CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains

Citation: Roggenkamp E, Giersch RM, Wedeman E, Eaton M, Turnquist E, Schrock MN, Alkotami L, Jirakittisonthon T, Schluter-Pascua SE, Bayne GH, Wasko C, Halloran M and Finnigan GC (2017) CRISPR-UnLOCK: Multipurpose Cas9-Based Strategies for Conversion of Yeast Libraries and Strains. Front. Microbiol. 8:1773. doi: 10.3389/fmicb.2017.01773Saccharomyces cerevisiae continues to serve as a powerful model system for both basic biological research and industrial application. The development of genome-wide collections of individually manipulated strains (libraries) has allowed for high-throughput genetic screens and an emerging global view of this single-celled Eukaryote. The success of strain construction has relied on the innate ability of budding yeast to accept foreign DNA and perform homologous recombination, allowing for efficient plasmid construction (in vivo) and integration of desired sequences into the genome. The development of molecular toolkits and “integration cassettes” have provided fungal systems with a collection of strategies for tagging, deleting, or over-expressing target genes; typically, these consist of a C-terminal tag (epitope or fluorescent protein), a universal terminator sequence, and a selectable marker cassette to allow for convenient screening. However, there are logistical and technical obstacles to using these traditional genetic modules for complex strain construction (manipulation of many genomic targets in a single cell) or for the generation of entire genome-wide libraries. The recent introduction of the CRISPR/Cas gene editing technology has provided a powerful methodology for multiplexed editing in many biological systems including yeast. We have developed four distinct uses of the CRISPR biotechnology to generate yeast strains that utilizes the conversion of existing, commonly-used yeast libraries or strains. We present Cas9-based, marker-less methodologies for (i) N-terminal tagging, (ii) C-terminally tagging yeast genes with 18 unique fusions, (iii) conversion of fluorescently-tagged strains into newly engineered (or codon optimized) variants, and finally, (iv) use of a Cas9 “gene drive” system to rapidly achieve a homozygous state for a hypomorphic query allele in a diploid strain. These CRISPR-based methods demonstrate use of targeting universal sequences previously introduced into a genome

Wirtschaftsbeziehungen zwischen Ost und West: Perspektiven und Probleme

Vorwort 3 I. Einleitung 5 II. Ost-West-Handel 8 a. Entwicklung 8 b. Aussichten für die nähere Zukunft 12 c. Der Handel mit China 14 d. Der sowjetische Ferne Osten 15 e. Komparative Vorteile im Ost-West-Süd-Handel . . 16 f. Gewinne aus dem Handel 17 III. Technologietransfer 20 IV. Die Finanzierung des Handels: Das Problem der Verschuldung . 26 V. Institutionelle Probleme 33 VI. Zusammenfassung und Schlußfolgerungen 37 Verzeichnis der Tabellen ^9 Literaturverzeichnis 40 --

Die Weltwirtschaft im Wandel: Symposium 1975

Seit einigen Jahren verstärkt sich der Eindruck, daß die Weltwirtschaft in einem raschen Wandel begriffen ist. Die Entwicklungsländer fordern eine neue Weltwirtschaftsordnung. Auf die weltweite Hochkonjunktur ist eine scharfe und recht lange anhaltende Rezession gefolgt; aber noch immer dominieren inflationäre Tendenzen. Den Konjunktureinbruch überlagert hat die Vervielfachung des Ölpreises Ende 1973. Sie wurde von vielen Beobachtern als Beginn einer Phase mit zunehmendem Rohstoffmangel und nie gekannten Ungleichgewichten in den Zahlungsbilanzen gedeutet. Von den vielfach geäußerten Befürchtungen haben sich manche als übertrieben erwiesen; denn viele Schwierigkeiten konnte der Markt viel besser als erwartet und ziemlich geräuschlos bewältigen. Gleichwohl erscheint es geraten, die alten und die neuen Probleme der Weltwirtschaft ständig zu beobachten. Vom 23. -27. Juni, traf sich in Kiel eine Gruppe unabhängiger Ökonomen aus Japan, Nordamerika und der Europäischen Gemeinschaft in der Absicht, die Situation der Weltwirtschaft zu analysieren und Vorschläge für wirtschaftspolitische Maßnahmen auf nationaler und internationaler Ebene auszuarbeiten. Das Institut für Weltwirtschaft war Gastgeber. Das Kieler Symposium ist ein Glied in einer Reihe trilateraler Konferenzen über weltwirtschaftliche Fragen, die von der Brookings Institution in Washington, dem Japan Economic Research Center in Tokio und dem Institut für Weltwirtschaft gemeinsam veranstaltet werden. Die Ergebnisse werden jeweils in englischer, japanischer und deutscher Sprache veröffentlicht; frühere Berichte in der deutschen Fassung sind als Kieler Diskussionsbeiträge Nr. 31 "Welthandel und Strukturanpassung in den Industrieländern" und Nr. 36 "Weltrohstoffversorgung: Konflikt oder Kooperation?" erschienen. --

