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Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni

Overview of attention for article published in BMC Microbiology, October 2015
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Title
Chromosomal integration vectors allowing flexible expression of foreign genes in Campylobacter jejuni
Published in
BMC Microbiology, October 2015
DOI 10.1186/s12866-015-0559-5
Pubmed ID
Authors

Adrian J. Jervis, Jonathan A. Butler, Brendan W. Wren, Dennis Linton

Abstract

Campylobacter jejuni is a major cause of human gastroenteritis yet there is limited knowledge of how disease is caused. Molecular genetic approaches are vital for research into the virulence mechanisms of this important pathogen. Vectors that allow expression of genes in C. jejuni via recombination onto the chromosome are particularly useful for genetic complementation of insertional knockout mutants and more generally for expression of genes in particular C. jejuni host backgrounds. A series of three vectors that allow integration of genes onto the C. jejuni chromosome were constructed by standard cloning techniques with expression driven from three different strong promoters. Following integration onto the C. jejuni chromosome expression levels were quantified by fluorescence measurements and cells visualized by fluorescence microscopy. We have created plasmid, pCJC1, designed for recombination-mediated delivery of genes onto the C. jejuni chromosome. This plasmid contains a chloramphenicol resistance cassette (cat) with upstream and downstream restriction sites, flanked by regions of the C. jejuni pseudogene Cj0223. Cloning of genes immediately upstream or downstream of the cat gene allows their subsequent introduction onto the C. jejuni chromosome within the pseudogene. Gene expression can be driven from the native gene promoter if included, or alternatively from the cat promoter if the gene is cloned downstream of, and in the same transcriptional orientation as cat. To provide increased and variable expression of genes from the C. jejuni chromosome we modified pCJC1 through incorporation of three relatively strong promoters from the porA, ureI and flaA genes of C. jejuni, Helicobacter pylori and Helicobacter pullorum respectively. These promoters along with their associated ribosome binding sites were cloned upstream of the cat gene on pCJC1 to create plasmids pCJC2, pCJC3 and pCJC4. To test their effectiveness, a green fluorescent protein (gfp) reporter gene was inserted downstream of each of the three promoters and following integration of promoter-gene fusions onto the C. jejuni host chromosome, expression levels were quantified. Expression from the porA promoter produced the highest fluorescence, from flaA intermediate levels and from ureI the lowest. Expression of gfp from the porA promoter enabled visualization by fluorescent microscopy of intracellular C. jejuni cells following invasion of HeLa cells. The plasmids constructed allow stable chromosomal expression of genes in C. jejuni and, depending on the promoter used, different expression levels were obtained making these plasmids useful tools for genetic complementation and high level expression.

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Mendeley readers

The data shown below were compiled from readership statistics for 36 Mendeley readers of this research output. Click here to see the associated Mendeley record.

Geographical breakdown

Country Count As %
United Kingdom 1 3%
Unknown 35 97%

Demographic breakdown

Readers by professional status Count As %
Student > Ph. D. Student 12 33%
Researcher 7 19%
Student > Bachelor 4 11%
Student > Master 4 11%
Lecturer 2 6%
Other 5 14%
Unknown 2 6%
Readers by discipline Count As %
Agricultural and Biological Sciences 15 42%
Biochemistry, Genetics and Molecular Biology 8 22%
Immunology and Microbiology 5 14%
Environmental Science 1 3%
Unspecified 1 3%
Other 1 3%
Unknown 5 14%

Attention Score in Context

This research output has an Altmetric Attention Score of 1. This is our high-level measure of the quality and quantity of online attention that it has received. This Attention Score, as well as the ranking and number of research outputs shown below, was calculated when the research output was last mentioned on 27 October 2015.
All research outputs
#5,614,876
of 7,421,206 outputs
Outputs from BMC Microbiology
#866
of 1,258 outputs
Outputs of similar age
#165,718
of 240,504 outputs
Outputs of similar age from BMC Microbiology
#52
of 85 outputs
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So far Altmetric has tracked 1,258 research outputs from this source. They receive a mean Attention Score of 2.9. This one is in the 19th percentile – i.e., 19% of its peers scored the same or lower than it.
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