One-step generation of error-prone PCR libraries using Gateway® technology

<p>Abstract</p> <p>Background</p> <p>Error-prone PCR (epPCR) libraries are one of the tools used in directed evolution. The Gateway^®technology allows constructing epPCR libraries virtually devoid of any background (<it>i.e</it>., of insert-free plasmid), but requires two steps: the BP and the LR reactions and the associated <it>E. coli </it>cell transformations and plasmid purifications.</p> <p>Results</p> <p>We describe a method for making epPCR libraries in Gateway^®plasmids using an LR reaction without intermediate BP reaction. We also describe a BP-free and LR-free sub-cloning method for in-frame transferring the coding sequence of selected clones from the plasmid used to screen the library to another one devoid of tag used for screening (such as the green fluorescent protein). We report preliminary results of a directed evolution program using this method.</p> <p>Conclusions</p> <p>The one-step method enables producing epPCR libraries of as high complexity and quality as does the regular, two-step, protocol for half the amount of work. In addition, it contributes to preserve the original complexity of the epPCR product.</p

Split-GFP Reassembly Assay: Strengths and Caveats from a Multiparametric Analysis

The split-Green Fluorescent Protein (GFP) reassembly assay is a powerful approach to study protein-protein interactions (PPIs). In this assay, two proteins, respectively, fused to the first seven and the last four β-strands of GFP are co-expressed in E. coli where they can bind to each other, which reconstitutes the full-length GFP. Thus, the fluorescence of the bacteria co-expressing the two fusion proteins accounts for the interaction of the two proteins of interest. The first split-GFP reassembly assay was devised in the early 2000s in Regan's lab. During the last ten years, we have been extensively using this assay to study the interactions of an intrinsically disordered protein (IDP) with two globular partners. Over that period, in addition to accumulating molecular information on the specific interactions under study, we progressively modified the original technique and tested various parameters. In those previous studies, however, we focused on the mechanistic insights provided by the approach, rather than on the method itself. Since methodological aspects deserve attention and the best bipartite reporter to study PPIs involving IDPs remains to be identified, we herein focus on technical aspects. To this end, we first revisit our previous modifications of the original method and then investigate the impact of a panel of additional parameters. The present study unveiled a few critical parameters that deserve consideration to avoid pitfalls and obtain reliable results

Recombinant protein production facility for fungal biomass-degrading enzymes using the yeast Pichia pastoris

International audienceFilamentous fungi are the predominant source of lignocellulolytic enzymes used in industry for the transformation of plant biomass into high-value molecules and biofuels. The rapidity with which new fungal genomic and post-genomic data are being produced is vastly outpacing functional studies. This underscores the critical need for developing platforms dedicated to the recombinant expression of enzymes lacking confident functional annotation, a prerequisite to their functional and structural study. In the last decade, the yeast Pichia pastoris has become increasingly popular as a host for the production of fungal biomass-degrading enzymes, and particularly carbohydrate-active enzymes (CAZymes). This study aimed at setting-up a platform to easily and quickly screen the extracellular expression of biomass-degrading enzymes in P. pastoris. We first used three fungal glycoside hydrolases (GHs) that we previously expressed using the protocol devised by Invitrogen to try different modifications of the original protocol. Considering the gain in time and convenience provided by the new protocol, we used it as basis to setup the facility and produce a suite of fungal CAZymes (GHs, carbohydrate esterases and auxiliary activity enzyme families) out of which more than 70% were successfully expressed. The platform tasks range from gene cloning to automated protein purifications and activity tests, and is open to the CAZyme users' community

GREENET - An Early Stage Training Network in Enabling Technologies for Green Radio

International audienceIn this paper, we describe GREENET (an early stage training network in enabling technologies for green radio), which is a new project recently funded by the European Commission under the auspices of the 2010 Marie Curie People Programme. Through the recruitment and personalized training of 17 Early Stage Researchers (ESRs), in GREENET we are committed to the development of new disruptive technologies to address all aspects of energy efficiency in wireless networks, from the user devices to the core network infrastructure, along with the ways the devices and equipment interact with one another. Novel techniques at the physical, link, and network layers to reduce the energy consumption and carbon footprint of 4G devices will be investigated, such as Spatial Modulation (SM) for Multiple-Input-Multiple-Output (MIMO) systems, Cooperative Automatic Repeat reQuest (C-ARQ) protocols, and Network Coding (NC) for lossy networks. Furthermore, cooperation and cognition paradigms will be exploited as additional assets to improve the energy efficiency of wireless networks with the challenging but indispensable constraint of optimizing the system capacity without degrading the user's Quality-of-Service (QoS)

Quantime - A miniature cesium atomic clock using CPT technique for telecom application

The Quantime project aims at developing a miniature atomic clock suited for the telecom market, requiring a wide operating temperature range (from -40 to +85°C), and a low production cost. The CPT (Coherent Population Trapping) technique for atomic interrogation is used for miniaturization and low power consumption. In the first phase of the project, the clock architecture was chosen, and the main sub-systems were developed. A clock breadboarding demonstrator was assembled and the measured Allan deviation of 1E-11 at 400 s confirms the operation of all the sub-systems

La dynamique de comblement du vallon et son paléoenvironnement

Picq Christophe, Laporte Luc, Cammas Cecilia, Marambat Laurence, Gruet Yves, Genre Christian. La dynamique de comblement du vallon et son paléoenvironnement. In: Gallia préhistoire, tome 44, 2002. pp. 8-25

Dissecting Partner Recognition by an Intrinsically Disordered Protein Using Descriptive Random Mutagenesis

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Les occupations du Ve millénaire avant J.C. et la question du Néolithique ancien sur la façade atlantique de la France

Laporte Luc, Picq Christophe, Oberlin Christine, Fabre Laurent, Marambat Laurence, Gruet Yves, Marchand Grégor. Les occupations du Ve millénaire avant J.C. et la question du Néolithique ancien sur la façade atlantique de la France. In: Gallia préhistoire, tome 44, 2002. pp. 27-86

The influence of wearing compression stockings on performance indicators and physiological responses following a prolonged trail running exercise

International audienceThe objective of this study was to investigate the effects of wearing compression socks (CS) on performance indicators and physiological responses during prolonged trail running. Eleven trained runners completed a 15.6 km trail run at a competition intensity whilst wearing or not wearing CS. Counter movement jump, maximal voluntary contraction and the oxygenation profile of vastus lateralis muscle using near-infrared spectroscopy (NIRS) method were measured before and following exercise. Run time, heart rate (HR), blood lactate concentration and ratings of perceived exertion were evaluated during the CS and non-CS sessions. No significant difference in any dependent variables was observed during the run sessions. Run times were 5681.1±503.5 and 5696.7±530.7 s for the non-CS and CS conditions, respectively. The relative intensity during CS and non-CS runs corresponded to a range of 90.5–91.5% HRmax. Although NIRS measurements such as muscle oxygen uptake and muscle blood flow significantly increased following exercise (+57.7% and + 42.6%,+59.2% and + 32.4%, respectively for the CS and non-CS sessions, P<0.05), there was no difference between the run conditions. The findings suggest that competitive runners do not gain any practical or physiological benefits from wearing CS during prolonged off-road running