Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine tuning

Research output: Contribution to journalJournal article – Annual report year: 2008Researchpeer-review

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Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine tuning. / Braatsch, Stephan; Helmark, Søren; Kranz, Harald; Købmann, Brian Jensen; Jensen, Peter Ruhdal.

In: BioTechniques, Vol. 45, No. 3, 2008, p. 335-337.

Research output: Contribution to journalJournal article – Annual report year: 2008Researchpeer-review

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@article{0743126ee8be4793b71562d431e65559,
title = "Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine tuning",
abstract = "System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional, fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression. Red/ET recombination perfectly complements SPL technology, since it enables easy modification of the Escherichia Coli genome and can be accomplished with linear DNA (i.e., the SPL). To demonstrate the synergistic use of Red/ET and SPL for metabolic engineering applications, we replaced the native promoter of a genomic localized phosphoglucose isomerase (pgi)-lacZ reporter construct by all SPL. Using these technologies together we were able to rapidly identify synthetic promoter sequences that resulted in activity range of 25{\%} to 570{\%} of the native pgi-promoter.",
keywords = "EXPRESSION",
author = "Stephan Braatsch and S{\o}ren Helmark and Harald Kranz and K{\o}bmann, {Brian Jensen} and Jensen, {Peter Ruhdal}",
year = "2008",
doi = "10.2144/000112907",
language = "English",
volume = "45",
pages = "335--337",
journal = "BioTechniques",
issn = "0736-6205",
publisher = "Informa Healthcare",
number = "3",

}

RIS

TY - JOUR

T1 - Escherichia coli strains with promoter libraries constructed by Red/ET recombination pave the way for transcriptional fine tuning

AU - Braatsch, Stephan

AU - Helmark, Søren

AU - Kranz, Harald

AU - Købmann, Brian Jensen

AU - Jensen, Peter Ruhdal

PY - 2008

Y1 - 2008

N2 - System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional, fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression. Red/ET recombination perfectly complements SPL technology, since it enables easy modification of the Escherichia Coli genome and can be accomplished with linear DNA (i.e., the SPL). To demonstrate the synergistic use of Red/ET and SPL for metabolic engineering applications, we replaced the native promoter of a genomic localized phosphoglucose isomerase (pgi)-lacZ reporter construct by all SPL. Using these technologies together we were able to rapidly identify synthetic promoter sequences that resulted in activity range of 25% to 570% of the native pgi-promoter.

AB - System-oriented applications of genetic engineering, such as metabolic engineering, often require the serial optimization of enzymatic reaction steps, which can be achieved by transcriptional, fine-tuning. However, approaches to changing gene expression are usually limited to deletion and/or strong overexpression and rarely match the desired optimal transcript levels. A solution to this all-or-nothing approach has been the use of a synthetic promoter library (SPL) that is based on randomized sequences flanking the consensus -10 and -35 promoter regions and allows for fine-tuning of bacterial gene expression. Red/ET recombination perfectly complements SPL technology, since it enables easy modification of the Escherichia Coli genome and can be accomplished with linear DNA (i.e., the SPL). To demonstrate the synergistic use of Red/ET and SPL for metabolic engineering applications, we replaced the native promoter of a genomic localized phosphoglucose isomerase (pgi)-lacZ reporter construct by all SPL. Using these technologies together we were able to rapidly identify synthetic promoter sequences that resulted in activity range of 25% to 570% of the native pgi-promoter.

KW - EXPRESSION

U2 - 10.2144/000112907

DO - 10.2144/000112907

M3 - Journal article

VL - 45

SP - 335

EP - 337

JO - BioTechniques

JF - BioTechniques

SN - 0736-6205

IS - 3

ER -