Dataset Information

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

ABSTRACT: E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage ? and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of genetic circuits. We characterised integration and expression at four candidate loci, fliD, fliS, fliT, and fliY, of the E. coli flagellar region 3a. The integration efficiency and expression from the four integrations varied considerably. Integration into fliD and fliS significantly decreased motility, while integration into fliT and fliY had only a minor effect on the motility. None of the integrations had negative effects on the growth of the bacteria. Overall, we found that fliT was the most suitable integration site.

SUBMITTER: Juhas M

PROVIDER: S-EPMC4211737 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

Juhas Mario M Evans Lewis D B LD Frost Joe J Davenport Peter W PW Yarkoni Orr O Fraser Gillian M GM Ajioka James W JW

PloS one 20141028 10

E. coli is a model platform for engineering microbes, so genetic circuit design and analysis will be greatly facilitated by simple and effective approaches to introduce genetic constructs into the E. coli chromosome at well-characterised loci. We combined the Red recombinase system of bacteriophage λ and Isothermal Gibson Assembly for rapid integration of novel DNA constructs into the E. coli chromosome. We identified the flagellar region as a promising region for integration and expression of g ...[more]

PMID: 25350000

Similar Datasets

Project description:Background:Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results:By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF ? factor/anti-? switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions:The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

Project description:Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown with aeration to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with a significance level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins. Keywords: Steady State

Project description:Gene expression profiles of Escherichia coli K-12 W3110 were compared as a function of steady-state external pH. Cultures were grown to an optical density at 600 nm of 0.3 in potassium-modified Luria-Bertani medium buffered at pH 5.0, 7.0, and 8.7. For each of the three pH conditions, cDNA from RNA of five independent cultures was hybridized to Affymetrix E. coli arrays. Analysis of variance with an alpha level of 0.001 resulted in 98% power to detect genes showing a twofold difference in expression. Normalized expression indices were calculated for each gene and intergenic region (IG). Differential expression among the three pH classes was observed for 763 genes and 353 IGs. Hierarchical clustering yielded six well-defined clusters of pH profiles, designated Acid High (highest expression at pH 5.0), Acid Low (lowest expression at pH 5.0), Base High (highest at pH 8.7), Base Low (lowest at pH 8.7), Neutral High (highest at pH 7.0, lower in acid or base), and Neutral Low (lowest at pH 7.0, higher at both pH extremes). Flagellar and chemotaxis genes were repressed at pH 8.7 (Base Low cluster), where the cell's transmembrane proton potential is diminished by the maintenance of an inverted pH gradient. High pH also repressed the proton pumps cytochrome o (cyo) and NADH dehydrogenases I and II. By contrast, the proton-importing ATP synthase F1Fo and the microaerophilic cytochrome d (cyd), which minimizes proton export, were induced at pH 8.7. These observations are consistent with a model in which high pH represses synthesis of flagella, which expend proton motive force, while stepping up electron transport and ATPase components that keep protons inside the cell. Acid-induced genes, on the other hand, were coinduced by conditions associated with increased metabolic rate, such as oxidative stress. All six pH-dependent clusters included envelope and periplasmic proteins, which directly experience external pH. Overall, this study showed that (i) low pH accelerates acid consumption and proton export, while coinducing oxidative stress and heat shock regulons; (ii) high pH accelerates proton import, while repressing the energy-expensive flagellar and chemotaxis regulons; and (iii) pH differentially regulates a large number of periplasmic and envelope proteins.

Dataset Information

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

Publications

Escherichia coli flagellar genes as target sites for integration and expression of genetic circuits.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets