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Bioscience Reports Dec 2017The use of ultrasound has gained great interest for nucleic acids delivery. Ultrasound can reach deep tissues in non-invasive manner. The process of sonoporation is... (Review)
Review
The use of ultrasound has gained great interest for nucleic acids delivery. Ultrasound can reach deep tissues in non-invasive manner. The process of sonoporation is based on the use of low-frequency ultrasound combined with gas-filled microbubbles (MBs) allowing an improved delivery of molecules including nucleic acids in the insonified tissue. For gene transfer, the engineering of cationic MBs is essential for creating strong electrostatic interactions between MBs and nucleic acids leading to their protection against nucleases degradation and high concentration within the target tissue. Cationic MBs must be stable enough to withstand nucleic acids interaction, have a good size distribution for administration, and enough acoustic activity to be detected by echography. This review aims to summarize the basic principles of ultrasound-based delivery and new knowledge acquired in these recent years about this method. A focus is made on gene delivery by discussing reported studies made with cationic MBs including ours. They have the ability for efficient delivery of plasmid DNA (pDNA), mRNA or siRNA. Last, we discuss about the key challenges that have to be faced for a fine use of this delivery system.
Topics: Animals; Cations; Gases; Gene Transfer Techniques; Humans; Microbubbles; Nucleic Acids; Plasmids; Ultrasonic Waves
PubMed: 29180378
DOI: 10.1042/BSR20160619 -
BioEssays : News and Reviews in... Sep 2021Plasmids are a major type of mobile genetic elements (MGEs) that mediate horizontal gene transfer. The stable maintenance of plasmids plays a critical role in the...
Plasmids are a major type of mobile genetic elements (MGEs) that mediate horizontal gene transfer. The stable maintenance of plasmids plays a critical role in the functions and survival for microbial populations. However, predicting and controlling plasmid persistence and abundance in complex microbial communities remain challenging. Computationally, this challenge arises from the combinatorial explosion associated with the conventional modeling framework. Recently, a plasmid-centric framework (PCF) has been developed to overcome this computational bottleneck. This framework enables the derivation of a simple metric, the persistence potential, to predict plasmid persistence and abundance. Here, we discuss how PCF can be extended to account for plasmid interactions. We also discuss how such model-guided predictions of plasmid fates can benefit from the development of new experimental tools and data-driven computational methods.
Topics: Gene Transfer, Horizontal; Microbiota; Plasmids
PubMed: 34278591
DOI: 10.1002/bies.202100084 -
Bioinformatics (Oxford, England) Jul 2023With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining...
SUMMARY
With recent advances in sequencing technologies, it is now possible to obtain near-perfect complete bacterial chromosome assemblies cheaply and efficiently by combining a long-read-first assembly approach with short-read polishing. However, existing methods for assembling bacterial plasmids from long-read-first assemblies often misassemble or even miss bacterial plasmids entirely and accordingly require manual curation. Plassembler was developed to provide a tool that automatically assembles and outputs bacterial plasmids using a hybrid assembly approach. It achieves increased accuracy and computational efficiency compared to the existing gold standard tool Unicycler by removing chromosomal reads from the input read sets using a mapping approach.
AVAILABILITY AND IMPLEMENTATION
Plassembler is implemented in Python and is installable as a bioconda package using 'conda install -c bioconda plassembler'. The source code is available on GitHub at https://github.com/gbouras13/plassembler. The full benchmarking pipeline can be found at https://github.com/gbouras13/plassembler_simulation_benchmarking, while the benchmarking input FASTQ and output files can be found at https://doi.org/10.5281/zenodo.7996690.
Topics: Sequence Analysis, DNA; High-Throughput Nucleotide Sequencing; Software; Plasmids; Benchmarking
PubMed: 37369026
DOI: 10.1093/bioinformatics/btad409 -
Philosophical Transactions of the Royal... Jan 2022Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has...
Conjugative plasmids are extrachromosomal mobile genetic elements pervasive among bacteria. Plasmids' acquisition often lowers cells' growth rate, so their ubiquity has been a matter of debate. Chromosomes occasionally mutate, rendering plasmids cost-free. However, these compensatory mutations typically take hundreds of generations to appear after plasmid arrival. By then, it could be too late to compete with fast-growing plasmid-free cells successfully. Moreover, arriving plasmids would have to wait hundreds of generations for compensatory mutations to appear in the chromosome of their new host. We hypothesize that plasmid-donor cells may use the plasmid as a 'weapon' to compete with plasmid-free cells, particularly in structured environments. Cells already adapted to plasmids may increase their inclusive fitness through plasmid transfer to impose a cost to nearby plasmid-free cells and increase the replication opportunities of nearby relatives. A mathematical model suggests conditions under which the proposed hypothesis works, and computer simulations tested the long-term plasmid maintenance. Our hypothesis explains the maintenance of conjugative plasmids not coding for beneficial genes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.
Topics: Bacteria; Biological Evolution; Conjugation, Genetic; Plasmids
PubMed: 34839709
DOI: 10.1098/rstb.2020.0473 -
BMC Biotechnology Mar 2021The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning...
BACKGROUND
The ability to clone DNA sequences quickly and precisely into plasmids is essential for molecular biology studies. The recent development of seamless cloning technologies has made significant improvements in plasmid construction, but simple and reliable tools are always desirable for time- and labor-saving purposes.
RESULTS
We developed and standardized a plasmid cloning protocol based on a universal MCS (Multiple Cloning Site) design and bacterial in vivo assembly. With this method, the vector is linearized first by PCR (Polymerase Chain Reaction) or restriction digestion. Then a small amount (10 ~ 20 ng) of this linear vector can be mixed with a PCR-amplified insert (5× molar ratio against vector) and transformed directly into competent E. coli cells to obtain the desired clones through in vivo assembly. Since we used a 36-bp universal MCS as the homologous linker, any PCR-amplified insert with ~ 15 bp compatible termini can be cloned into the vector with high fidelity and efficiency. Thus, the need for redesigning insert-amplifying primers according to various vector sequences and the following PCR procedures was eliminated.
