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The use of a CRISPR-Cas9 system to protect probiotic strains from transferrable drug resistance genesLundberg, Sara January 2021 (has links)
The discovery of antibiotics have revolutionized modern medicine, facilitating the treatment of a variety of bacterial infections, enabled surgeries otherwise impossible to perform and increased life expectancy in all countries. However, the rapid development of resistance among microorganisms and the increasing numbers of clinical outbreaks caused by multiresistant bacteria have accelerated the need for new alternatives to antibiotics. Probiotic bacteria armed with defense systems have been studied as potential substitutes of antibiotics. These probiotic competitors can still contribute to the spread of resistance genes among microorganisms through horizontal gene transfer. The aim of this study was to investigate whether constructed CRISPR-Cas9 systems have the potential to protect probiotic bacteria against horizontal transfer of antibiotic resistance genes. Transformation, transduction and conjugation assays in strains carrying or not carrying a plasmid-bourne CRISPR-Cas9 system were performed in order to compare the frequencies of transfer of the most common resistance genes. The transformation and transduction assays demonstrated that the constructed CRISPR-Cas9 system entails a decrease in efficiency of transfer for targeted resistance genes. Moreover, it can be concluded that potentially increasing Cas9 levels by reducing its degradation results in increased prevention of horizontal gene transfer through transformation and transduction. Finally, we state that the CRISPRCas9 system does not result in protection against antibiotic resistance genes entering the cells through conjugation.
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Molekularbiologische und physiologische Untersuchungen zur Prozessoptimierung der lichtgetriebenen Wasserstofferzeugung mit Rhodobacter sphaeroidesWappler, Nadine Christina 25 April 2022 (has links)
Durch die vorliegende Arbeit wurde gezeigt, dass Rhodobacter sphaeroides das Potenzial besitzt, umweltverträglich photoheterotroph Wasserstoff als alternativer, erneuerbarer Energieträger zu erzeugen. Aus genomischen und transkriptomischen Erkenntnissen konnten Rückschlüsse auf Ansatzpunkte für weitere Optimierungen getroffen werden. Durch ein neues Minimalmedium, welches zukünftig sogar einen Beitrag zur Abfallbeseitigung leisten kann, wurde ein wichtiger Schritt hinsichtlich der industriellen Anwendbarkeit von R. sphaeroides für die biologische Wasserstoffproduktion gemacht.:Danksagung
Datenverfügbarkeit
Inhaltsverzeichnis
Abbildungsverzeichnis
Tabellenverzeichnis
Abkürzungsverzeichnis
1. Einleitung
1.1 Wasserstoff
1.1.1 Wasserstoff als Energieträger
1.1.2 Herstellung von Wasserstoff
1.1.2.1 Konventionelle Wasserstoffproduktion
1.1.2.2 Biologische Wasserstoffproduktion
1.1.2.3 Biologische Wasserstoffproduktion aus Abfällen
1.2 Photosynthetische Bakterien
1.2.1 Rhodobacter sphaeroides im Kontext der biologischen Wasserstoffproduktion
1.2.2 An der Wasserstoffproduktion beteiligte Enzyme
1.3 Third Generation-Sequencing Technologien
2. Zielstellung
3. Material
3.1 Chemikalien
3.2 Medien und Pufferlösungen
3.2.1 Van Niel´s Yeast Medium
3.2.2 Medium nach Krujatz et al. (2014)
3.2.3 RÄ-Medium nach Mougiakos et al. (2019)
3.2.4 PY (Peptone Yeast) Agarmedium
3.2.5 2x YT Medium
3.2.6 LB Medium
3.2.7 GYCC Medium
3.2.8 SOB Medium
3.2.9 SOC Medium
3.2.10 Pufferlösungen
3.3 Mikroorganismen
3.4 Molekularbiologische Reagenzien und Primer
3.5 Plasmide
3.5.1 pCas9
3.5.2 pRKPOL2
3.5.3 pSUPPOL2Sca
3.5.4 pBBRBB-Ppuf843-1200-DsRed
3.5.5 pBBR_cas9_NT
3.6 Geräte
4. Methoden
4.1 Rhodobacter sphaeroides Dauerkultur in Van Niel´s Yeast Medium 112 (ohne Wasserstoffproduktion)
4.2 Rhodobacter sphaeroides Batch-Kultivierung
4.2.1 Kultivierung in Medium nach Krujatz et al. (2014); Vollmedium mit Wasserstoffproduktion
4.2.2 Kultivierung in Fruchtsaftmedium
4.3 Rhodobacter sphaeroides Kultivierung mit kontinuierlicher Aufzeichnung von Temperatur, pH, optischer Dichte, Wasserstoffproduktion und Gasanalyse
4.4 Zellernte
