Encapsulation enhances protoplast fusant stability

A barrier to cost-efficient biomanufacturing is the instability of engineered genetic elements, such as plasmids. Instability can also manifest at the whole-genome level, when fungal dikaryons revert to parental species due to nuclear segregation during cell division. Here, we show that by encapsulating Saccharomyces cerevisiae-Pichia stipitis dikaryons in an alginate matrix, we can limit cell division and preserve their expanded metabolic capabilities. As a proxy to cellulosic ethanol production, we tested the capacity of such cells to carry out ethanologenic fermentation of glucose and xylose, examining substrate use, ploidy, and cell viability in relation to planktonic fusants, as well as in relation to planktonic and encapsulated cell cultures consisting of mixtures of these species. Glucose and xylose consumption and ethanol production by encapsulated dikaryons were greater than planktonic controls. Simultaneous co-fermentation did not occur; rather the order and kinetics of glucose and xylose catabolism by encapsulated dikaryons were similar to cultures where the two species were encapsulated together. Over repeated cycles of fed-batch culture, encapsulated S. cerevisiae-P. stipitis fusants exhibited a dramatic increase in genomic stability, relative to planktonic fusants. Encapsulation also increased the stability of antibiotic-resistance plasmids used to mark each species and preserved a fixed ratio of S. cerevisiae to P. stipitis cells in mixed cultures. Our data demonstrate how encapsulating cells in an extracellular matrix restricts cell division and, thereby, preserves the stability and biological activity of entities ranging from genomes to plasmids to mixed populations, each of which can be essential to cost-efficient biomanufacturing

Experimental Investigation of a 7 by 7 Nozzle Jet Array for Dynamic Impingement Cooling

Dynamic impingement cooling is a promising way for more efficient exploitation of cooling air in highly heat charged environments. In many applications the deployed impinging jets are subjected to crossflow superimposed on the flow field of the transverse jets. The present study describes the initial experimental investigations regarding dynamic heat transfer between a flat surface and an array of 49 impingement jets, which are dynamically controlled by changing frequency, duty cycle and phasing. A new test rig was designed and manufactured in order to investigate the interactions of impingement jets and their impact on heat transfer. The test rig satisfies the needs of different measurement techniques. Surface measurements using pressure sensors, thermocouples, hot wires, hot films and liquid crystal thermography are planned for investigating the interactions near the wall. Furthermore, the test rig is suitable for efficient flow field measurements between jet orifices and impingement plate using particle image velocimetry. Parallel to the test rig development, time resolved PIV measurements have been performed in the test section of a recirculating free-surface water tunnel in order to investigate the influence of cross flow superimposed to periodically generated vortex rings impinging on a flat plate. The central goal of these preliminary testing is to understand under which formation conditions periodically generated vortex rings in cross flow have the ability to maximize the transport of vorticity close to the wall. First results are presented and discussed.DFG, 200291049, SFB 1029: TurbIn - Signifikante Wirkungsgradsteigerung durch gezielte, interagierende Verbrennungs- und Strömungsinstationaritäten in Gasturbine

Diverse conditions support near-zero growth in yeast: Implications for the study of cell lifespan

Baker’s yeast has a finite lifespan and ages in two ways: a mother cell can only divide so many times (its replicative lifespan), and a non-dividing cell can only live so long (its chronological lifespan). Wild and laboratory yeast strains exhibit natural variation for each type of lifespan, and the genetic basis for this variation has been generalized to other eukaryotes, including met-azoans. To date, yeast chronological lifespan has chiefly been studied in relation to the rate and mode of functional decline among non-dividing cells in nutrient-depleted batch culture. However, this culture method does not accurately capture two major classes of long-lived metazoan cells: cells that are terminally differentiated and metabolically active for periods that approximate animal lifespan (e.g. cardiac myocytes), and cells that are pluripotent and metabolically quiescent (e.g. stem cells). Here, we consider alternative ways of cultivating Saccharomyces cerevisiae so that these different metabolic states can be explored in non-dividing cells: (i) yeast cultured as giant colonies on semi-solid agar, (ii) yeast cultured in retentostats and provided sufficient nutrients to meet minimal energy requirements, and (iii) yeast encapsulated in a semisolid matrix and fed ad libitum in bioreactors. We review the physiology of yeast cultured under each of these conditions, and explore their potential to provide unique insights into determinants of chronological lifespan in the cells of higher eukaryotes

Werkstattbericht zum Lehr-Lern-Projekt "Monarchie und Adel - Forschungsorientiertes Projektlernen in Kleingruppen"

