シンポジウム

Additional file 14. A table with description of strains and plasmids generated and used in this study and graphical representation of over-expressed operons/genes

Rule-Based Design of Plant Expression Vectors Using GenoCAD

<div><p>Plant synthetic biology requires software tools to assist on the design of complex multi-genic expression plasmids. Here a vector design strategy to express genes in plants is formalized and implemented as a grammar in GenoCAD, a Computer-Aided Design software for synthetic biology. It includes a library of plant biological parts organized in structural categories and a set of rules describing how to assemble these parts into large constructs. Rules developed here are organized and divided into three main subsections according to the aim of the final construct: protein localization studies, promoter analysis and protein-protein interaction experiments. The GenoCAD plant grammar guides the user through the design while allowing users to customize vectors according to their needs. Therefore the plant grammar implemented in GenoCAD will help plant biologists take advantage of methods from synthetic biology to design expression vectors supporting their research projects.</p></div

Example of three different designs obtained following <i>ppi</i> route.

<p>A) <i>bifc</i> route. B) <i>coip</i> route using MYC and HA as epitope tags. C) <i>coip</i> route using GFP and HIS as epitope tags.</p

Example of three different designs for localization studies purposes as developed with the plant grammar.

<p>A. Scheme of the most basic structure we can design, where the expression cassette includes the GEN fused to a FTG by means of a LNK domain on the N terminal. B. Sample design includes an expression cassette with 2 PRO and a GEN fused to a FTG on the N terminal and to an ETG on the C terminal. C. Same as B but with the expression cassette in reverse orientation.</p

Example of two different designs for promoter analysis studies.

<p>A. The expression cassette includes a FTG under the control of a NPRO and fused with an ETG by means of a LNK. B. The expression cassette has reverse orientation and double TER.</p

Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

<div><p>In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and <i>hmg</i> maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.</p> </div

La Petite presse : journal quotidien... / [rédacteur en chef : Balathier Bragelonne]

25 novembre 18841884/11/25 (A18,N6776)

Adaptation of the MapMan ontology to biotic stress responses: application in solanaceous species-1

<p><b>Copyright information:</b></p><p>Taken from "Adaptation of the MapMan ontology to biotic stress responses: application in solanaceous species"</p><p>http://www.plantmethods.com/content/3/1/10</p><p>Plant Methods 2007;3():10-10.</p><p>Published online 4 Sep 2007</p><p>PMCID:PMC2018691.</p><p></p>he pathogen which is recognized by the related receptors (putative R genes), transcription of the cascade of the plant defence mechanism is triggered, including oxidative stress changes. Inside the cell, signals are transmitted to lead to the production of defence molecules (PR-proteins, heat shock proteins and secondary metabolites). Genes with experimental indication of involvement in the biotic stress are gathered on the main panel (coloured with dark grey), while genes and pathways that are putatively involved in biotic stress pathway are shown on the left and right sides (coloured in light grey). ) Potato samples 30 minutes after inoculation with potato virus . ) Tobacco samples 24 hours after inoculation with . In both cases, the signal after infection is expressed as a ratio relative to the signal in unifected controls, converted to a scale, and displayed. The scale is shown in the figures

Summary table of qPCR, ddPCR and cdPCR performance for MON810 detection and quantification.

<p>qPCR: data produced in this study, or obtained from the literature, when indicated <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Burns1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-EuropeanUnionReferenceLaboratoryforGM1" target="_blank">[22]</a></p><p>ddPCR: data produced in this study.</p><p>cdPCR: data produced on a BioMark System (Fluidigm, South San Francisco) using the 12.765 digital arrays (Fluidigm) and obtained from the literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Corbisier1" target="_blank">[11]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Burns1" target="_blank">[18]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062583#pone.0062583-Bhat1" target="_blank">[19]</a>.</p><p>Repeatability through the dynamic range: assessed through the coefficient of variation (Cv) of the target copy numbers or the MON810 content between repeats.</p><p>Trueness: assessed through the calculation of the bias between the MON810 content measured and the target MON810 content. * For our study, trueness is indicated only when qPCR and ddPCR results could be compared to a third, independent value (obtained from the CRM provider or proficiency test organizer).</p><p>Time for results/96 well plate: Total time needed from DNA pipetting to the analysis of the results; reaction mixes are already prepared.</p><p>Price/sample if 96-well plate: Price based on material and reagent costs available at NIB, including labor cost.</p><p>N.A.: not evaluated.</p

Precision of the duplex ddPCR assay as a function of the target concentration.

<p>MON810 content measured by ddPCR in five series of seven target concentrations. The target MON810 content (3.85%) is indicated by a dotted line. Acceptance criterion for precision is ±25% of the target content (from 2.89% to 4.81%) represented by the dashed lines. Error bars represent the standard deviation of the measured MON810% by ddPCR at each target concentration (five replicates per target concentration).</p