<?xml version='1.0' encoding='UTF-16' ?> <!DOCTYPE composition PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> <html xmlns="http://www.w3.org/1999/xhtml" xmlns:ui="http://java.sun.com/jsf/facelets" xmlns:h="http://java.sun.com/jsf/html" xmlns:p="http://primefaces.prime.com.tr/ui" xmlns:f="http://java.sun.com/jsf/core"> <ui:composition template="./resources/Templates/home_Template.xhtml"> <ui:define name="page"> <div class="post"> <h1 class="title">Symbiosis -Workpackage 6 <br /> Understanding Regulation of Metabolites </h1> <div class="entry"> <p> <h:form> <!-- <img align="left" src="../resources/images/SORF/puzzle-home.png"/>--> <div id="menuItems"> <h3> <h3> <p>It was agreed to start with growth experiments of Pseudomonas strains (focussing P. putida and at a later stage to include P. fluorescens and P. fragi) and to study gene expression at various growth stages in broth at different temperatures by microarray analysis.</p> <p>Partner 1 was responsible for the growth experiments of P. putida KT2440 which was acquired from ATCC collection. The growth kinetics of KT2440 were determined in Luria- Bertani broth at 10 C and 30 C. The concentration of glucose was monitored during growth.</p> <p>Partner 4 was responsible for ordering the P. putida KT2440 microarrays and to set up protocols for the RNA isolation, microarray hybridizations and data analysis.</p> <p>A training of one month was scheduled in September 2009 for partner 1 at the laboratory of Partner 4. The training comprised the performance of RNA isolations, microarray hybridizations and data analysis. Both partners were involved in the determination of the list of key genes (D6.1 and D6.2). Partner 1 focussed on key genes in relation to metabolic pathways for malodorous end-product production in P. putida, whereas Partner 4 concentrated on key genes in relation to stress and virulence in Salmonella and Listeria.</p> <p>Initially gene expression will be examined of different samples taken during the growth of P. fluorescens and/or P. putida in broth under aerobic conditions at different temperatures starting with 10 C. AUA has started to determined the growth curves for P. fluorescens and P. putida under these conditions and will provide RIKILT with either cells, RNA or cDNA depending on what is most convenient. Samples will be taken from late log, early stationary, mid stationary and late stationary fase. During growth additional parameters such as pH, glucose concentration, production of end-products (proteolysis may be included also) will also be monitored to provide a broader 'picture' of the environmental expression.</p> <p>RIKILT has checked for commercially available microarrays for P. fluorescens and P. putida. Two companies have been found who sell oligonucleotide microarrays for the Pseudomonas species of interest (P. fluorescens PfO-1 and P. putida KT2440). Based on the description of the two different platforms and the quotations it was decided to initially focus on one species and order P. putida KT2440 oligonucleotide microarrays from Progenika Biopharma, S.A. (Spain).</p> <p>Literature is currently being checked for key genes involved in spoilage metabolite production among Pseudomonads and will be continued for the next period.</p> <p>RIKILT did an extensive comparative search of the available genome sequences of the Pseudomonads of interest; i.e. P. fluorescens PfO-1, P. fluorescens Pf-5, P. putida F1, P. putida GB-1, P. putida KT2440, and P. putida W619. Genes have been categorized according to primary function based on the Cluster of Orthologous Groups (COG). The categorization will be completed in the next period. Additional, alignments were made for various genes after blast searches and retrieval of the DNA sequences from the public databases. The outcome of this categorization, blast and literature searches revealed that the genus Pseudomonas is very heterogeneous. Moreover, a rather large genomic diversity within the species P. fluorescens was observed, like also among the 4 P. putida strain analyzed. Additionaly, some genes/orthologs are even completely absent in either fluorescens or putida. Although, other genes are only missing in one of the available fluorescens and putida genome sequences. This inherent heterogeneity of the P. fluorescens and P. putida grouping will have to be taken in to account during the hybridizations of the microarray platforms derived from strain KT2440 or PfO-1 with non-sequenced Pseudomonas str</p> </h3> </h3> </div> </h:form> </p> </div> </div> </ui:define> </ui:composition> </html>