title: Development of a plasmid-mediated reporter system for in vivo monitoring of gene expression in the archaeon Methanosarcina acetivorans.
abstract: A plasmid-based gene reporter system has been developed to construct lacZ gene fusions for monitoring intrinsic promoter expression in Methanosarcina acetivorans. Constructs transform with high efficiency that can be readily screened by color selection on plates and exhibit a consistent copy number on different substrates negating the need for gene copy normalization. Expression of the CO dehydrogenase-acetyl coenzyme A synthase promoter fusion to lacZ revealed 18- to 54-fold down-regulation in cells grown on methylotrophic substrates compared with acetate-grown cells, which is up to an order of magnitude greater than the range of regulation previously reported by enzyme activity assays. This system complements and expands the current techniques for studying genetics of the methanosarcinal Archaea by providing a rapid method for monitoring and quantifying gene expression.
authors.affiliation: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, MD 21202, USA.
authors.forename: Ethel E
authors.initials: EE
authors.lastname: Apolinario
authors.forename: Karin M
authors.initials: KM
authors.lastname: Jackson
authors.forename: Kevin R
authors.initials: KR
authors.lastname: Sowers
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D003470:Culture Media; D019848:Gene Expression Regulation, Archaeal; D017930:Genes, Reporter; D007763:Lac Operon; D017020:Methanosarcina / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D019843:Archaeal Proteins; D003470:Culture Media; D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D000445:Aldehyde Oxidoreductases; C019823:carbon monoxide dehydrogenase