A method for estimation of elasticities in metabolic networks using steady state and dynamic metabolomics data and linlog kinetics

BACKGROUND: Dynamic modeling of metabolic reaction networks under in vivo conditions is a crucial step in order to obtain a better understanding of the (dis)functioning of living cells. So far dynamic metabolic models generally have been based on mechanistic rate equations which often contain so many parameters that their identifiability from experimental data forms a serious problem. Recently, approximative rate equations, based on the linear logarithmic (linlog) format have been proposed as a suitable alternative with fewer parameters. RESULTS: In this paper we present a method for estimation of the kinetic model parameters, which are equal to the elasticities defined in Metabolic Control Analysis, from metabolite data obtained from dynamic as well as steady state perturbations, using the linlog kinetic format. Additionally, we address the question of parameter identifiability from dynamic perturbation data in the presence of noise. The method is illustrated using metabolite data generated with a dynamic model of the glycolytic pathway of Saccharomyces cerevisiae based on mechanistic rate equations. Elasticities are estimated from the generated data, which define the complete linlog kinetic model of the glycolysis. The effect of data noise on the accuracy of the estimated elasticities is presented. Finally, identifiable subset of parameters is determined using information on the standard deviations of the estimated elasticities through Monte Carlo (MC) simulations. CONCLUSION: The parameter estimation within the linlog kinetic framework as presented here allows the determination of the elasticities directly from experimental data from typical dynamic and/or steady state experiments. These elasticities allow the reconstruction of the full kinetic model of Saccharomyces cerevisiae, and the determination of the control coefficients. MC simulations revealed that certain elasticities are potentially unidentifiable from dynamic data only. Addition of steady state perturbation of enzyme activities solved this problem

Overview of the PALM model system 6.0

In this paper, we describe the PALM model system 6.0. PALM (formerly an abbreviation for Parallelized Largeeddy Simulation Model and now an independent name) is a Fortran-based code and has been applied for studying a variety of atmospheric and oceanic boundary layers for about 20 years. The model is optimized for use on massively parallel computer architectures. This is a follow-up paper to the PALM 4.0 model description in Maronga et al. (2015). During the last years, PALM has been significantly improved and now offers a variety of new components. In particular, much effort was made to enhance the model with components needed for applications in urban environments, like fully interactive land surface and radiation schemes, chemistry, and an indoor model. This paper serves as an overview paper of the PALM 6.0 model system and we describe its current model core. The individual components for urban applications, case studies, validation runs, and issues with suitable input data are presented and discussed in a series of companion papers in this special issue

Enhanced Visual Temporal Resolution in Autism Spectrum Disorders

Cognitive functions that rely on accurate sequencing of events, such as action planning and execution, verbal and nonverbal communication, and social interaction rely on well-tuned coding of temporal event-structure. Visual temporal event-structure coding was tested in 17 high-functioning adolescents and adults with autism spectrum disorder (ASD) and mental- and chronological-age matched typically-developing (TD) individuals using a perceptual simultaneity paradigm. Visual simultaneity thresholds were lower in individuals with ASD compared to TD individuals, suggesting that autism may be characterised by increased parsing of temporal event-structure, with a decreased capability for integration over time. Lower perceptual simultaneity thresholds in ASD were also related to increased developmental communication difficulties. These results are linked to detail-focussed and local processing bias