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Methods in Molecular Biology (Clifton,... 2014Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product...
Plasmid DNA for immunization applications must be of the highest purity and quality. The ability of downstream purification to efficiently produce a pure final product is directly influenced by the performance of the upstream fermentation process. While several clinical manufacturing facilities already have validated fermentation processes in place to manufacture plasmid DNA for use in humans, a simple and inexpensive laboratory-scale fermentation process can be valuable for in-house production of plasmid DNA for use in animal efficacy studies. This chapter describes a simple fed-batch fermentation process for producing bacterial cell paste enriched with high-quality plasmid DNA. A constant feeding strategy results in a medium cell density culture with continuously increasing plasmid amplification towards the end of the process. Cell banking and seed culture preparation protocols, which can dramatically influence final product yield and quality, are also described. These protocols are suitable for production of research-grade plasmid DNA at the 100 mg-to-1.5 g scale from a typical 10 L laboratory benchtop fermentor.
Topics: Batch Cell Culture Techniques; Fermentation; Immunization; Plasmids; Vaccines, DNA
PubMed: 24715290
DOI: 10.1007/978-1-4939-0410-5_13 -
Current Opinion in Biotechnology Oct 2002In vivo electrotransfer is a physical technique for gene delivery in various mammalian tissues, which involves the injection of plasmid DNA into a target tissue and... (Review)
Review
In vivo electrotransfer is a physical technique for gene delivery in various mammalian tissues, which involves the injection of plasmid DNA into a target tissue and administration of an electric field. Its ease of performance, as well as recent understanding of its mechanism and applications to different mammalian tissues such as skeletal muscle, liver, brain and tumors, makes it a powerful technique. It could be used in gene therapy and as a laboratory tool to study gene functions.
Topics: Animals; DNA; Electromagnetic Fields; Electrophoresis; Electroporation; Gene Expression Profiling; Gene Expression Regulation; Gene Transfer Techniques; Genetic Therapy; Humans; Muscle, Skeletal; Neoplasms; Plasmids
PubMed: 12459335
DOI: 10.1016/s0958-1669(02)00377-4 -
Plasmid Jul 2015
Topics: Plasmids
PubMed: 26256501
DOI: 10.1016/S0147-619X(15)00068-2 -
Acta Microbiologica Et Immunologica... 2004Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory... (Review)
Review
Various bacterial plasmids can be eliminated from bacterial species cultured as pure or mixed bacterial cultures by non-mutagenic heterocyclic compounds at subinhibitory concentrations. For plasmid curing, the replication should be inhibited at three different levels simultaneously: the intracellular replication of plasmid DNA, partition and intercellular transconjugal transfer. The antiplasmid action of the compounds depends on the chemical structure. The targets for antiplasmid compounds were analysed in detail. It was found that amplified extrachromosomal DNA in the superhelical state binds more drug molecules than does the linear or open-circular form of the plasmid or the chromosome, without stereospecificity which leads to functional inactivation of the extrachromosomal genetic code. Plasmid elimination also occurs in ecosystems containing numerous bacterial species simultaneously, but the elimination of antibiotic resistance-encoding plasmids from all individual cells of the population is never complete. The medical significance of plasmid elimination in vitro is, it provides a method to isolate plasmid-free bacteria for biotechnology without any risk of mutations, and it opens up a new perspective in rational drug design against bacterial plasmids. Hypothetically, the combination of antiplasmid drugs and antibiotics may improve the effectivity of antibiotics against resistant bacteria; therefore, the results cannot be exploited until the curing efficiency reaches 100%. Inhibition of the conjugational transfer of antibiotic resistance plasmids can be exploited to reduce the spreading of these plasmids in ecosystems.
Topics: Anti-Bacterial Agents; Bacteria; Conjugation, Genetic; Drug Resistance, Multiple; Plasmids; Proton Pump Inhibitors
PubMed: 15571073
DOI: 10.1556/AMicr.51.2004.3.11 -
Acta Biochimica Polonica 2001The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active... (Review)
Review
The stable inheritance of bacterial plasmids is achieved by a number of different mechanisms. Among them are resolution of plasmid oligomers into monomers, active plasmid partitioning into dividing cells and selective killing of plasmid-free segregants. A special focus is given to the last mechanism. It involves a stable toxin and an unstable antidote. The antidotes neutralize their cognate toxins or prevent their synthesis. The different decay rates of the toxins and the antidotes underlie molecular mechanisms of toxin activation in plasmid-free cells. By eliminating of plasmid-free cells from the population of plasmid-bearing ones the toxin-antidote couples therefore act as plasmid addiction systems.
Topics: Bacteriophages; Models, Genetic; Oligonucleotides, Antisense; Operon; Plasmids; Recombination, Genetic
PubMed: 11995964
DOI: No ID Found -
Trends in Biotechnology Feb 2024Plasmids that replicate independently from chromosomes are valuable genetic tools for biological research. Dynamic control of plasmid copy number facilitates flexible...
Plasmids that replicate independently from chromosomes are valuable genetic tools for biological research. Dynamic control of plasmid copy number facilitates flexible regulation of the gene of interest or the genetic circuit installed in the plasmid. This useful strategy is being integrated into synthetic biology for metabolic reprogramming and biosensing applications.
