title: A phosphate-regulated promoter for fine-tuned and reversible overexpression in Ostreococcus: application to circadian clock functional analysis.
abstract: BACKGROUND
  The green picoalga Ostreococcus tauri (Prasinophyceae), which has been described as the smallest free-living eukaryotic organism, has minimal cellular ultra-structure and a very small genome. In recent years, O. tauri has emerged as a novel model organism for systems biology approaches that combine functional genomics and mathematical modeling, with a strong emphasis on light regulated processes and circadian clock. These approaches were made possible through the implementation of a minimal molecular toolbox for gene functional analysis including overexpression and knockdown strategies. We have previously shown that the promoter of the High Affinity Phosphate Transporter (HAPT) gene drives the expression of a luciferase reporter at high and constitutive levels under constant light.
  
  
  RESULTS
  Here we report, using a luciferase reporter construct, that the HAPT promoter can be finely and reversibly tuned by modulating the level and nature of phosphate in culture medium. This HAPT regulation was additionally used to analyze the circadian clock gene Time of Cab expression 1 (TOC1). The phenotype of a TOC1ox/CCA1:Luc line was reverted from arrhythmic to rhythmic simply by adding phosphate to the culture medium. Furthermore, since the time of phosphate injection had no effect on the phase of CCA1:Luc expression, this study suggests further that TOC1 is a central clock gene in Ostreococcus.
  We have developed a phosphate-regulated expression system that allows fine gene function analysis in Ostreococcus. Recently, there has been a growing interest in microalgae as cell factories. This non-toxic phosphate-regulated system may prove useful in tuning protein expression levels quantitatively and temporally for biotechnological applications.
authors.affiliation: Universite Pierre et Marie Curie, Paris 06, Observatoire Océanologique, Banyuls/mer, France.
authors.forename: El Batoul
authors.initials: el B
authors.lastname: Djouani-Tahri
authors.forename: Frédéric
authors.initials: F
authors.lastname: Sanchez
authors.forename: Jean-Claude
authors.initials: JC
authors.lastname: Lozano
authors.forename: François-Yves
authors.initials: FY
authors.lastname: Bouget
mesh_terms: D001483:Base Sequence; D000460:Chlorophyta / Q000166:cytology / Q000187:drug effects* / Q000235:genetics* / Q000254:growth & development; D057906:Circadian Clocks / Q000187:drug effects / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects*; D007700:Kinetics; D008969:Molecular Sequence Data; D028061:Phosphate Transport Proteins / Q000235:genetics / Q000378:metabolism; D010710:Phosphates / Q000494:pharmacology*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D013997:Time Factors
chemical_list: D028061:Phosphate Transport Proteins; D010710:Phosphates