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Genome Research May 2023Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and...
Recombinant plasmid vectors are versatile tools that have facilitated discoveries in molecular biology, genetics, proteomics, and many other fields. As the enzymatic and bacterial processes used to create recombinant DNA can introduce errors, sequence validation is an essential step in plasmid assembly. Sanger sequencing is the current standard for plasmid validation; however, this method is limited by an inability to sequence through complex secondary structure and lacks scalability when applied to full-plasmid sequencing of multiple plasmids owing to read-length limits. Although high-throughput sequencing does provide full-plasmid sequencing at scale, it is impractical and costly when used outside of library-scale validation. Here, we present Oxford nanopore-based rapid analysis of multiplexed plasmids (OnRamp), an alternative method for routine plasmid validation that combines the advantages of high-throughput sequencing's full-plasmid coverage and scalability with Sanger's affordability and accessibility by leveraging nanopore's long-read sequencing technology. We include customized wet-laboratory protocols for plasmid preparation along with a pipeline designed for analysis of read data obtained using these protocols. This analysis pipeline is deployed on the OnRamp web app, which generates alignments between actual and predicted plasmid sequences, quality scores, and read-level views. OnRamp is designed to be broadly accessible regardless of programming experience to facilitate more widespread adoption of long-read sequencing for routine plasmid validation. Here we describe the OnRamp protocols and pipeline and show our ability to obtain full sequences from pooled plasmids while detecting sequence variation even in regions of high secondary structure at less than half the cost of equivalent Sanger sequencing.
Topics: Genome, Bacterial; Sequence Analysis, DNA; Plasmids; High-Throughput Nucleotide Sequencing; Proteomics
PubMed: 37156622
DOI: 10.1101/gr.277369.122 -
Microbiology (Reading, England) Jul 2023Plasmids, extrachromosomal DNA molecules commonly found in bacterial and archaeal cells, play an important role in bacterial genetics and evolution. Our understanding of... (Review)
Review
Plasmids, extrachromosomal DNA molecules commonly found in bacterial and archaeal cells, play an important role in bacterial genetics and evolution. Our understanding of plasmid biology has been furthered greatly by the development of mathematical models, and there are many questions about plasmids that models would be useful in answering. In this review, we present an introductory, yet comprehensive, overview of the biology of plasmids suitable for modellers unfamiliar with plasmids who want to get up to speed and to begin working on plasmid-related models. In addition to reviewing the diversity of plasmids and the genes they carry, their key physiological functions, and interactions between plasmid and host, we also highlight selected plasmid topics that may be of particular interest to modellers and areas where there is a particular need for theoretical development. The world of plasmids holds a great variety of subjects that will interest mathematical biologists, and introducing new modellers to the subject will help to expand the existing body of plasmid theory.
Topics: Humans; Plasmids; Bacteria; Biology; Gene Transfer, Horizontal
PubMed: 37505810
DOI: 10.1099/mic.0.001362 -
IEEE/ACM Transactions on Computational... 2022Plasmids are extra-chromosomal genetic materials with important markers that affect the function and behaviour of the microorganisms supporting their environmental...
Plasmids are extra-chromosomal genetic materials with important markers that affect the function and behaviour of the microorganisms supporting their environmental adaptations. Hence the identification and recovery of such plasmid sequences from assemblies is a crucial task in metagenomics analysis. In the past, machine learning approaches have been developed to separate chromosomes and plasmids. However, there is always a compromise between precision and recall in the existing classification approaches. The similarity of compositions between chromosomes and their plasmids makes it difficult to separate plasmids and chromosomes with high accuracy. However, high confidence classifications are accurate with a significant compromise of recall, and vice versa. Hence, the requirement exists to have more sophisticated approaches to separate plasmids and chromosomes accurately while retaining an acceptable trade-off between precision and recall. We present GraphPlas, a novel approach for plasmid recovery using coverage, composition and assembly graph topology. We evaluated GraphPlas on simulated and real short read assemblies with varying compositions of plasmids and chromosomes. Our experiments show that GraphPlas is able to significantly improve accuracy in detecting plasmid and chromosomal contigs on top of popular state-of-the-art plasmid detection tools. The source code is freely available at: https://github.com/anuradhawick/GraphPlas.
Topics: Genome, Bacterial; Metagenomics; Plasmids; Sequence Analysis, DNA; Software
PubMed: 34029192
DOI: 10.1109/TCBB.2021.3082915 -
Journal of Cellular Biochemistry Dec 2018Gene therapy is considered as a promising approach for treating cardiac dysfunction. In this review, we evaluated the clinical trials assessing gene therapy in... (Review)
Review
Gene therapy is considered as a promising approach for treating cardiac dysfunction. In this review, we evaluated the clinical trials assessing gene therapy in cardiovascular diseases (CVD) from 2000 to 2017. PubMed and ClinicalTrials.gov (only English language) were searched for clinical trials published between January 2000 and May 2017, using the search terms "gene transfer" OR "gene therapy" and "cardiovascular diseases" and related terms. The trials with sample size lower than 10 patients were excluded. Twenty-six clinical trials on human and animals, including 1543 patients were listed and evaluated. The sample size in 14 trials was lower than 100 patients and in seven trials lower than 20 patients. Eleven trials used plasmid DNA and eight trials used adenovirus, one study used plasmid DNA, adenovirus, and liposome. We detected that gene therapy was a safe approach and improved the symptoms of CVD; however, the effect of gene therapy on the cardiac dysfunction is controversial.