4.5 Nukleinsäureextraktion mit dem MasterPureTM Complete RNA and DNA Purification Kit
4.6 DNase-Abbau
4.7 RNase-Abbau
4.8 Qualitätskontrolle der RNA und DNA mit dem Agilent 2100 Bioanalyzer
4.9 Reverse Transkription und Probenaufreinigung
4.10 qRT-Polymerasekettenreaktion
4.11 Etablierung der CRISPR-Cas9- Methodik bei Rhodobacter sphaeroides – Gen-Knockout der Hydrogenase Untereinheit hupL mit CRISPR-Cas9
4.11.1 Anzucht der Escherichia coli Stämme mit und ohne Plasmid
4.11.2 Plasmid Extraktion mit GeneJET Plasmid Miniprep Kit (#K0502, Thermo Scientific)
4.11.3 Restriktionsverdau zur Vektorlinearisierung
4.11.4 Design der guideRNA
4.11.5 Phosphorylierung der guideRNA
4.11.6 Ligation der guideRNA in pCas9
4.11.7 Transformation pCas9_hupL1/hupL2 in Escherichia coli JM109 durch chemische Kompetenz
4.11.8 Colony-PCR zum Insertnachweis hupL1&2 in pCas9 mit GoTaq® G2 Green Master Mix (Promega)
4.11.9 Konstruktion weiterer Vektoren mit CRISPR-Cas9 Maschinerie aus pCas9_hupL1/2
4.12 Genomeditierung in Rhodobacter sphaeroides
4.12.1 Transformation durch chemische Kompetenz mit PEG-Methode
4.12.2 Transformation durch chemische Kompetenz nach Hanahan et al. (1991)
4.12.3 Konjugation mit Escherichia coli S17-1
4.12.4 Elektroporation
4.12.5 Bioballistische Genomeditierung mit PDS-1000/He Particle Delivers System (BIORAD)
4.12.6 Konjugation mit Escherichia coli S17-1 nach Mougiakos et al. (2019) 65
4.13 Probenvorbereitung für Sequenzierungen
4.13.1 Illumina MiSeq (Genomsequenzierung)
4.13.2 MinION (Genomsequenzierung)
4.13.3 Illumina HiSeq (Transkriptomsequenzierung)
4.14 Bioinformatische Methoden
4.14.1 Genomsequenzierung (Re-Sequenzierung)
4.14.2 Transkriptom-Datenanalyse
5. Ergebnisse und Diskussion
5.1 Schrittweise Reduktion des Vollmediums nach Krujatz et al. (2014) zum Fruchtsaft-Minimalmedium
5.2 Untersuchung der Wasserstoffproduktion in Fruchtsaft-Minimalmedium
5.3 Kontinuierliche Aufzeichnung von Prozessdaten im 1,2 L Bioreaktor
5.3.1 Vergleich der Reaktorläufe in Vollmedium nach Krujatz et al. (2014), Trauben- und Ananas-Minimalmedium der Stämme DSM 158 und SubH2
5.3.2 Prozessgasanalyse
5.4 Analyse des Genoms
5.4.1 Multiples Sequenzalignment der kompletten genomischen Assemblies von Rhodobacter sphaeroides
5.4.2 MiSeq-Sequenzierung des Stammes Rhodobacter sphaeroides 2.4.1. SubH2
5.4.2.1 Bioinformatische Funktionsanalyse von SNPs
5.4.2.2 SNP-Analyse mittels Homology-Modeling
5.4.3 Genomische Architekturanalyse mittels MinION Sequenzierung der Rhodobacter sphaeroides Stämme DSM 158 und 2.4.1. SubH2
5.4.4 Vergleich der MiSeq- und MinION Genomanalysen
5.5 Analyse des Transkriptoms
5.6 Analyse der Genexpression mit qRT-PCR im Vergleich mit der Wasserstoffproduktion
5.7 CRISPR-Cas9 zum Plasmid-basierten hupL Knock-out
5.7.1 Erstellung der Plasmide pCas9_hupL1 und pCas9_hupL2
5.7.2 PEG-basierte Transformation nach Fornari et al. (1982)
5.7.3 Transformation mittels Elektroporation
5.7.4 Erstellung weiterer Vektoren mit CRISPR-Cas9_Maschinerie aus pCas9_hupL1&2
5.7.5 Transformation mittels Konjugation I
5.7.6 Bioballistische Transformation
5.7.7 Problembehandlung zur Transformation
5.7.8 Transformation mittels Konjugation II
6 Zusammenfassung
7 Ausblick
8 Summary
Literaturverzeichnis
Anhangsverzeichnis
Anhang
Versicherung
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Vitamin D and its in vitro therapeutic action mediated through VDR rather than PDIA3Pyburn, Jaeden 01 May 2022 (has links)
Brain calcification is a common occurrence in the aging process, with >20% of individuals over the age of 65 showing hardened plaques in the basal ganglia. Loss of the vitamin D receptor (VDR) in transgenic mice leads to formation of calcified plaques in the basal ganglia and thalamus within the mice. Vitamin D signals through two known vitamin D responsive proteins, protein disulfide isomerase A3 (PDIA3) and VDR. In vitro, vitamin D has been demonstrated to suppress calcification in osteoblast-like cells. Here, we aim to elucidate which of either PDIA3 or VDR transduce vitamin D mediated suppression of calcification in vitro. PDIA3 or VDR were selectively knocked out in human osteosarcoma (SaOs) cells using CRISPR-Cas9 technology to generate