62 research outputs found
Phenotypic heterogeneity during heterologous protein production in Bacillus megaterium
Bei Bacillus megaterium handelt es sich um einen attraktiven Wirt für die heterologe Protein- produktion, dessen Expressionssystem mit Hilfe eines Xylose-induzierbaren Promotors (PxylA) immer mehr zu einer Produktionsmaschinerie für rekombinante Proteine entwickelt wurde. Das System basiert auf einem multicopy Plasmid, dessen Bestandteile aus dem genomischen xylABT-Operon stammen. In Abwesenheit von Xylose wird die Expression des Operons durch den Xylose-Repressor xylR verhindert. In Gegenwart von Xylose bindet diese an den Repressor, was die Repression aufhebt und die Expression des Operons bewirkt. Zur Validierung wurde das mittels Fluoreszenzmessung leicht nachweisbare eGFP, an den PxylA-Promotor fusioniert.
Obwohl Vektorsysteme entwickelt wurden, die bis zu 1,25 g/l eGFP produzierten, zeigte ein Teil der Kultur auf Einzelzellebene eine Heterogenität bezüglich der Proteinproduktion. Daten aus qRT-PCR-Analysen von sortierten Zellen deckten die relativen Expressionsverhältnisse der beteiligten Gene auf.
Daraufhin wurden drei mögliche Faktoren untersucht. Durch die Induktion des XylR-Repressors könnte zum einen freie Xylose wegtitriert werden, was die Gleichgewichte in der Zelle verschieben würde (1). Zum anderen könnten ungleichmäßige Plasmidverteilungen als Folge der Zellteilung (2) oder Effekte beim Altern von Zellen (3) eine maßgebliche Rollen spielen.
Um dies zu überprüfen, wurde XylR mit dem Fluoreszenzprotein mCherry fusioniert und Zeitraffer-Filme einer wachsenden Mikrokolonie aufgenommen. Dabei wurde sowohl die Produktion von eGFP, als auch von mCherry als Reaktion auf Xylose aufgezeichnet. Des Weiteren wurde ein Plasmid konstruiert, dem das xylR-Gen fehlt. Um konkrete molekulare Informationen über den Einfluss der Plasmidkopienzahl zu gewinnen, wurde das Plasmid durch zusätzliche XylR-Bindestellen, die mit mCherry fusioniert wurden, visualisiert. Analysen mittels Fluoreszenz in situ Hybridisierung (FISH) wurden zur direkten zellulären Lokalisation des Plasmids eingesetzt.
Die gewonnen Daten legten nahe, dass selbst bei selektiven Bedingungen die beobachtete bimodale Verteilung kein Resultat von Gen-Regelkreisen war, sondern ein Phänomen der ungleichen Plasmidsegregation zwischen Tochterzellen. Die Beobachtungen wurden in ein mathematisches Modell integriert, um das bistabile Produktionsverhalten über Computersimulationen zu reproduzieren.Within the last years Bacillus megaterium was systemically developed for the gram per liter production of recombinant proteins using the strong xylose-inducible promoter system PxylA. The expression system is based on a multicopy plasmid containing the functional elements of the system – the gene encoding the repressor XylR and the promoter PxylA. For a deeper understanding, PxylA was fused to the coding sequence of the green fluorescent protein (GFP). In absence of xylose its expression is repressed by the xylose repressor XylR while in the presence of xylose the expression is derepressed. Although high yields of GFP were produced the culture showed a significant level of heterogeneity at the single cell level during the recombinant protein production process with up to 30 % of low-producing cells leading to three speculative working models or combinations of them – (1) an imbalanced repressor inducer equilibrium, (2) an unequal distribution of plasmids during cell division or (3) cell aging effects. In order to validate these models, new plasmids carrying different modifications were generated. For an estimation of the single cell XylR abundance and intracellular distribution the plasmid encoded xylR gene was translationally fused to mCherry. Further, the intracellular ratio of repressor molecules and DNA binding sites was modified by deleting this xylR gene and introducing additional XylR binding sites. Finally, alternative origins of replication were introduced to change and affect the plasmid copy number. All new plasmid strains were analyzed with respect to their overall GFP production behavior grown under different conditions. Single cell analyses were performed using time-lapse microscopy and flow cytometry to get an insight in the production dynamics of distinct cell lineages and subpopulation distributions. In addition, fluorescence in situ hybridization (FISH) experiments were performed to localize the plasmids in the cells. Derived results led to the hypothesis, that heterogeneous production behavior is probably a consequence of unequal plasmid distribution. Finally, the observations were incorporated into an extended mathematical model in order to reproduce the bistable production behavior via computer simulations
Recommended from our members
Signatures of slip in dewetting polymer films
Thin liquid polymer films on hydrophobic substrates are susceptable to
rupture and formation of holes, which in turn initiate a complex dewetting
process that eventually evolves into characteristic stationary droplet
patterns. Experimental and theoretical studies suggest that the specific type
of droplet pattern largely depends on the nature of the polymer-substrate