Topics: Adenoviridae; Animals; Cardiovascular Diseases; Clinical Trials as Topic; Genetic Therapy; Genetic Vectors; Humans; Plasmids
PubMed: 30129172
DOI: 10.1002/jcb.27303 -
The ISME Journal Oct 2021Plasmids are autonomous genetic elements that can be exchanged between microorganisms via horizontal gene transfer (HGT). Despite the central role they play in...
Plasmids are autonomous genetic elements that can be exchanged between microorganisms via horizontal gene transfer (HGT). Despite the central role they play in antibiotic resistance and modern biotechnology, our understanding of plasmids' natural ecology is limited. Recent experiments have shown that plasmids can spread even when they are a burden to the cell, suggesting that natural plasmids may exist as parasites. Here, we use mathematical modeling to explore the ecology of such parasitic plasmids. We first develop models of single plasmids and find that a plasmid's population dynamics and optimal infection strategy are strongly determined by the plasmid's HGT mechanism. We then analyze models of co-infecting plasmids and show that parasitic plasmids are prone to a "tragedy of the commons" in which runaway plasmid invasion severely reduces host fitness. We propose that this tragedy of the commons is averted by selection between competing populations and demonstrate this effect in a metapopulation model. We derive predicted distributions of unique plasmid types in genomes-comparison to the distribution of plasmids in a collection of 17,725 genomes supports a model of parasitic plasmids with positive plasmid-plasmid interactions that ameliorate plasmid fitness costs or promote the invasion of new plasmids.
Topics: Animals; Drug Resistance, Microbial; Gene Transfer, Horizontal; Parasites; Plasmids
PubMed: 33833414
DOI: 10.1038/s41396-021-00954-6 -
Biotechnology & Genetic Engineering... Apr 2019Vaccination is the most effective and least expensive technique used for human diseases prevention and eradication. The need for more vaccine doses and the rapid... (Review)
Review
Vaccination is the most effective and least expensive technique used for human diseases prevention and eradication. The need for more vaccine doses and the rapid establishment of facilities for the development of new vaccines are stimulating significate changes in the vaccine industry, which is gradually moving towards cell culture production. One approach is the third generation of vaccines, which are based on the use of plasmid DNA (pDNA) containing transgenes that encode an antigen capable of mimicking intracellular pathogenic infection and triggering both humoral and cellular immune responses. Plasmid DNA vaccination has distinct advantages over other vaccine technologies in terms of safety, ease of fabrication and stability. The effectiveness of pDNA vaccines against viruses, bacteria, parasites and cancer cells has been demonstrated in preclinical and clinical assays. Furthermore, currently there are a few veterinary pDNA vaccines in the market. The application of a simple formulation of naked pDNA as a vaccine is attractive, but a low transfection efficiency is often obtained. The use of nanoparticles to increase transfection efficiency is an approach that has been tested clinically. This review provides a summary of vaccine production, advances and major challenges associated with pDNA lipid and polymeric nanovaccines applications.
Topics: Animals; Drug Development; Genetic Vectors; Humans; Lipids; Nanoparticles; Plasmids; Polymers; Transgenes; Vaccines, DNA
PubMed: 30587085
DOI: 10.1080/02648725.2018.1560552 -
The American Naturalist Oct 2021AbstractPlasmids are extrachromosomal segments of DNA that can transfer genes between bacterial cells. Many plasmid genes benefit bacteria but cause harm to human health...
AbstractPlasmids are extrachromosomal segments of DNA that can transfer genes between bacterial cells. Many plasmid genes benefit bacteria but cause harm to human health by granting antibiotic resistance to pathogens. Transfer rate is a key parameter for predicting plasmid dynamics, but observed rates are highly variable, and the effects of selective forces on their evolution are unclear. We apply evolutionary analysis to plasmid conjugation models to investigate selective pressures affecting plasmid transfer rate, emphasizing host versus plasmid control, the costs of plasmid transfer, and the role of recipient cells. Our analyses show that plasmid-determined transfer rates can be predicted with three parameters (host growth rate, plasmid loss rate, and the cost of plasmid transfer on growth) under some conditions. We also show that low-frequency genetic variation in transfer rate can accumulate, facilitating rapid adaptation to changing conditions. Furthermore, reduced transfer rates due to host control have limited effects on plasmid prevalence until low enough to prevent plasmid persistence. These results provide a framework to predict plasmid transfer rate evolution in different environments and demonstrate the limited impact of host mechanisms to control the costs incurred when plasmids are present.