PDIA3 KO or VDR KO cells. Knockout for PDIA3 or VDR was confirmed by RT-qPCR assay or western blot analysis. The calcification of SaOs-2 cells was induced with treatment of β-glycerophosphate along with ascorbic acid allowing for determination of whether loss of PDIA3 or VDR would lead to altered calcium deposition. Cells null for PDIA3 but not VDR grew at a significantly slower rate than wild-type (WT) cells. Intriguingly, PDIA3 and VDR KO cells displayed significantly more calcification relative to WT control cells. Calcitriol or the synthetic analogue EB1089 suppressed calcification in vitro in WT and PDIA3 KO but not VDR KO cells as measured by alizarin red staining. These data suggest VDR is critical for mediating vitamin D’s inhibition of calcification in vitro, and that PDIA3 has a role in suppressing calcification. This study provides novel insights into vitamin D signaling and provides a foundation for further study and understanding of vitamin D related pathologies.
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Therapeutic Potential of Piperlongumine for Pancreatic Ductal AdenocarcinomaMohammad, Jiyan Mageed January 2019 (has links)
Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal malignancies because it is often diagnosed at a late disease stage and has a poor response rate to currently available treatments. Therefore, it is critical to develop new therapeutic approaches that will enhance the efficacy and reduce the toxicity of currently used therapies. Here we aimed to evaluate the therapeutic potential and mechanisms of action for piperlongumine (PL), an alkaloid from long pepper, in PDAC models. We postulated that PL causes PDAC cell death through oxidative stress and complements the therapeutic efficacy of chemotherapeutic agents in PDAC cells. First, we determined that PL is one of the most abundant alkaloids with antitumor properties in the long pepper plant. We also showed PL in combination with gemcitabine, a chemotherapy agent used to treat advanced pancreatic cancer, reduced tumor weight and volume compared to vehicle-control and individual treatments. Further, biochemical analysis, including RNA sequencing and immunohistochemistry, suggested that the antitumor activity of PL was associated with decreased cell proliferation, induction of cell cycle arrest, and oxidative stress-induced cell death. Moreover, we identified that c-Jun N-terminal kinase (JNK) inhibition blocks PL-induced cell death, translocation of Nrf2, and transcriptional activation of HMOX1 in PDAC. Finally, high-throughput drug and CRISPR screenings identified potential targets that could be used in combination with PL to treat PDAC cells. Collectively, our data suggests that cell cycle regulators in combination with PL might be an effective approach to combat pancreatic cancer. / NIH
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High-efficiency plant genome engineering via CRISPR/Cas9 systemEid, Ayman 04 1900 (has links)
Precise engineering of genomes holds great promise to advance our understanding of gene function and biotechnological applications. DNA double strand breaks are repaired via imprecise non-homologous end joining repair or via precise homology-directed repair processes. Therefore, we could harness the DSBs to engineer the genomes with a variety of genetic outcomes and with singlebase- level precision. The major barrier for genome engineering was the generation of site-specific DNA DSBs. Programmable DNA enzymes capable of making a complete and site-specific cut in the genome do not exist in nature. However, these enzymes can be made in in vitro as chimeric fusions of two modules, a DNA binding module and a DNA cleaving module. The DNA cleaving module can be programmed to bind to any user-defined sequence and the DNA cleaving module would generate DSBs in the target sequence. These enzymes called molecular scissors include zinc finger nucleases (ZFNs) and transcriptional activator like effector nucleases (TALENs). The programmability of these enzymes depends on protein engineering for DNA binding specificity which may be complicated, recourse intensive and suffer from reproducibility issues.
