Experimental and computational validation of models of fluorescent and luminescent reporter genes in bacteria

<p>Abstract</p> <p>Background</p> <p>Fluorescent and luminescent reporter genes have become popular tools for the real-time monitoring of gene expression in living cells. However, mathematical models are necessary for extracting biologically meaningful quantities from the primary data.</p> <p>Results</p> <p>We present a rigorous method for deriving relative protein synthesis rates (mRNA concentrations) and protein concentrations by means of kinetic models of gene expression. We experimentally and computationally validate this approach in the case of the protein Fis, a global regulator of transcription in <it>Escherichia coli</it>. We show that the mRNA and protein concentration profiles predicted from the models agree quite well with direct measurements obtained by Northern and Western blots, respectively. Moreover, we present computational procedures for taking into account systematic biases like the folding time of the fluorescent reporter protein and differences in the half-lives of reporter and host gene products. The results show that large differences in protein half-lives, more than mRNA half-lives, may be critical for the interpretation of reporter gene data in the analysis of the dynamics of regulatory systems.</p> <p>Conclusions</p> <p>The paper contributes to the development of sound methods for the interpretation of reporter gene data, notably in the context of the reconstruction and validation of models of regulatory networks. The results have wide applicability for the analysis of gene expression in bacteria and may be extended to higher organisms.</p

Mécanisme du contrôle de la décision lyse-lysogénie du bactériophage transposable Mu

Doctorat en Sciencesinfo:eu-repo/semantics/nonPublishe

Characterization of a temperate phage hosted by Alcaligenes eutrophus strain A5

Nineteen strains of Alcaligenes eutrophus were tested for the presence of prophages. One strain that lysed upon mitomycin C treatment produced a phage which could not form plaques on any of the strains available. DNA extracted from partially purified phage lysates was digested with various restriction enzymes which showed that the 42 kb long viral double-stranded DNA circularizes by means of cohesive ends. To our knowledge, this is the first description of a phage for the genus Alcaligenes.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Control of bacteriophage Mu lysogenic repression

The transposable and temperate phage Mu infects Escherichia coli where it can enter the lytic life-cycle or reside as a repressed and integrated prophage. The repressor protein Rep is the key element in the lysis-lysogeny decision. We have analyzed the fate of Rep in different mutants by Western blotting under two conditions that can induce a lysogen: high temperature and stationary phase. We show that, unexpectedly, Rep accumulates under all conditions where the prophage is completely derepressed, and that this accumulation is ClpX-dependent. An analysis of the degradation kinetics shows that Rep is a target of two protease systems: inactivation of either the clpP or lon gene results in a stabilization of Rep. Such a reaction scheme explains the counterintuitive observation that derepression is correlated with high repressor concentration. We conclude that under all conditions of phage induction the repressor is sequestered in a non-active form. A quantitative simulation accounts for our experimental data. It provides a model that captures the essential features of Mu induction and explains some of the mechanisms by which the physiological signals affecting the lysis-lysogeny decision converge onto Rep. © 2005 Elsevier Ltd. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Cobalt stress in Escherichia coli: the effect on the iron-sulfur proteins

International audienc

E. coli chromosomal-driven expression of NADK2 from A. thaliana: A preferable alternative to plasmid-driven expression for challenging proteins

International audienceL'expression et la purification de grandes protéines recombinantes ou complexes protéiques est problématique pour certains laboratoires de biotechnologie. En effet, il est souvent difficile d'obtenir suffisamment de protéines actives pour effectuer une caractérisation biologique ou atteindre la commercialisation, lorsque de grandes protéines ou complexes protéiques sont exprimés via le système d'expression populaire basé sur le plasmide T7 . E. coliIl existe également une demande industrielle pour réduire notre dépendance à l'expression basée sur les plasmides, en raison de ses inconvénients, tels que : i) l'utilisation courante d'antibiotiques pour maintenir le plasmide, ii) le problème du nombre de copies de plasmide et iii)le risque de surcharger le système d'expression. Malgré tous ces problèmes, les solutions alternatives, telles que l'intégration de gènes dans le chromosome bactérien , sont rarement employées et leurs avantages font encore débat.Arabidopsis thalianaIci, nous montrons que l'expression axée sur les chromosomes a permis la récupération de la protéine NADK2 plus active que les systèmes d'expression T7 classiques, ainsi qu'une meilleure production, confirmant ainsi que l'expression à partir d'une seule copie chromosomique est préférable à l'expression axée sur les plasmides et pourrait être attrayante pour les deux bases. et la recherche appliquée

A genome-wide screen for identifying all regulators of a target gene

International audienceWe have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs, encoding the acetyl coenzyme A synthetase, in Escherichia coli. We confirm most of the known genetic regulators, including CRP-cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA, sucB and lpdA, encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC. The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library