Topics: Adaptation, Physiological; Bacteria; Drug Resistance, Microbial; Gene Transfer, Horizontal; Humans; Plasmids
PubMed: 34559608
DOI: 10.1086/716063 -
Molecular Biology and Evolution Mar 2019The ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes....
The ubiquity of plasmids in all prokaryotic phyla and habitats and their ability to transfer between cells marks them as prominent constituents of prokaryotic genomes. Many plasmids are found in their host cell in multiple copies. This leads to an increased mutational supply of plasmid-encoded genes and genetically heterogeneous plasmid genomes. Nonetheless, the segregation of plasmid copies into daughter cells during cell division is considered to occur in the absence of selection on the plasmid alleles. We investigate the implications of random genetic drift of multicopy plasmids during cell division-termed here "segregational drift"-to plasmid evolution. Performing experimental evolution of low- and high-copy non-mobile plasmids in Escherichia coli, we find that the evolutionary rate of multicopy plasmids does not reflect the increased mutational supply expected according to their copy number. In addition, simulated evolution of multicopy plasmid alleles demonstrates that segregational drift leads to increased loss frequency and extended fixation time of plasmid mutations in comparison to haploid chromosomes. Furthermore, an examination of the experimentally evolved hosts reveals a significant impact of the plasmid type on the host chromosome evolution. Our study demonstrates that segregational drift of multicopy plasmids interferes with the retention and fixation of novel plasmid variants. Depending on the selection pressure on newly emerging variants, plasmid genomes may evolve slower than haploid chromosomes, regardless of their higher mutational supply. We suggest that plasmid copy number is an important determinant of plasmid evolvability due to the manifestation of segregational drift.
Topics: Biological Evolution; Chromosomes, Bacterial; Escherichia coli; Gene Frequency; Genetic Drift; Models, Genetic; Plasmids
PubMed: 30517696
DOI: 10.1093/molbev/msy225 -
Mathematical Biosciences and... Mar 2022Bacteria, in contrast to eukaryotic cells, contain two types of genes: chromosomal genes that are fixed to the cell, and plasmids, smaller loops of DNA capable of being...
Bacteria, in contrast to eukaryotic cells, contain two types of genes: chromosomal genes that are fixed to the cell, and plasmids, smaller loops of DNA capable of being passed from one cell to another. The sharing of plasmid genes between individual bacteria and between bacterial lineages has contributed vastly to bacterial evolution, allowing specialized traits to 'jump ship' between one lineage or species and the next. The benefits of this generosity from the point of view of both recipient cell and plasmid are generally understood: plasmids receive new hosts and ride out selective sweeps across the population, recipient cells gain new traits (such as antibiotic resistance). Explaining this behavior from the point of view of donor cells is substantially more difficult. Donor cells pay a fitness cost in order to share plasmids, and run the risk of sharing advantageous genes with their competition and rendering their own lineage redundant, while seemingly receiving no benefit in return. Using both compartment based models and agent based simulations we demonstrate that 'secretive' genes which restrict horizontal gene transfer are favored over a wide range of models and parameter values, even when sharing carries no direct cost. 'Generous' chromosomal genes which are more permissive of plasmid transfer are found to have neutral fitness at best, and are generally disfavored by selection. Our findings lead to a peculiar paradox: given the obvious benefits of keeping secrets, why do bacteria share information so freely?
Topics: Bacteria; Drug Resistance, Microbial; Gene Transfer, Horizontal; Phenotype; Plasmids
PubMed: 35603365
DOI: 10.3934/mbe.2022257 -
Plasmid Jan 2021Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification...
Multicopy plasmids play an important role in bacterial ecology and evolution by accelerating the rate of adaptation and providing a platform for rapid gene amplification and evolutionary rescue. Despite the relevance of plasmids in bacterial evolutionary dynamics, evaluating the population-level consequences of randomly segregating and replicating plasmids in individual cells remains a challenging problem, both in theory and experimentally. In recent years, technological advances in fluorescence microscopy and microfluidics have allowed studying temporal changes in gene expression by quantifying the fluorescent intensity of individual cells under controlled environmental conditions. In this paper, we will describe the manufacture, experimental setup, and data analysis pipeline of different microfluidic systems that can be used to study plasmid dynamics, both in single-cells and in populations. To illustrate the benefits and limitations of microfluidics to study multicopy plasmid dynamics, we will use an experimental model system consisting on Escherichia coli K12 carrying non-conjugative, multicopy plasmids (19 copies per cell, in average) encoding different fluorescent markers and β-lactam resistance genes. First, we will use an image-based flow cytometer to estimate changes in the allele distribution of a heterogeneous population under different selection regimes. Then we will use a mothermachine microfluidic device to obtain time-series of fluorescent intensity of individual cells to argue that plasmid segregation and replication dynamics are inherently stochastic processes. Finally, using a microchemostat, we track thousands of cells in time to reconstruct bacterial lineages and evaluate the allele frequency distributions that emerge in response to a range of selective pressures.
Topics: Bacteria; Computational Biology; Microfluidics; Plasmids; beta-Lactam Resistance
PubMed: 32535165
DOI: 10.1016/j.plasmid.2020.102517