Recently, clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated endonuclease 9 (Cas9) an adaptive immune system of bacterial and archaeal species has been developed for genome engineering applications. CRISPR/Cas9 is an RNA-guided DNA endonuclease and can be reprogrammed through the engineering of single guide RNA molecule (sgRNA). CRISPR/Cas9 activity has been shown across eukaryotic species including plants. Although the engineering of CRISPR/Cas9 is quite predictable and reproducible, there are many technological challenges and improvements that need to be made to achieve robust, specific, and efficient plant genome engineering. Here in this thesis, I developed a number of technologies to improve specificity, delivery and expression and heritability of CRISRP/Cas9-modification in planta. Moreover, I used these technologies to answer basic questions to understand the molecular underpinning of the interplay between splicing and abiotic stress.
To improve Cas9 specificity, I designed and constructed a chimeric fusion between catalytically dead Cas9 (dCas9) and FOKI catalytic DNA cleaving domain (dCas9.FoKI). This synthetic chimeric fusion enzyme improved Cas9 specificity which enable precision genome engineering. Delivery of genome engineering reagents into plant cells is quite challenging, I developed a virus-based system to deliver sgRNAs into plants which facilitates plant genome engineering and could bypass the need for tissue culture in engineering plant genomes. To improve the expression of the CRISPR/Cas9 machinery in plant species, I developed a meiotically-driven expression of CRISPR/Cas9 which improved genome editing and heritability of editing in seed progeny, thereby facilitating robust genome engineering applications.
To understand the molecular basis of the interplay between splicing stress and abiotic stress, I used the CRISPR/Cas9 machinery to engineer components of the U2snRNP complex coupled which chemical genomics to understand the splicing stress regulation in response to abiotic stress conditions. Finally, I harnessed the technological improvements and developments I have achieved with CRISPR/Cas9 system to develop a directed evolution platform for targeted trait engineering which expands and accelerates trait discovery and engineering of plant species resilient to climate change conditions.
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Patenting CRISPR-Cas9 therapeutic applications: Legal Framework for Human welfare- based applications in USA and EU. : Applicability of patent laws in modern biology and their limits on CRISPR- Cas9 genome engineering.Priyanka, Priyanka January 2020 (has links)
CRISPR-Cas9 based genome engineering has emerged as a revolutionary biological technique in the past decade with multifarious therapeutic and biotechnological applications for human welfare. It is anticipated that the global CRISPR-Cas9 market will exceed a revenue of USD 3 billion by 2024. In the past few years, product and process oriented CRISPR-Cas9 applications have led to extensive patenting efforts, resulting in some major patent disputes. Some of CRISPR-Cas9 based applications for human welfare are directly linked to modifications within the human cells as well as their possible misuse on the ethical grounds, there is a fundamental divide in the United States and the European Union about various patenting legal provisions. In the proposed study, I will categorically investigate the legal frameworks on the patentability of various human welfare related CRISPR-Cas9 applications in the United States and the European Union as well as their socio-economic impact.
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Spiderworms: Using Silkworms as Hosts to Produce a Hybrid Silkworm-Spider Silk FiberLicon, Ana Laura 01 August 2019 (has links)
Spider silk has received significant attention due to its fascinating mechanical properties. Given the solitary and cannibalistic behavior of spiders, spider silk farming is impractical. Unlike spiders, silkworms are capable of producing large quantities of a fibrous product in a manner mimetic to spiders, and there already exists an industry to process cocoons into threads and textiles for many applications. The combination of silk farming (sericulture), a millennia old practice, and modern advancements in genetic engineering has given rise to an innovative biomaterial inspired by nature; transgenic silkworm silk.
This project focuses on the creation of chimeric silkworm-spider silk fibers through the genetic modification of silkworms. Advanced genetic engineering techniques were used to introduce the minor ampullate spider silk (MiSp) genes into the silkworm genome. A subset of these transgenic silkworms was cross-bred with other transgenic silkworms containing the same spider silk gene in a different section of the silkworm genome to create hybrid, dual-transgenic silkworms. The transgenic silk samples showed increased mechanical properties compared to native silkworm fibers, with the strongest fibers approaching or surpassing the mechanical properties of native spider silk. The transgenic silk retained the elasticity of the native silkworm silk and gained the strength of the spider silk. Ultimately, genetic engineering opens the door to mass produce synthetic spider silk in an established organism and industry, and the results of this project demonstrate that the properties of silkworm silk can be predictably altered through this technology.
