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Journal of Applied Physiology... Jul 2022The strategy of gene delivery into skeletal muscles has provided exciting avenues in identifying new potential therapeutics toward muscular disorders and addressing... (Review)
Review
The strategy of gene delivery into skeletal muscles has provided exciting avenues in identifying new potential therapeutics toward muscular disorders and addressing basic research questions in muscle physiology through overexpression and knockdown studies. In vivo electroporation methodology offers a simple, rapidly effective technique for the delivery of plasmid DNA into postmitotic skeletal muscle fibers and the ability to easily explore the molecular mechanisms of skeletal muscle plasticity. The purpose of this review is to describe how to robustly electroporate plasmid DNA into different hindlimb muscles of rodent models. Furthermore, key parameters (e.g., voltage, hyaluronidase, and plasmid concentration) that contribute to the successful introduction of plasmid DNA into skeletal muscle fibers will be discussed. In addition, details on processing tissue for immunohistochemistry and fiber cross-sectional area (CSA) analysis will be outlined. The overall goal of this review is to provide the basic and necessary information needed for successful implementation of in vivo electroporation of plasmid DNA and thus open new avenues of discovery research in skeletal muscle physiology.
Topics: Animals; DNA; Electroporation; Gene Transfer Techniques; Genetic Therapy; Mice; Muscle, Skeletal; Plasmids
PubMed: 35511722
DOI: 10.1152/japplphysiol.00088.2022 -
International Journal of Molecular... Mar 2020Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate...
Although many methods have been reported, plasmid construction compromises transformant efficiency (number of transformants per ng of DNAs) with plasmid accuracy (rate of scarless plasmids). An efficient method is two-step PCR serving DNA amplification. An accurate method is ExnaseII cloning serving homology recombination (HR). We combine DNA amplification and HR to develop an intra-molecular HR by amplifying plasmid DNAs to contain homology 5'- and 3'-terminus and recombining the plasmid DNAs in vitro. An example was to construct plasmid pET20b-. The generality was checked by constructing plasmid pET21a- and pET22b- in parallel. The DNAs having 30-bp homology arms were optimal for intra-molecular HR, and transformation of which created 14.2 transformants/ng and 90% scarless plasmids, more than the two-step PCR and the ExnaseII cloning. Transformant efficiency correlated with the component of nicked circular plasmid DNAs of HR products, indicating nick modification in vivo leads to scar plasmids.
Topics: Cloning, Molecular; Escherichia coli; Genetic Engineering; Homologous Recombination; Plasmids; Transformation, Bacterial
PubMed: 32131382
DOI: 10.3390/ijms21051697 -
Nature Microbiology Jul 2023Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated...
Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated conjugation has been well-studied in bacteria, information is sparse in Archaea and known representatives exist only in the Sulfolobales order of Crenarchaeota. Here we present the first self-transmissible plasmid identified in a Euryarchaeon, Thermococcus sp. 33-3. The 103 kbp plasmid, pT33-3, is seen in CRISPR spacers throughout the Thermococcales order. We demonstrate that pT33-3 is a bona fide conjugative plasmid that requires cell-to-cell contact and is dependent on canonical, plasmid-encoded T4SS-like genes. Under laboratory conditions, pT33-3 transfers to various Thermococcales and transconjugants propagate at 100 °C. Using pT33-3, we developed a genetic toolkit that allows modification of phylogenetically diverse Archaeal genomes. We demonstrate pT33-3-mediated plasmid mobilization and subsequent targeted genome modification in previously untransformable Thermococcales species, and extend this process to interphylum transfer to a Crenarchaeon.
Topics: Archaea; Plasmids; DNA; Bacteria; Genome, Archaeal
PubMed: 37277532
DOI: 10.1038/s41564-023-01387-x -
Biodegradation Aug 2021Plasmids, circular DNA that exist and replicate outside of the host chromosome, have been important in the spread of non-essential genes as well as the rapid evolution... (Review)
Review
Plasmids, circular DNA that exist and replicate outside of the host chromosome, have been important in the spread of non-essential genes as well as the rapid evolution of prokaryotes. Recent advances in environmental engineering have aimed to utilize the mobility of plasmids carrying degradative genes to disseminate them into the environment for cost-effective and environmentally friendly remediation of harmful contaminants. Here, we review the knowledge surrounding plasmid transfer and the conditions needed for successful transfer and expression of degradative plasmids. Both abiotic and biotic factors have a great impact on the success of degradative plasmid transfer and expression of the degradative genes of interest. Properties such as ecological growth strategies of bacteria may also contribute to plasmid transfer and may be an important consideration for bioremediation applications. Finally, the methods for detection of conjugation events have greatly improved and the application of these tools can help improve our understanding of conjugation in complex communities. However, it remains clear that more methods for in situ detection of plasmid transfer are needed to help detangle the complexities of conjugation in natural environments to better promote a framework for precision bioremediation.
Topics: Bacteria; Biodegradation, Environmental; Conjugation, Genetic; Gene Transfer, Horizontal; Plasmids
PubMed: 34046775
DOI: 10.1007/s10532-021-09950-1 -
Methods in Molecular Biology (Clifton,... 2018The introduction of ectopic DNA, such as plasmids, into yeast cells has for decades been a critical protocol for the study of this eukaryotic model system. We describe... (Review)
Review
The introduction of ectopic DNA, such as plasmids, into yeast cells has for decades been a critical protocol for the study of this eukaryotic model system. We describe here an efficient transformation procedure for use in the fission yeast Schizosaccharomyces pombe. This method relies on chemical agents (lithium acetate, and polyethylene glycol) and temperature stresses, which ultimately facilitate transfer of the genetic material through the cell wall and plasma membrane without significant impact on the transferred DNA or the recipient cell. Using this protocol, we consistently see transformation efficiencies between 1.0 × 10 and 1.0 × 10 transformants per microgram of the plasmid with 10 S. pombe cells. The principal benefits and advantages of this method are its simplicity, efficiency, and relative speed of completion.
