{"@context":{"@vocab":"https://cir.nii.ac.jp/schema/1.0/","rdfs":"http://www.w3.org/2000/01/rdf-schema#","dc":"http://purl.org/dc/elements/1.1/","dcterms":"http://purl.org/dc/terms/","foaf":"http://xmlns.com/foaf/0.1/","prism":"http://prismstandard.org/namespaces/basic/2.0/","cinii":"http://ci.nii.ac.jp/ns/1.0/","datacite":"https://schema.datacite.org/meta/kernel-4/","ndl":"http://ndl.go.jp/dcndl/terms/","jpcoar":"https://github.com/JPCOAR/schema/blob/master/2.0/"},"@id":"https://cir.nii.ac.jp/crid/1363670318596514432.json","@type":"Article","productIdentifier":[{"identifier":{"@type":"DOI","@value":"10.1186/1471-2199-12-18"}},{"identifier":{"@type":"URI","@value":"https://link.springer.com/content/pdf/10.1186/1471-2199-12-18.pdf"}}],"dc:title":[{"@value":"Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR"}],"description":[{"type":"abstract","notation":[{"@value":"<jats:title>Abstract</jats:title>\n          <jats:sec>\n            <jats:title>Background</jats:title>\n            <jats:p>Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in <jats:italic>E. coli</jats:italic>. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in <jats:italic>E. coli</jats:italic>.</jats:p>\n          </jats:sec>\n          <jats:sec>\n            <jats:title>Results</jats:title>\n            <jats:p>In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various <jats:italic>E. coli</jats:italic> strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, <jats:italic>rrsA</jats:italic> encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in <jats:italic>E. coli</jats:italic> cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes <jats:italic>cysG</jats:italic>, <jats:italic>hcaT</jats:italic>, and <jats:italic>idnT</jats:italic> but not <jats:italic>rrsA</jats:italic> and <jats:italic>ihfB</jats:italic> as highly invariant in two <jats:italic>E. coli</jats:italic> strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression.</jats:p>\n          </jats:sec>\n          <jats:sec>\n            <jats:title>Conclusion</jats:title>\n            <jats:p>This study identified <jats:italic>cysG/hcaT/idnT</jats:italic> to be reliable novel reference genes for transcription analysis in recombinant protein producing <jats:italic>E. coli</jats:italic>.</jats:p>\n          </jats:sec>"}]}],"creator":[{"@id":"https://cir.nii.ac.jp/crid/1383670318596514562","@type":"Researcher","foaf:name":[{"@value":"Kang Zhou"}]},{"@id":"https://cir.nii.ac.jp/crid/1383670318596514432","@type":"Researcher","foaf:name":[{"@value":"Lihan Zhou"}]},{"@id":"https://cir.nii.ac.jp/crid/1383670318596514560","@type":"Researcher","foaf:name":[{"@value":"Qing 'En Lim"}]},{"@id":"https://cir.nii.ac.jp/crid/1383670318596514561","@type":"Researcher","foaf:name":[{"@value":"Ruiyang Zou"}]},{"@id":"https://cir.nii.ac.jp/crid/1383670318596514564","@type":"Researcher","foaf:name":[{"@value":"Gregory Stephanopoulos"}]},{"@id":"https://cir.nii.ac.jp/crid/1383670318596514563","@type":"Researcher","foaf:name":[{"@value":"Heng-Phon Too"}]}],"publication":{"publicationIdentifier":[{"@type":"EISSN","@value":"14712199"}],"prism:publicationName":[{"@value":"BMC Molecular Biology"}],"dc:publisher":[{"@value":"Springer Science and Business Media LLC"}],"prism:publicationDate":"2011-04-23","prism:volume":"12","prism:number":"1","prism:startingPage":"18"},"reviewed":"false","url":[{"@id":"https://link.springer.com/content/pdf/10.1186/1471-2199-12-18.pdf"}],"createdAt":"2011-04-26","modifiedAt":"2021-09-01","relatedProduct":[{"@id":"https://cir.nii.ac.jp/crid/1360005514532325120","@type":"Article","resourceType":"学術雑誌論文(journal article)","relationType":["isReferencedBy"],"jpcoar:relatedTitle":[{"@value":"Light‐inducible flux control of triosephosphate isomerase on glycolysis in 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