Journal
SYNTHETIC BIOLOGY
Volume 4, Issue 1, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/synbio/ysz026
Keywords
transcriptional regulatory elements; genetic circuit engineering; biological techniques; gene expression regulation; synthetic biology
Funding
- Alberta Innovates Technology Futures (AITF) Postdoctoral Fellowship
- AITF Strategic Chair in RNA Bioengineering [SC60-T2]
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Synthetic biology and the rational design and construction of biological devices require vast numbers of characterized biological parts, as well as reliable design tools to build increasingly complex, multigene architectures. Design principles for intrinsic terminators have been established; however, additional sequence-structure studies are needed to refine parameters for termination-based genetic devices. We report a rapid single-pot method to generate libraries of thousands of randomized bidirectional intrinsic terminators and a modified quantitative Term-Seq (qTerm-Seq) method to simultaneously identify terminator sequences and measure their termination efficiencies (TEs). Using qTerm-Seq, we characterize hundreds of additional strong terminators (TE > 90%) with some terminators reducing transcription read-through by up to 1000-fold in Escherichia coli. Our terminator library and qTerm-Seq pipeline constitute a flexible platform enabling identification of terminator parts that can achieve transcription termination not only over a desired range but also to investigate their sequence-structure features, including for specific genetic and application contexts beyond the common in vivo systems such as E. coli.
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