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Methods in Molecular Biology (Clifton,... 2018DNA supercoiling plays critical roles in several essential DNA metabolic pathways, such as replication, transcription and recombination. Typically plasmid DNA molecules...
DNA supercoiling plays critical roles in several essential DNA metabolic pathways, such as replication, transcription and recombination. Typically plasmid DNA molecules are used to measure DNA supercoiling status inside bacterial cells. In this chapter, we describe how to isolate plasmid DNA molecules from E. coli cells and determine DNA supercoiling density by 1% agarose gel electrophoresis containing chloroquine using plasmid pACYC184 as an example.
Topics: Chloroquine; DNA Replication; DNA, Superhelical; Electrophoresis, Agar Gel; Escherichia coli; Nucleic Acid Conformation; Plasmids
PubMed: 29177733
DOI: 10.1007/978-1-4939-7459-7_4 -
Electrophoresis Jun 2022In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as...
In the production of novel biological products, plasmids are often engineered into delivery vectors for target genes, which can be used directly as vaccines or as intermediate products for gene/cell therapy. Plasmid DNA exists in several topological forms such as supercoiled, linear, and open circular. As supercoiled plasmid shows the highest efficiency in transfecting eukaryotic cells, the content of supercoiled plasmids becomes an important indicator of plasmid quality. CGE is an effective analysis method for separating different topological structures of plasmids. For the purpose of providing plasmid manufacturers and regulatory agencies with an efficient and readily used tool for monitoring the quality of plasmids, this article identifies the optimal separation and detection conditions of CGE, presents a platform-based plasmid analytical method, and uses plasmid of different sizes to verify the feasibility of this method. In terms of detector, the LIF detector has obvious advantages over the ultraviolet detector in sensitivity and resolution. Using the optimal CE condition (10× gel buffer), baseline separation of different topological forms and impurities can be achieved for different plasmid sizes (5.9, 7.8, 15.4 kb). In addition, 6.5 kb plasmid was used to compare the different separation technologies such as CGE-LIF, ion exchange chromatography and agarose gel electrophoresis. The result shows that CGE-LIF can provide better resolution and quantitation accuracy than ion exchange chromatography and agarose gel electrophoresis. CGE-LIF, as a quick and convenient method to separate and quantify plasmids, has the advantages of high sensitivity, high resolution, and high quantitative accuracy. Therefore, it is ideal for analysis of plasmids with different sizes, and it can also be used as a platform method for manufacturers and regulatory agencies to monitor the purity and stability of plasmids.
Topics: Chromatography, Ion Exchange; Electrophoresis, Agar Gel; Electrophoresis, Capillary; Plasmids; Protein Isoforms
PubMed: 35289414
DOI: 10.1002/elps.202100343 -
Methods in Molecular Biology (Clifton,... 2022Gene editing is increasing its popularity day by day especially as an essential tool for the research. It is based on two recombination mechanisms in mammalian cells:...
Gene editing is increasing its popularity day by day especially as an essential tool for the research. It is based on two recombination mechanisms in mammalian cells: nonhomologous end-joining (NHEJ) and homology-directed repair (HDR). The first one can be used to silence a specific gene or a portion of it and the second one to insert new DNA, in presence of a donor template, in a targeted position in the genome. In order to exploit one of these two mechanisms, three major targeted nucleases have been developed: zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas (clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein). The last one seems to be the most promising tool among the others for gene editing. By using the properties and versatility of the Cell Penetrating Peptide (CPP) PepFect14, we developed a protocol to deliver a plasmid encoding for CRISPR-Cas9 and Green Fluorescent Protein (GFP) in BHM cell line expressing luciferase (Bomirsky Hamster Melanoma pLuc). Aiming to knocking down the luciferase gene in the cell line and to expressing GFP. Having two fast and easy read-outs of the plasmid's activity at the same time. Furthermore, by labeling the CRISPR plasmid with Cy5 it is possible to have a visual confirmation of the cellular uptake of the pDNA/CPP complex, via fluorescent microscopy, as described.
