32 research outputs found
Strategies to control plasmid copy number
Die Gentherapie erscheint viel versprechend, fehlerhafte Gene auf transkriptioneller oder translationeller Ebene stillzulegen oder diese durch Rekombination mit funktionellen Genen zu korrigieren. Vor nicht allzu langer Zeit revolutionierte die Gentherapie die moderne Medizin und mehr als tausend vorgeschlagene oder laufende klinische Versuche wurden weltweit genehmigt. Um das Potential der medizinischen Gentherapie und DNA-Impfung in die Praxis umzusetzen, muss die Herstellung hochreiner Plasmid-DNA (pDNA) sichergestellt werden, die frei von jedweder bakteriell chromosomaler DNA, RNA, Proteine und Endotoxine sein muss. Es ist bekannt, dass die Plasmidkopienzahl und Plasmid-Stabilität wesentliche Determinanten sind, die die Gendosis in der Wirtszelle beeinflussen. Daher ist das rationale Design eines pDNA Vektors ein großes Ziel im Rahmen der Gentherapie. Weiters ist bekannt, dass die Regulierung der PCN entscheidend ist, um eine metabolische Belastung der Wirtszelle zu verhindern, da sich eine übermäßige Plasmidreplikation wachstumshemmend auswirkt oder gar bis hin zum Zelltod führen kann. In dieser Arbeit sollte die Konzentration von Plasmiden des ColE1-Typs durch Überexpression des Gens rssB sowie tRNAAlaU in Escherichia Coli (E. coli) reguliert werden, die sich in der Literatur als erfolgreich erwiesen haben. Im ersten Ansatz wurde das für die tRNAAlaU codierende Gen, das unter die Kontrolle des T7 Promoters gebracht wurde, in das E. coli Genom inseriert. In Schüttelkolbenexperimenten wurde die Überexpression des tRNAAlaU-Gens durch Zugabe von IPTG induziert und anschließend wurde dessen Auswirkung auf drei verschiedene Plasmide des ColE1-Typs getestet. In einem zweiten Ansatz sollte die Überexpression des Gens rssB, das ebenso unter der Kontrolle des T7 Promotors stand, zu einer Erhöhung der Plasmidkonzentration nach IPTG-Zugabe führen, wobei der biologische Mechanismus bisher noch nicht vollständig geklärt ist. In Schüttelkolbenexperimenten wurde eine rssB-Überexpression durch Zugabe von IPTG induziert. Insgesamt konnte in dieser Arbeit gezeigt werden, dass eine Überexpression von rssB und tRNAAlaU zu einer sichtbaren Erhöhung der Plasmidkonzentration des ColE1-Typs geführt hat.Gene therapy holds its promises to silence dysfunctional genes on either transcriptional or translational level or by replacement with functional genes. Not long ago it has revolutionized modern medicine and since then thousand clinical trials have been authorized worldwide. To put the medical profit of gene therapy and DNA vaccination into practice, methods to produce highly pure plasmid DNA (pDNA) free from bacterial chromosomal DNA, RNA, proteins and endotoxins have to be developed. Since it is known that plasmid copy number (PCN) and plasmid stability are affecting the success of gene therapy to a large extent, the rational design of an optimized vector is an ultimate ambition. To prevent excessive plasmid replication, posing metabolic burden resulting in growth inhibition right up to cell death to the workhorse Escherichia coli (E. coli), regulation of PCN is to be considered in the rational design of any vector as well.
In this work the main goal was to elaborate a system that regulates plasmid replication of ColE1-type plasmids in Escherichia Coli (E. coli) by overexpressing tRNAAlaU and rssB encoding genes. In the first approach the wt and mutated tRNAAlaU gene, whose expression was controlled by the T7 promoter, was inserted into the E. coli chromosome. In shake flask experiments overexpression of the tRNAAlaU gene was induced by addition of IPTG and subsequently its effect on three different ColE1-type plasmids has been tested. In the second approach, overexpression of rssB, whose expression was also controlled by the T7 promoter, should result in enhanced plasmid concentration after IPTG induction whereas the biological mechanism remains unclear. In shake flask experiments, rssB overexpression was also induced by adding IPTG. All in all, the experimentally gained data have demonstrated clearly that overexpression of rssB and tRNAAlaU encoding genes resulted in enhanced plasmid concentration of ColE1-type plasmids
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Mathematical and experimental approaches to the dimer catastrophe theory
Multicopy plasmids rely on random distribution for stable inheritance by daughter cells at division. Threats to plasmid copy number increase the probability of plasmid loss, which can be detrimental to both plasmid and host. Plasmid dimers emerge through homologous recombination. Dimers have two independent origins of replication and thus have a replicative advantage and reduced copy number. Models of plasmid behaviour suggest that dimers would overtake a cell population, but that this can be prevented if they impose a small metabolic load, which has been observed in vivo. Plasmid ColE1 also contains a cer site, which allows for dimer resolution by XerCD site-specific recombination. A small RNA, Rcd, is expressed from the cer site in dimers and interacts with tryptophanase to increase the concentration of indole in the cell. It is proposed that, as indole inhibits cell division, Rcd imposes a checkpoint on the cell until plasmid dimers are resolved.