Topics: Synthetic Biology; DNA Copy Number Variations; Plasmids
PubMed: 37689527
DOI: 10.1016/j.tibtech.2023.08.004 -
Plasmid Sep 2017As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of...
As shown in the previous article, two distinct conjugative plasmids sometimes interact within bacterial cells, implicating changes of transfer rates. In most cases of interactions within bacteria, the transfer of one of the plasmids decreases. Less frequently, the transfer rate of one of the plasmids increases. Here we analyse what happens if three distinct conjugative plasmids colonize the same bacterial cell. Our aim is to understand how interactions between two plasmids affect the transfer rate of the third plasmid. After showing that plasmids interact in 59 out of 84 possible interactions we show that, with some exceptions, if the transfer rate of a plasmid decreases in the presence of a second plasmid, a decrease is also observed in the presence of a third plasmid. Moreover, if the conjugation rate of a plasmid increases in the presence of another, an increase is also observed if there is a third plasmid in the cell. Both types of interactions are mostly independent of the third plasmid's identity, even if sometimes the third plasmid quantitatively distorts the interaction of the other two plasmids. There is a bias towards negative intensifying interactions, which provide good news concerning the spread conjugative plasmids encoding antibiotic-resistance genes and virulence factors.
Topics: Bacteria; Conjugation, Genetic; Drug Resistance, Microbial; Plasmids
PubMed: 28842133
DOI: 10.1016/j.plasmid.2017.08.002 -
Trends in Biotechnology Apr 1999Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One... (Review)
Review
Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, acquired immunodeficiency syndrome (AIDS) and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amounts of plasmid DNA. There are several problems and bottlenecks associated with the design and operation of large-scale processes for the production of pharmaceutical-grade plasmid DNA for gene therapy.
Topics: Biopharmaceutics; Biotechnology; Chemistry, Pharmaceutical; Drug Contamination; Drug Industry; Enzymes; Fermentation; Genetic Therapy; Plasmids; Quality Control; Solvents
PubMed: 10203776
DOI: 10.1016/s0167-7799(98)01291-8 -
Methods in Molecular Biology (Clifton,... 2021Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas...
Therapeutic applications of plasmid DNA (pDNA) have significantly advanced during the last years. Currently, several pDNA-based drugs are already in the market, whereas several others have entered phases 2 and 3 of clinical trials. The present and future demand for pDNA requires the development of efficient bioprocesses to produce it. Commonly, pDNA is produced by cultures of Escherichia coli. It has been previously demonstrated that specific strains of E. coli with a modified substrate transport system can be able to attain high cell densities in batch mode, due to the very low overflow metabolism displayed. However, the large amounts of oxygen demanded can lead to microaerobic conditions after some hours of cultivation, even at small scale. Typically, the inherent problems for these cultures are the high oxygen demand and the accumulation of acetate, a metabolic byproduct that is synthesized aerobically when the glucose rate exceeds the limits.In recent years, several researches have been focused on the study of induction of plasmid DNA as well as strategies for fermentation using semi-defined mediums. These studies conceived relevant results that allow us to design a production platform for enhanced plasmid DNA. So, the main goal of this chapter is to show how the development of an experimental design directed to aromatic amino acids pathway can improve the yield of a therapeutic plasmid DNA by culture of a new strain of Escherichia coli VH33.
Topics: Bioreactors; Escherichia coli; Fermentation; Plasmids; Research Design; Transformation, Genetic; Vaccines, DNA
PubMed: 32827135
DOI: 10.1007/978-1-0716-0872-2_7 -
Microbiology (Reading, England) Jun 1997Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction...
Chlamydiae exhibit low interspecies DNA homology and plasmids from different chlamydial species can be readily distinguished by Southern blot analysis and restriction enzyme profiling. In contrast, available plasmid sequence data from within the species Chlamydia trachomatis indicate that plasmids from human isolates are highly conserved. To evaluate the nature and extent of plasmid variation, the complete nucleotide sequences were determined for novel plasmids from three diverse non-human chlamydial isolates: pCpA1 from avian Chlamydia psittaci (N352); pCpnE1 from equine Chlamydia pneumoniae (N16); and pMoPn from C. trachomatis mouse pneumonitis. Comparison of the sequence data did not identify an overall biological function for the plasmid but did reveal considerable sequence conservation (> 60%) and a remarkably consistent genomic arrangement comprising eight major ORFs and four 22 bp tandem repeats. The plasmid sequences were close to 7500 nucleotides in length (pCpA1, 7553 bp; pMoPn, 7502 bp) however the equine C. pneumoniae plasmid was smaller (7362 bp) than all other chlamydial plasmids. The reduced size of this plasmid was due to a single large deletion occurring within ORF 1; this potentially generates two smaller ORFs. The disruption of ORF 1 is the only significant variation identified amongst the chlamydial plasmids and could prove important for future vector development studies.
Topics: Amino Acid Sequence; Animals; Chlamydia; Cloning, Molecular; Conserved Sequence; Genome, Bacterial; Humans; Mice; Molecular Sequence Data; Open Reading Frames; Phylogeny; Plasmids; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Sequence Homology, Amino Acid
PubMed: 9202459
DOI: 10.1099/00221287-143-6-1847