Topics: Animals; CRISPR-Cas Systems; Cell-Penetrating Peptides; Gene Editing; Plasmids; Transcription Activator-Like Effector Nucleases
PubMed: 34766316
DOI: 10.1007/978-1-0716-1752-6_38 -
Cellular Physiology and Biochemistry :... 2018The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The...
BACKGROUND/AIMS
The target genome editing technology not only plays an important role in basic biology studies but also holds a great promise for potential clinical applications. The new generation of engineered nuclease RGEN (RNA Guided EndoNuclease) is much easier to construct and modify, and attracts more attentions. In the current study, we compared different plasmid construction strategies of Cas9-gRNA (guide RNA).
METHODS
Different plasmid construction strategies of Cas9-gRNA were compared. And more modifications were introduced into the plasmid construction strategy.
RESULTS
The plasmid construction efficiency of expressing the gRNA and Cas9 in one plasmid was lower than expressing them in two separate plasmids. However, they showed the similar genome editing efficiency. We further introduced the Golden-gate assembly and blue-white screening approaches into the Cas9-gRNA construction procedures, without the process of vector digestion and gel purification.
CONCLUSIONS
Combing with the optimized gRNA structure (gRNA-BL) we identified before, we established one more cost-effective, time-saving and efficient plasmid construction strategy for Cas9-gRNA.
Topics: CRISPR-Cas Systems; Genes, Reporter; HEK293 Cells; Humans; Plasmids; RNA, Guide, CRISPR-Cas Systems
PubMed: 30001549
DOI: 10.1159/000491669 -
Microbiology (Reading, England) Jul 2023This short primer is intended to give an overview of bacterial plasmids for those not yet familiar with these fascinating genetic elements. It covers their basic... (Review)
Review
This short primer is intended to give an overview of bacterial plasmids for those not yet familiar with these fascinating genetic elements. It covers their basic properties but does not attempt to cover the diversity of phenotypic properties that can be encoded by plasmids, and includes suggestions for further reading.
Topics: Bacteria; Conjugation, Genetic; DNA, Bacterial; Gene Transfer, Horizontal; Logic; Plasmids
PubMed: 37395112
DOI: 10.1099/mic.0.001336 -
PLoS Genetics Jul 2021Extra-chromosomal genetic elements are important drivers of evolutionary transformations and ecological adaptations in prokaryotes with their evolutionary success often...
Extra-chromosomal genetic elements are important drivers of evolutionary transformations and ecological adaptations in prokaryotes with their evolutionary success often depending on their 'utility' to the host. Examples are plasmids encoding antibiotic resistance genes, which are known to proliferate in the presence of antibiotics. Plasmids carrying an essential host function are recognized as permanent residents in their host. Essential plasmids have been reported in several taxa where they often encode essential metabolic functions; nonetheless, their evolution remains poorly understood. Here we show that essential genes are rarely encoded on plasmids; evolving essential plasmids in Escherichia coli we further find that acquisition of an essential chromosomal gene by a plasmid can lead to plasmid extinction. A comparative genomics analysis of Escherichia isolates reveals few plasmid-encoded essential genes, yet these are often integrated into plasmid-related functions; an example is the GroEL/GroES chaperonin. Experimental evolution of a chaperonin-encoding plasmid shows that the acquisition of an essential gene reduces plasmid fitness regardless of the stability of plasmid inheritance. Our results suggest that essential plasmid emergence leads to a dose effect caused by gene redundancy. The detrimental effect of essential gene acquisition on plasmid inheritance constitutes a barrier for plasmid-mediated lateral gene transfer and supplies a mechanistic understanding for the rarity of essential genes in extra-chromosomal genetic elements.
Topics: Biological Evolution; Chromosomes; Escherichia coli; Evolution, Molecular; Gene Transfer, Horizontal; Genes, Essential; Genomics; Plasmids
PubMed: 34252089
DOI: 10.1371/journal.pgen.1009656 -
Methods in Molecular Biology (Clifton,... 2021Rapid and efficient protocols aimed at the isolation and purification of DNA for the purpose of downstream applications, such as cloning, PCR, Southern blotting, or...
