Dataset Information

Design and synthesis of synthetic UP elements for modulation of gene expression in Escherichia coli.

ABSTRACT: Metabolic engineering requires fine-tuned gene expression for most pathway optimization applications. To develop a suitable suite of promoters, traditional bacterial promoter engineering efforts have focused on modifications to the core region, especially the -10 and -35 regions, of native promoters. Here, we demonstrate an alternate, unexplored route of promoter engineering through randomization of the UP element of the promoter-a region that contacts the alpha subunit carboxy-terminal domain instead of the sigma subunit of the RNA polymerase holoenzyme. Through this work, we identify five novel UP element sequences through library-based searches in Escherichia coli. The resulting elements were used to activate the E. coli core promoter, rrnD promoter, to levels on par and higher than the prevalent strong bacterial promoter, OXB15. These relative levels of expression activation were transferrable when applied upstream of alternate core promoter sequences, including rrnA and rrnH. This work thus presents and validates a novel strategy for bacterial promoter engineering with transferability across varying core promoters and potential for transferability across bacterial species.

SUBMITTER: Presnell KV

PROVIDER: S-EPMC6501063 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Design and synthesis of synthetic UP elements for modulation of gene expression in <i>Escherichia coli</i>.

Presnell Kristin V KV Flexer-Harrison Madeleine M Alper Hal S HS

Synthetic and systems biotechnology 20190502 2

Metabolic engineering requires fine-tuned gene expression for most pathway optimization applications. To develop a suitable suite of promoters, traditional bacterial promoter engineering efforts have focused on modifications to the core region, especially the -10 and -35 regions, of native promoters. Here, we demonstrate an alternate, unexplored route of promoter engineering through randomization of the UP element of the promoter-a region that contacts the alpha subunit carboxy-terminal domain i ...[more]

PMID: 31080900

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Project description:Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription. Since their discovery, archaea have not only proven a fascinating domain in their own right, but also helped us understand the evolution and function of molecular components they share with bacteria or eukaryotes. Archaeal histones are homologous to their eukaryotic counterparts, but operate in a less constrained bacterial-like cellular environment and their role in transcription and genome function remains obscure. In order to understand how archaeal histones affect transcriptional processes, we induced expression of the two histones from the archaeon Methanothermus fervidus in a naive bacterial system (E. coli) that has not evolved to integrate this kind of proteins. We show, using a series of MNase digestion experiments, that these histones bind the bacterial genome and wrap DNA in vivo in a pattern consistent with a previously proposed multimerisation model, in a similar pattern observed natively. We correlate genome-wide occupancy maps and gene expression profiles in different phases of growth to show that – although expression of archaeal histones triggers morphological changes in E. coli – there appears to only be an indirect effect on transcription.

Dataset Information

Design and synthesis of synthetic UP elements for modulation of gene expression in Escherichia coli.

Publications

Design and synthesis of synthetic UP elements for modulation of gene expression in <i>Escherichia coli</i>.

Similar Datasets

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