Im Wintersemester 2013/2014 führten der Dresdener Lehrstuhl für Neuere deutsche Literatur und Kulturgeschichte und der Chemnitzer Lehrstuhl für Europäische Geschichte des 19. und 20. Jahrhunderts das gemeinsame interdisziplinäre Lehrprojekt Monarchie und Adel durch. Themen waren die Entwicklung monarchischer Repräsentationskultur vom späten 18. bis zum frühen 20. Jahrhundert zum einen, die künstlerische und insbesondere die literarische Auseinandersetzung mit monarchischen Herrschaftstypen, welche die europäische Aufklärung und ihre Gegenbewegungen begleitete, zum anderen. Kernziel der Veranstaltung war die Vorbereitung der Studierenden auf ein eigenständiges, forschungsorientiertes Arbeiten nach dem Studium. Dabei wurde besonderer Wert darauf gelegt, Methoden im Dialog mit der Forschung erfahrbar zu machen; die systematische und vollständige Erschließung des Forschungsstandes zu einer je spezifischen Fragestellung war unabdingbar; der Weg zu den Quellen wurde damit geöffnet. Dieses zweifellos aufwendigere, am Anspruch genuiner Forschungsarbeit orientierte Vorgehen sollte gegenüber dem Rückgriff auf Handbuchwissen etabliert werden, der gerade in den zeitlich knapp strukturierten modularisierten Studiengängen allzu oft eine gründliche Recherchearbeit ersetzt. Die studentischen Beiträge, die als Studienleistung den Abschluss des Lernprojektes bildeten, wurden an der Fähigkeit zu selbständigem Arbeiten innerhalb des Forschungsfeldes gemessen. Das konnte durch die Leistung umfassender Überblicksdarstellungen, aber – in einigen Fällen – auch durch die eigenständige Arbeit an Desideraten geschehen

\u3ci\u3eEx Uno Plures\u3c/i\u3e: Clonal Reinforcement Drives Evolution of a Simple Microbial Community

A major goal of genetics is to define the relationship between phenotype and genotype, while a major goal of ecology is to identify the rules that govern community assembly. Achieving these goals by analyzing natural systems can be difficult, as selective pressures create dynamic fitness landscapes that vary in both space and time. Laboratory experimental evolution offers the benefit of controlling variables that shape fitness landscapes, helping to achieve both goals. We previously showed that a clonal population of E. coli experimentally evolved under continuous glucose limitation gives rise to a genetically diverse community consisting of one clone, CV103, that best scavenges but incompletely utilizes the limiting resource, and others, CV101 and CV116, that consume its overflow metabolites. Because this community can be disassembled and reassembled, and involves cooperative interactions that are stable over time, its genetic diversity is sustained by clonal reinforcement rather than by clonal interference. To understand the genetic factors that produce this outcome, and to illuminate the community’s underlying physiology, we sequenced the genomes of ancestral and evolved clones. We identified ancestral mutations in intermediary metabolism that may have predisposed the evolution of metabolic interdependence. Phylogenetic reconstruction indicates that the lineages that gave rise to this community diverged early, as CV103 shares only one Single Nucleotide Polymorphism with the other evolved clones. Underlying CV103’s phenotype we identified a set of mutations that likely enhance glucose scavenging and maintain redox balance, but may do so at the expense of carbon excreted in overflow metabolites. Because these overflow metabolites serve as growth substrates that are differentially accessible to the other community members, and because the scavenging lineage shares only one SNP with these other clones, we conclude that this lineage likely served as an ‘‘engine’’ generating diversity by creating new metabolic niches, but not the occupants themselves

Starvation-Associated Genome Restructuring Can Lead to Reproductive Isolation in Yeast

Knowledge of the mechanisms that lead to reproductive isolation is essential for understanding population structure and speciation. While several models have been advanced to explain post-mating reproductive isolation, experimental data supporting most are indirect. Laboratory investigations of this phenomenon are typically carried out under benign conditions, which result in low rates of genetic change unlikely to initiate reproductive isolation. Previously, we described an experimental system using the yeast Saccharomyces cerevisiae where starvation served as a proxy to any stress that decreases reproduction and/or survivorship. We showed that novel lineages with restructured genomes quickly emerged in starved populations, and that these survivors were more fit than their ancestors when re-starved. Here we show that certain yeast lineages that survive starvation have become reproductively isolated from their ancestor. We further demonstrate that reproductive isolation arises from genomic rearrangements, whose frequency in starving yeast is several orders of magnitude greater than an unstarved control. By contrast, the frequency of point mutations is less than 2-fold greater. In a particular case, we observe that a starved lineage becomes reproductively isolated as a direct result of the stress-related accumulation of a single chromosome. We recapitulate this result by demonstrating that introducing an extra copy of one or several chromosomes into naïve, i.e. unstarved, yeast significantly diminishes their fertility. This type of reproductive barrier, whether arising spontaneously or via genetic manipulation, can be removed by making a lineage euploid for the altered chromosomes. Our model provides direct genetic evidence that reproductive isolation can arise frequently in stressed populations via genome restructuring without the precondition of geographic isolation