Topics: Acetates; DNA; Plasmids; Schizosaccharomyces; Transfection; Transformation, Genetic
PubMed: 29423856
DOI: 10.1007/978-1-4939-7546-4_15 -
BMC Bioinformatics Jul 2021Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust...
BACKGROUND
Plasmids are mobile genetic elements, key in the dissemination of antibiotic resistance, virulence determinants and other adaptive traits in bacteria. Obtaining a robust method for plasmid classification is necessary to better understand the genetics and epidemiology of many pathogens. Until now, plasmid classification systems focused on specific traits, which limited their precision and universality. The definition of plasmid taxonomic units (PTUs), based on average nucleotide identity metrics, allows the generation of a universal plasmid classification scheme, applicable to all bacterial taxa. Here we present COPLA, a software able to assign plasmids to known and novel PTUs, based on their genomic sequence.
RESULTS
We implemented an automated pipeline able to assign a given plasmid DNA sequence to its cognate PTU, and assessed its performance using a sample of 1000 unclassified plasmids. Overall, 41% of the samples could be assigned to a previously defined PTU, a number that reached 63% in well-known taxa such as the Enterobacterales order. The remaining plasmids represent novel PTUs, indicating that a large fraction of plasmid backbones is still uncharacterized.
CONCLUSIONS
COPLA is a bioinformatic tool for universal, species-independent, plasmid classification. Offered both as an automatable pipeline and an open web service, COPLA will help bacterial geneticists and clinical microbiologists to quickly classify plasmids.
Topics: Drug Resistance, Microbial; Gene Transfer, Horizontal; Genomics; Plasmids; Virulence Factors
PubMed: 34332528
DOI: 10.1186/s12859-021-04299-x -
The ISME Journal Jun 2023The rise of β-lactam resistance among pathogenic bacteria, due to the horizontal transfer of plasmid-encoded β-lactamases, is a current global health crisis....
The rise of β-lactam resistance among pathogenic bacteria, due to the horizontal transfer of plasmid-encoded β-lactamases, is a current global health crisis. Importantly, β-lactam hydrolyzation by β-lactamases, not only protects the producing cells but also sensitive neighboring cells cooperatively. Yet, how such cooperative traits affect plasmid transmission and maintenance is currently poorly understood. Here we experimentally show that KPC-2 β-lactamase expression and extracellular activity were higher when encoded on plasmids compared with the chromosome, resulting in the elevated rescue of sensitive non-producers. This facilitated efficient plasmid transfer to the rescued non-producers and expanded the potential plasmid recipient pool and the probability of plasmid transfer to new genotypes. Social conversion of non-producers by conjugation was efficient yet not absolute. Non-cooperative plasmids, not encoding KPC-2, were moderately more competitive than cooperative plasmids when β-lactam antibiotics were absent. However, in the presence of a β-lactam antibiotic, strains with non-cooperative plasmids were efficiently outcompeted. Moreover, plasmid-free non-producers were more competitive than non-producers imposed with the metabolic burden of a plasmid. Our results suggest that cooperative antibiotic resistance especially promotes the fitness of replicons that transfer horizontally such as conjugative plasmids.
Topics: Gene Transfer, Horizontal; Drug Resistance, Microbial; Plasmids; beta-Lactamases; Genotype; Conjugation, Genetic; Chromosomes, Bacterial; beta-Lactams; Anti-Bacterial Agents; Bacteria
PubMed: 36949153
DOI: 10.1038/s41396-023-01393-1 -
Theoretical Population Biology Dec 2023Plasmids may carry genes coding for beneficial traits and thus contribute to adaptation of bacterial populations to environmental stress. Conjugative plasmids can...
Plasmids may carry genes coding for beneficial traits and thus contribute to adaptation of bacterial populations to environmental stress. Conjugative plasmids can horizontally transfer between cells, which a priori facilitates the spread of adaptive alleles. However, if the potential recipient cell is already colonized by another incompatible plasmid, successful transfer may be prevented. Competition between plasmids can thus limit horizontal transfer. Previous modeling has indeed shown that evolutionary rescue by a conjugative plasmid is hampered by incompatible resident plasmids in the population. If the rescue plasmid is a mutant variant of the resident plasmid, both plasmids transfer at the same rates. A high conjugation rate then has two, potentially opposing, effects - a direct positive effect on spread of the rescue plasmid and an increase in the fraction of resident plasmid cells. This raises the question whether a high conjugation rate always benefits evolutionary rescue. In this article, we systematically analyze three models of increasing complexity to disentangle the benefits and limits of increasing horizontal gene transfer in the presence of plasmid competition and plasmid costs. We find that the net effect can be positive or negative and that the optimal transfer rate is thus not always the highest one. These results can contribute to our understanding of the many facets of plasmid-driven adaptation and the wide range of transfer rates observed in nature.
Topics: Conjugation, Genetic; Plasmids; Biological Evolution; Gene Transfer, Horizontal; Bacteria
PubMed: 37923145
DOI: 10.1016/j.tpb.2023.10.001 -
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