boundary condition. To follow the morphological evolution numerically over
long time scales and for the multiple length scales involved has so far been
a major challenge. In this study a highly adaptive finite-element based
numerical scheme is presented that allows for large-scale simulations to
follow the evolution of the dewetting process deep into the nonlinear regime
of the model equations, capturing the complex dynamics including shedding of
droplets. In addition, the numerical results predict the previouly unknown
shedding of satellite droplets during the destabilisation of liquid ridges,
that form during the late stages of the dewetting process. While the
formation of satellite droplets is well-known in the context of elongating
fluid filaments and jets, we show here that for dewetting liquid ridges this
property can be dramatically altered by the interfacial condition between
polymer and substrate, namely slip
Signatures of slip in dewetting polymer films
Thin liquid polymer films on hydrophobic substrates are susceptable to rupture and formation of holes, which in turn initiate a complex dewetting process that eventually evolves into characteristic stationary droplet patterns. Experimental and theoretical studies suggest that the specific type of droplet pattern largely depends on the nature of the polymer-substrate boundary condition. To follow the morphological evolution numerically over long time scales and for the multiple length scales involved has so far been a major challenge. In this study a highly adaptive finite-element based numerical scheme is presented that allows for large-scale simulations to follow the evolution of the dewetting process deep into the nonlinear regime of the model equations, capturing the complex dynamics including shedding of droplets. In addition, the numerical results predict the previouly unknown shedding of satellite droplets during the destabilisation of liquid ridges, that form during the late stages of the dewetting process. While the formation of satellite droplets is well-known in the context of elongating fluid filaments and jets, we show here that for dewetting liquid ridges this property can be dramatically altered by the interfacial condition between polymer and substrate, namely slip
Recommended from our members
Influence of slip on the Rayleigh-Plateau rim instability in dewetting viscous films
A dewetting viscous film develops a characteristic fluid rim at its
receding edge due to mass conservation. In the course of the dewetting
process the rim becomes unstable via an instability of Rayleigh-Plateau type.
An important difference exists between this classic instability of a liquid
column and the rim instability in the thin film as the growth of the rim is
continuously fueled by the receding film. We explain how the development and
macroscopic morphology of the rim instability are controlled by the slip of
the film on the substrate. A single thin-film model captures quantitatively
the characteristics of the evolution of the rim observed in our experiments
Influence of slip on the Rayleigh--Plateau rim instability in dewetting viscous films
A dewetting viscous film develops a characteristic fluid rim at its receding edge due to mass conservation. In the course of the dewetting process the rim becomes unstable via an instability of Rayleigh-Plateau type. An important difference exists between this classic instability of a liquid column and the rim instability in the thin film as the growth of the rim is continuously fueled by the receding film. We explain how the development and macroscopic morphology of the rim instability are controlled by the slip of the film on the substrate. A single thin-film model captures quantitatively the characteristics of the evolution of the rim observed in our experiments
Hypersurface Bohm-Dirac models
We define a class of Lorentz invariant Bohmian quantum models for N entangled
but noninteracting Dirac particles. Lorentz invariance is achieved for these
models through the incorporation of an additional dynamical space-time
structure provided by a foliation of space-time. These models can be regarded
as the extension of Bohm's model for N Dirac particles, corresponding to the
foliation into the equal-time hyperplanes for a distinguished Lorentz frame, to
more general foliations. As with Bohm's model, there exists for these models an
equivariant measure on the leaves of the foliation. This makes possible a
simple statistical analysis of position correlations analogous to the
equilibrium analysis for (the nonrelativistic) Bohmian mechanics.Comment: 17 pages, 3 figures, RevTex. Completely revised versio
Klimatische Stabilität von Mittelgebirgsmooren
Inwieweit bisherige und prognostizierte Klimaänderungen Mittelgebirgsmoore beeinflussen, wurde am Beispiel der Mothhäuser Haide im Mittleren Erzgebirge untersucht. Mit den angewandten moorkundlichen Methoden und dem Vergleich von drei Landnutzungsszenarien (IST, pnV, Paläovegetation) mit drei Klimaszenarien (Messdaten 1981 - 2000, WEREX IV 2041-60, Paläoklima) lassen sich die Ökotopveränderungen der Moorfläche ableiten.