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Neo-morphic missense mutant p53 proteins and the co-drivers promoting cell invasionJanuary 2019 (has links)
abstract: Phenotypic and molecular profiling demonstrates a high degree of heterogeneity in the breast tumors. TP53 tumor suppressor is mutated in 30% of all breast tumors and the mutation frequency in basal-like subtype is as high as 80% and co-exists with several other somatic mutations in different genes. It was hypothesized that tumor heterogeneity is a result of a combination of neo-morphic functions of specific TP53 driver mutations and distinct co-mutations or the co-drivers for each type of TP53 mutation. The 10 most common p53 missense mutant proteins found in breast cancer patients were ectopically expressed in normal-like mammary epithelial cells and phenotypes associated with various hallmarks of cancer examined. Supporting the hypothesis, a wide spectrum of phenotypic changes in cell survival, resistance to apoptosis and anoikis, cell migration, invasion and polarity was observed in the mutants compared to wildtype p53 expressing cells. The missense mutants R248W, R273C and Y220C were most aggressive. Integrated analysis of ChIP and RNA seq showed distinct promoter binding profiles of the p53 mutant proteins different than wildtype p53, implying altered transcriptional activity of mutant p53 proteins and the phenotypic heterogeneity of tumors. Enrichment and model-based pathway analyses revealed dysregulated adherens junction and focal adhesion pathways associated with the aggressive p53 mutants. As several somatic mutations co-appear with mutant TP53, we performed a functional assay to fish out the relevant collaborating driver mutations, the co-drivers. When PTEN was deleted by CRISPR-Cas9 in non-invasive p53-Y234C mutant cell, an increase in cell invasion was observed justifying the concept of co-drivers. A genome wide CRISPR library-based screen on p53-Y234C and R273C cells identified separate candidate co-driver mutations that promoted cell invasion. The top candidates included several mutated genes in breast cancer patients harboring TP53 mutations and were associated with cytoskeletal and apoptosis resistance pathways. Overall, the combined approach of molecular profiling and functional genomics screen highlighted distinct sets of co-driver mutations that can lead to heterogeneous phenotypes and promote aggressiveness in cells with different TP53 mutation background, which can guide development of novel targeted therapies. / Dissertation/Thesis / Doctoral Dissertation Biochemistry 2019
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Blokování inhibičních receptorů při imunoterapii nádorů / Checkpoint blockade in cancer immunotherapyVacková, Julie January 2021 (has links)
The immune checkpoint blockade is a novel approach of cancer therapy, which markedly enhanced treatment efficacy of several cancer types. However, the frequency of cancer patients non-responding to this treatment is high. Establishment of predictive markers to distinguish patients suitable for the immune checkpoint blockade would enhance the number of patients receiving benefit from the therapy. This dissertation thesis focuses on the enhancement of efficacy of immune checkpoint inhibitors (ICIs) and predictive markers in experimental models of mouse tumours induced by TC-1 and TC-1/A9 cell lines and its clones with deactivation of interferon (IFN)-γ signalling (TC-1/dIfngr1 and TC-1/A9/dIfngr1), or CD80 molecule (TC-1/dCD80-1). IFN-γ is presumed to be the main inducer of programmed death ligand 1 (PD-L1) and a major histocompatibility complex I (MHC-I). Moreover, PD-L1 expression may predict sensitivity to PD-1/PD-L1 blockade. Non-functional IFN-γ signalling or downregulated MHC-I expression has been associated with resistance to ICIs in some patients. We found that IFNs type I (IFN-α and IFN-β) induced the expression of PD-L1 and MHC-I on TC-1/A9/dIfngr1 tumour cells with reversible downregulation of both molecules. We also showed that deactivation of IFN-γ signalling in TC-1/A9 cells was not a...
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Single-step generation of gene knockout-rescue system in pluripotent stem cells by promoter insertion with CRISPR/Cas9 / CRISPR/Cas9を用いたプロモーター配列挿入による簡便なノックアウト・レスキューシステムの構築Matsunaga, Taichi 24 March 2014 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18177号 / 医博第3897号 / 新制||医||1004(附属図書館) / 31035 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 中畑 龍俊, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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