Rapid and efficient protocols aimed at the isolation and purification of DNA for the purpose of downstream applications, such as cloning, PCR, Southern blotting, or sequencing, are essential for genetic, biochemical, and molecular biological analyses of a given bacterium. The protocols herein presented provide a robust and efficient method for the isolation of chromosomal and plasmid DNA from Bifidobacterium strains by organic extraction. The methods are simple, and the yield, purity, and quality of the DNA are adequate to perform various downstream applications including next-generation sequencing.
Topics: Bifidobacterium; Chromosomes, Bacterial; DNA, Bacterial; High-Throughput Nucleotide Sequencing; Plasmids; Whole Genome Sequencing
PubMed: 33649945
DOI: 10.1007/978-1-0716-1274-3_3 -
Tissue Engineering. Part C, Methods Jun 2021Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired...
Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy drugs' usage became the subject of this study. To study plasmid biodistribution, toxicity, mutagenicity, and genotoxicity, we used previously described bicistronic genetic construct encoding human brain-derived neurotrophic factor (hBDNF) and human urokinase plasminogen activator (huPA) for nerve repair. Biodistribution studies were conducted in mice: a course of intramuscular plasmid injections was followed by the study of the content of the plasmid (real-time polymerase chain reaction) and recombinant proteins (enzyme-linked immunosorbent assay) in murine organs and tissues. The study of the plasmid chronic toxicity was carried out on rats with registration of their weight dynamics, neurological status, emotional state, and blood test parameters. The mutagenicity of the plasmid was studied in an DNA comet test in mice. Plasmid genotoxicity was investigated in the model of somatic recombination in Drosophila females. We have shown that plasmids can disseminate from the injection site, but do not mediate ectopic expression of growth factors upon repeated intramuscular injections. The studied plasmid also does not reveal toxic, mutagenic, or genotoxic effects. During the toxicological study on rats, we have shown that daily injections of this genetic construct, despite its ability to disseminate from the injection site, do not affect the physical, cognitive, and emotional state of experimental animals. We have demonstrated the safety of the bicistronic plasmid, encoding hBDNF and huPA, upon its repeated administration. The properties of genetic constructs strongly depend on their sequence and delivery approach, which requires conducting of their safety studies in each specific case. Impact statement Gene therapy is one of the promising approaches for regenerative medicine. Local and long-term expression of essential growth factors allows to achieve the desired therapeutic effect. However, some aspects of prolonged usage of genetic constructs encoding growth factors, such as toxicity, mutagenicity, genotoxicity, and ability to disseminate from the injection site and mediate ectopic expression of therapeutic proteins, are poorly investigated. These aspects of gene therapy became the subject of this study. To our knowledge, this is a unique study that provides a thorough safety investigation of a bicistronic plasmid after its readministration.
Topics: Animals; DNA; Female; Mice; Plasmids; Rats; Tissue Distribution
PubMed: 34015967
DOI: 10.1089/ten.TEC.2021.0033 -
The ISME Journal Oct 2023Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within...
Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids' ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.
Topics: Microbiota; Plasmids; Ecology; Anti-Bacterial Agents
PubMed: 37558861
DOI: 10.1038/s41396-023-01487-w -
Molecular Systems Biology Mar 2023The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical...
The molecular and ecological factors shaping horizontal gene transfer (HGT) via natural transformation in microbial communities are largely unknown, which is critical for understanding the emergence of antibiotic-resistant pathogens. We investigate key factors shaping HGT in a microbial co-culture by quantifying extracellular DNA release, species growth, and HGT efficiency over time. In the co-culture, plasmid release and HGT efficiency are significantly enhanced than in the respective monocultures. The donor is a key determinant of HGT efficiency as plasmids induce the SOS response, enter a multimerized state, and are released in high concentrations, enabling efficient HGT. However, HGT is reduced in response to high donor lysis rates. HGT is independent of the donor viability state as both live and dead cells transfer the plasmid with high efficiency. In sum, plasmid HGT via natural transformation depends on the interplay of plasmid properties, donor stress responses and lysis rates, and interspecies interactions.
Topics: Coculture Techniques; Plasmids; DNA; Anti-Bacterial Agents; Gene Transfer, Horizontal
PubMed: 36714980
DOI: 10.15252/msb.202211406