In this work, plasmid behaviour in a growing cell population was modelled stochastically in more detail than previous work. A plasmid replication model suggested that dimers replicate to more than half the average copy number of monomers, perhaps accounting for their increased metabolic load. A cell population model suggested that the presence of dimer-only cells decreased the average plasmid stability by less than in previous models, which used a fixed plasmid copy number. The rate of dimer resolution required to affect plasmid stability was unreasonably high, indicating the necessity of the Rcd checkpoint. The model thus suggested that the checkpoint may be an escape route for dimer-only cells rather than an immediate response to the emergence of an initial dimer.
The Rcd checkpoint itself was also subject to critical analysis. It was realised that neither inhibition of cell division nor cell growth were sufficient to assist dimer resolution; inhibition of plasmid replication was required. Experiments in vivo found that indole inhibited plasmid replication at a concentration that may be achievable endogenously. DNA gyrase was investigated as a component of the mechanism of this inhibition, and indole was found to inhibit its supercoiling activity in vitro.The author of this work was the recipient of a PhD studentship from the Medical Research Council (UK
Development of a Thermosensitive Endonuclease to Act as a Plasmid Kill-Switch
Biocontainment is an integral part of biomedical research that aims to protect the environment and human health by containing hazardous or invasive organisms in the laboratory. Containment systems often rely on elaborate genetic circuits; however, cells may escape containment by developing mutations that render the genetic circuits inviable or resistant to killing mechanisms. The aim of this thesis was to create a site-specific nuclease for biocontainment of plasmids in the mammalian gastrointestinal tract. LAGLIDADG homing endonucleases would be good candidate nucleases for a biocontainment system as they are resistant to mutations in their coding sequence and their target sequence in comparison to other nucleases, such as Cas9. Screening mutagenic libraries of the I-OnuI nuclease yielded a single variant that displays thermosensitive properties in vivo that may prove useful for biocontainment purposes. Using thermosensitive LAGLIDADG homing endonucleases for this purpose may prove to be a new, more robust approach to biocontainment
In vitro-Charakterisierung regulatorischer und sensorischer RNAs in Prokaryoten
Im Rahmen dieser Arbeit wurden eine cis-kodierte und eine trans-kodierte Antisense-RNA aus Bacillus subtilis sowie eine RNA-Thermometerstruktur aus Salmonella enterica in vitro strukturell und funktionell charakterisiert. In vitro-Analysen zur inhibitorischen Wirkung der cis-kodierten Antisense-RNA RNAIII haben gezeigt, dass die Ausbildung einer vollständigen Duplex mit ihrer Target-RNA (RNAII) nicht erforderlich ist. Ein Bindungsintermediat, das lediglich die simultane Beteiligung der Stem-Loops L3 und L4 von RNAIII erfordert, reicht als inhibitorischer Komplex für die Regulation der Replikation des Streptokokkenplasmides pIP501 aus. SR1 aus Bacillus subtilis ist eine 205 nt lange trans-kodierte Antisense-RNA, die durch direkte Basenpaarung regulierend auf ihr Target, ahrC-mRNA, einwirken kann. SR1 induziert strukturelle Veränderungen stromabwärts der Ribosomenbindungsstelle von ahrC-mRNA, wodurch die Translationsinitiation von AhrC verhindert wird. Die Translationskontrolle des agsA-Transkriptes in Salmonella enterica erfolgt über eine RNA-Thermometer-Struktur, die keinem der bisher identifizierten RNA-Thermometertypen entspricht
Importancia de los plásmidos ColE1 en la resistencia a antibióticos
Los antibióticos han salvado millones de vidas desde que en la década de 1940 se introdujeran en la práctica clínica. Desafortunadamente debido a la rápida aparición de resistencias, su fácil diseminación y la dificultad de su eliminación una vez que se han establecido en la población, nos encontramos ante un grave problema tanto para la salud humana como para la sanidad animal. Actualmente, se producen en torno a 700.000 muertes al año por bacterias resistentes a antibióticos, y se calcula que para 2050 se superarán las 10 millones asociadas a la resistencia a antibióticos. Los plásmidos juegan un papel determinante en la diseminación de la resistencia a antibióticos. Los plásmidos son moléculas de ADN que replican de forma independiente al cromosoma bacteriano y que tienen la capacidad de movilizar genes horizontalmente. En concreto, los plásmidos tipo ColE1 tienen una gran importancia en la resistencia a antibióticos. Estos pequeños replicones portan algunos determinantes de resistencia a los antibióticos más relevantes en la práctica clínica. Además son capaces de coexistir en una misma bacteria generando multirresistencia a varios antibióticos. Cuando una bacteria adquiere resistencia a antibióticos mediante la transferencia horizontal de un plásmido, compromete su crecimiento debido al coste biológico asociado al replicón. Esto genera que, en ausencia de presión selectiva, las bacterias que porten plásmidos serán menos competitivas comparado con las bacterias libres de replicones. Sin embargo, en presencia de antibióticos, las bacterias portando plásmidos serán seleccionadas en la población. La adaptación plásmido/hospedador es, por tanto, crucial para entender la presencia de los plásmidos portadores de resistencia en las poblaciones bacterianas..