Im Ergebnis wird ein Rückgang an nässeren Ökotopen prognostiziert, wobei der Charakter eines Moores aber erhalten bleibt. Anthropogene Einflüsse wie Straßenbau, Entwässerung und Torfabbau haben größere Veränderungen bewirkt als durch den Klimawandel zu erwarten sind.
Die Wiederherstellung der hydrologischen Durchgängigkeit des Moores, d.h. die Beseitigung von Gräben und Barrieren, kann die negativen Folgen eines zukünftig wärmeren Klimas abpuffern.
Hinweise des Herausgebers (Stand: 4. April 2011):
Zu Seite 18, Abbildung 4:
Die Temperaturreihe vom Hohenpeißenberg und die Temperaturrekonstruktionen nach Glaser (2001), welche diese Reihe mit berücksichtigt hat, weisen in der verwendeten Form Inhomogenitäten auf. Nach einer Homogenisierung durch den DWD wird deutlich, dass das mittlere Temperaturniveau um 1780 eher dem von 1970 entspricht. Die Folgejahre sind von einem Anstieg um ca.
1 °C gekennzeichnet (Glaser 2008).
Zu Seite 20, Abschnitt 4.3.2:
Die genannten Datenfehler wurden inzwischen behoben. Weil vergleichbare Unplausibilitäten nie ganz ausgeschlossen werden können, sind Qualitätsprüfungen von Beobachtungs- und Projektionsdaten vor jeder Datenanwendung durchzuführen.
Zu Seite 20, Abschnitt 4.3.3:
Das Regionalisierungsverfahren WEREX simuliert im Betrachtungszeitraum 10 Realisierungen. Statistische Kenngrößen sollten aus allen 10 Realisierungen für vorzugsweise 30-jährige Zeiträume abgeleitet werden. Abweichungen der statistischen Kenngrößen in Modelldaten von denen des beobachteten Datenkollektives sind modellimmanent und deshalb zu erwarten. Die dargestellten Ergebnisse ermöglichen keine abschließende Bewertung der statistischen Eigenschaften der Projektionsdaten.
Klimaprojektionen liefern generell nur Annahmen einer möglichen Klimazukunft. Neben der Plausibilitätsprüfung sollte die Bewertung der Aussagen eines Modells möglichst immer in die Bandbreite der Ergebnisse vieler Modelle erfolgen
In vitro interaction network of a synthetic gut bacterial community
A key challenge in microbiome research is to predict the functionality of microbial communities based on community membership and (meta)-genomic data. As central microbiota functions are determined by bacterial community networks, it is important to gain insight into the principles that govern bacteria-bacteria interactions. Here, we focused on the growth and metabolic interactions of the Oligo-Mouse-Microbiota (OMM12) synthetic bacterial community, which is increasingly used as a model system in gut microbiome research. Using a bottom-up approach, we uncovered the directionality of strain-strain interactions in mono- and pairwise co-culture experiments as well as in community batch culture. Metabolic network reconstruction in combination with metabolomics analysis of bacterial culture supernatants provided insights into the metabolic potential and activity of the individual community members. Thereby, we could show that the OMM12 interaction network is shaped by both exploitative and interference competition in vitro in nutrient-rich culture media and demonstrate how community structure can be shifted by changing the nutritional environment. In particular, Enterococcus faecalis KB1 was identified as an important driver of community composition by affecting the abundance of several other consortium members in vitro. As a result, this study gives fundamental insight into key drivers and mechanistic basis of the OMM12 interaction network in vitro, which serves as a knowledge base for future mechanistic in vivo studies
LUBAC assembles a ubiquitin signaling platform at mitochondria for signal amplification and transport of NF-κB to the nucleus
Mitochondria are increasingly recognized as cellular hubs to orchestrate signaling pathways that regulate metabolism, redox homeostasis, and cell fate decisions. Recent research revealed a role of mitochondria also in innate immune signaling; however, the mechanisms of how mitochondria affect signal transduction are poorly understood. Here, we show that the NF-κB pathway activated by TNF employs mitochondria as a platform for signal amplification and shuttling of activated NF-κB to the nucleus. TNF treatment induces the recruitment of HOIP, the catalytic component of the linear ubiquitin chain assembly complex (LUBAC), and its substrate NEMO to the outer mitochondrial membrane, where M1- and K63-linked ubiquitin chains are generated. NF-κB is locally activated and transported to the nucleus by mitochondria, leading to an increase in mitochondria-nucleus contact sites in a HOIP-dependent manner. Notably, TNF-induced stabilization of the mitochondrial kinase PINK1 furthermore contributes to signal amplification by antagonizing the M1-ubiquitin-specific deubiquitinase OTULIN. Overall, our study reveals a role for mitochondria in amplifying TNF-mediated NF-κB activation, both serving as a signaling platform, as well as a transport mode for activated NF-κB to the nuclear
Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis
Ustilago maydis is a ubiquitous pathogen of maize and a well-established model organism for the study of plant-microbe interactions. This basidiomycete fungus does not use aggressive virulence strategies to kill its host. U. maydis belongs to the group of biotrophic parasites (the smuts) that depend on living tissue for proliferation and development. Here we report the genome sequence for a member of this economically important group of biotrophic fungi. The 20.5-million-base U. maydis genome assembly contains 6,902 predicted protein-encoding genes and lacks pathogenicity signatures found in the genomes of aggressive pathogenic fungi, for example a battery of cell-wall-degrading enzymes. However, we detected unexpected genomic features responsible for the pathogenicity of this organism. Specifically, we found 12 clusters of genes encoding small secreted proteins with unknown function. A significant fraction of these genes exists in small gene families. Expression analysis showed that most of the genes contained in these clusters are regulated together and induced in infected tissue. Deletion of individual clusters altered the virulence of U. maydis in five cases, ranging from a complete lack of symptoms to hypervirulence. Despite years of research into the mechanism of pathogenicity in U. maydis, no 'true' virulence factors had been previously identified. Thus, the discovery of the secreted protein gene clusters and the functional demonstration of their decisive role in the infection process illuminate previously unknown mechanisms of pathogenicity operating in biotrophic fungi. Genomic analysis is, similarly, likely to open up new avenues for the discovery of virulence determinants in other pathogens. ©2006 Nature Publishing Group.J.K., M. B. and R.K. thank G. Sawers and U. Kämper for critical reading of the manuscript. The genome sequencing of Ustilago maydis strain 521 is part of the fungal genome initiative and was funded by National Human Genome Research Institute (USA) and BayerCropScience AG (Germany). F.B. was supported by a grant from the National Institutes of Health (USA). J.K. and R.K. thank the German Ministry of Education and Science (BMBF) for financing the DNA array setup and the Max Planck Society for their support of the manual genome annotation. F.B. was supported by a grant from the National Institutes of Health, B.J.S. was supported by the Natural Sciences and Engineering Research Council of Canada and the Canada Foundation for Innovation, J.W.K. received funding from the Natural Sciences and Engineering Research Council of Canada, J.R.-H. received funding from CONACYT, México, A.M.-M. was supported by a fellowship from the Humboldt Foundation, and L.M. was supported by an EU grant. Author Contributions All authors were involved in planning and executing the genome sequencing project. B.W.B., J.G., L.-J.M., E.W.M., D.D., C.M.W., J.B., S.Y., D.B.J., S.C., C.N., E.K., G.F., P.H.S., I.H.-H., M. Vaupel, H.V., T.S., J.M., D.P., C.S., A.G., F.C. and V. Vysotskaia contributed to the three independent sequencing projects; M.M., G.M., U.G., D.H., M.O. and H.-W.M. were responsible for gene model refinement, database design and database maintenance; G.M., J. Kämper, R.K., G.S., M. Feldbrügge, J.S., C.W.B., U.F., M.B., B.S., B.J.S., M.J.C., E.C.H.H., S.M., F.B., J.W.K., K.J.B., J. Klose, S.E.G., S.J.K., M.H.P., H.A.B.W., R.deV., H.J.D., J.R.-H., C.G.R.-P., L.O.-C., M.McC., K.S., J.P.-M., J.I.I., W.H., P.G., P.S.-A., M. Farman, J.E.S., R.S., J.M.G.-P., J.C.K., W.L. and D.H. were involved in functional annotation and interpretation; T.B., O.M., L.M., A.M.-M., D.G., K.M., N.R., V. Vincon, M. VraneŠ, M.S. and O.L. performed experiments. J. Kämper, R.K. and M.B. wrote and edited the paper with input from L.-J.M., J.G., F.B., J.W.K., B.J.S. and S.E.G. Individual contributions of authors can be found as Supplementary Notes
- …