characterizing and Tailoring the Substrate Profile of a γ-Glutamyltransferase Variant.ACS synthetic biology · 2024Article
- Logical regulation of endogenous gene expression using programmable, multi-input processing CRISPR guide RNAs.Nucleic acids research · 2024Article
- Complex Dependence ofACS synthetic biology · 2023Article
- T7Max transcription system.Journal of biological engineering · 2023Article
- Slight Variations in the Sequence Downstream of the Polyadenylation Signal Significantly Increase Transgene Expression in HEK293T and CHO Cells.International journal of molecular sciences · 2022Article
- Development of an expression-tunable multiple protein synthesis system in cell-free reactions using T7-promoter-variant series.Synthetic biology (Oxford, England) · 2022Article
- Scalable and automated CRISPR-based strain engineering using droplet microfluidics.Microsystems & nanoengineering · 2022Article
- Effective Use of Linear DNA in Cell-Free Expression Systems.Frontiers in bioengineering and biotechnology · 2021Review
- Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology.Microorganisms · 2019Review
- Optimized PCR conditions minimizing the formation of chimeric DNA molecules from MPRA plasmid libraries.BMC genomics · 2019Article
- Article
Corrections and comments
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Authors and funding
5 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.