DESIGN AND DEVELOPMENT OF MOLECULAR TOOLS FOR THE COMBINATORIAL MULTIVARIANT-MODULAR APPROACH TO ENGINEERING METABOLIC PATHWAYS IN E.COLI
Ph.DDOCTOR OF PHILOSOPH
Modélisation et simulation de processus de biologie moléculaire basée sur les réseaux de Pétri : une revue de littérature
Les réseaux de Pétri sont une technique
de simulation à événements discrets
développée pour la représentation de systèmes et plus particulièrement de
leurs propriétés de concurrence et de synchronisation.
Différentes extensions à
la théorie initiale de cette méthode ont
été utilisées pour la modélisation de
processus de biologie moléculaire et de
réseaux métaboliques. Il s’agit des
extensions stochastiques, colorées, hybrides et fonctionnelles. Ce document
fait une première revue des différentes
approches qui ont été employées et des
systèmes biologiques qui ont été modélisés grâce à celles-ci. De plus, le
contexte d’application et les objectif
s de modélisation de chacune sont
discutés
A New Approach to Plasmid Upstream Processing for Vaccine and Gene Therapy Applications
There has been a rise in the interest of plasmid DNA as therapeutics. The rise is evident in the number of ongoing clinical trials involving the use of plasmid DNA. To be useful as therapeutics, the DNA needs to be of high yield and high level of supercoiling. From the bioprocessing point of view, the level of supercoiling can potentially have an impact on the ease of downstream processing. We have approached meeting these requirements through plasmid engineering and developing an optimized fermentation strategy. Two different plasmids (small and large size) were developed. A 7.2kb plasmid was developed by insertion of Bacteriophage-Mu Strong gyrase-binding sequence (Mu-SGS) to 6.8kb pSVβ-Gal. Four E. coli strains were transformed with both the modified pSVβ-Gal398 plasmid and pSVβ-Gal. Small scale fermentation and analysis were carried out in triplicates cultures to screen for best-performing strains. There was over 20% increase in the total plasmid yield with pSVβ-Gal398 in two of the strains. The supercoiled topoisomer content was increased by 5% in both strains leading to a 27% increase in the overall yield. The two strains were investigated further, and an increase in supercoiling and plasmid yield was also observed. The extent of supercoiling was examined by superhelical density (σ) quantification with pSVβ-Gal398 maintaining a superhelical density of -0.022 and pSVβ-Gal -0.019 in both strains. The compactness of the plasmid DNA was also quantified by hydrodynamic diameter (Dh) measurement using the Nanoparticle Tracking Analysis (NTA), and it was observed that pSVβ-Gal398 was more compact with a Dh of 40-59 nm compared to pSVβ-Gal with Dh of 70-90 nm for both strains examined. In order to investigate this approach can be scaled on plasmid, a 27 kb plasmid pSTFλ398 was constructed with its respective control plasmid, 26.6 kb pSTFλ. There was an almost 2-fold increase in the plasmid yield for the SGS containing plasmid pSTFλ398 and a supercoiled content of 93%. In addition, SGS containing plasmid was maintained integrity when subjected to shear. An approach was also developed to increase the plasmid yield by developing a batch fermentation for high cell density which involves supplementation with minimal media with amino acids. The media was supplemented with histidine and glycine which have been reported to be implicated in increasing plasmid yield, and methionine for suppressing acetate inhibition. The supplementation allowed the use of initial glycerol concentration as high as 100 g/L with a volumetric yield and plasmid yield of twice as much as using 100 g/L glycerol in the media without supplementation. The report of this study has shown that plasmid modification with the Mu-phage SGS sequence and optimizing fermentation strategies have beneficial effects on improving not only the yield of total plasmid but also the supercoiled topoisomer content of therapeutic plasmid DNA during bioprocessing