CONCLUSIONS
Our protocol significantly reduced hands-on time for preparing transformation reactions, had excellent reliability, and was confirmed to be a rapid and versatile plasmid cloning technique. The protocol contains mostly mixing steps, making it an extremely automation-friendly and promising tool in modern biology studies.
Topics: Cloning, Molecular; DNA Primers; Escherichia coli; Genetic Vectors; Plasmids; Polymerase Chain Reaction
PubMed: 33722223
DOI: 10.1186/s12896-021-00679-6 -
Current Opinion in Microbiology Apr 2024Horizontal transfer of plasmids by conjugation is a fundamental mechanism driving the widespread dissemination of drug resistance among bacterial populations. The... (Review)
Review
Horizontal transfer of plasmids by conjugation is a fundamental mechanism driving the widespread dissemination of drug resistance among bacterial populations. The successful colonization of a new host cell necessitates the plasmid to navigate through a series of sequential steps, each dependent on specific plasmid or host factors. This review explores recent advancements in comprehending the cellular and molecular mechanisms that govern plasmid transmission, establishment, and long-term maintenance. Adopting a plasmid-centric perspective, we describe the critical steps and bottlenecks in the plasmid's journey toward a new host cell, encompassing exploration and contact initiation, invasion, establishment and control, and assimilation.
Topics: Conjugation, Genetic; Plasmids; Bacteria
PubMed: 38432159
DOI: 10.1016/j.mib.2024.102449 -
Bioinformatics (Oxford, England) May 2023As prevalent extrachromosomal replicons in many bacteria, plasmids play an essential role in their hosts' evolution and adaptation. The host range of a plasmid refers to...
MOTIVATION
As prevalent extrachromosomal replicons in many bacteria, plasmids play an essential role in their hosts' evolution and adaptation. The host range of a plasmid refers to the taxonomic range of bacteria in which it can replicate and thrive. Understanding host ranges of plasmids sheds light on studying the roles of plasmids in bacterial evolution and adaptation. Metagenomic sequencing has become a major means to obtain new plasmids and derive their hosts. However, host prediction for assembled plasmid contigs still needs to tackle several challenges: different sequence compositions and copy numbers between plasmids and the hosts, high diversity in plasmids, and limited plasmid annotations. Existing tools have not yet achieved an ideal tradeoff between sensitivity and precision on metagenomic assembled contigs.
RESULTS
In this work, we construct a hierarchical classification tool named HOTSPOT, whose backbone is a phylogenetic tree of the bacterial hosts from phylum to species. By incorporating the state-of-the-art language model, Transformer, in each node's taxon classifier, the top-down tree search achieves an accurate host taxonomy prediction for the input plasmid contigs. We rigorously tested HOTSPOT on multiple datasets, including RefSeq complete plasmids, artificial contigs, simulated metagenomic data, mock metagenomic data, the Hi-C dataset, and the CAMI2 marine dataset. All experiments show that HOTSPOT outperforms other popular methods.
AVAILABILITY AND IMPLEMENTATION
The source code of HOTSPOT is available via: https://github.com/Orin-beep/HOTSPOT.
Topics: Phylogeny; Plasmids; Software; Metagenome; Metagenomics; Bacteria
PubMed: 37086432
DOI: 10.1093/bioinformatics/btad283 -
MBio May 2021Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial...
Plasmids play an important role in bacterial evolution by transferring niche-adaptive functional genes between lineages, thus driving genomic diversification. Bacterial genomes commonly contain multiple, coexisting plasmid replicons, which could fuel adaptation by increasing the range of gene functions available to selection and allowing their recombination. However, plasmid coexistence is difficult to explain because the acquisition of plasmids typically incurs high fitness costs for the host cell. Here, we show that plasmid coexistence was stably maintained without positive selection for plasmid-borne gene functions and was associated with compensatory evolution to reduce fitness costs. In contrast, with positive selection, plasmid coexistence was unstable despite compensatory evolution. Positive selection discriminated between differential fitness benefits of functionally redundant plasmid replicons, retaining only the more beneficial plasmid. These data suggest that while the efficiency of negative selection against plasmid fitness costs declines over time due to compensatory evolution, positive selection to maximize plasmid-derived fitness benefits remains efficient. Our findings help to explain the forces structuring bacterial genomes: coexistence of multiple plasmids in a genome is likely to require either rare positive selection in nature or nonredundancy of accessory gene functions among the coexisting plasmids. Bacterial genomes often contain multiple coexisting plasmids that provide important functions like antibiotic resistance. Using lab experiments, we show that such plasmid coexistence within a genome is stable only in environments where the function they encode is useless but is unstable if the function is useful and beneficial for bacterial fitness. Where competing plasmids perform the same useful function, only the most beneficial plasmid is kept by the cell, a process that is similar to competitive exclusion in ecological communities. This process helps explain how bacterial genomes are structured: bacterial genomes expand in size by acquiring multiple plasmids when selection is relaxed but subsequently contract during periods of strong selection for the useful plasmid-encoded function.
Topics: Adaptation, Physiological; Bacteria; Genetic Fitness; Genome, Bacterial; Phenotype; Plasmids; Selection, Genetic
PubMed: 33975933
DOI: 10.1128/mBio.00558-21 -
Molecular Systems Biology Mar 2021Plasmid conjugation is a major mechanism responsible for the spread of antibiotic resistance. Plasmid fitness costs are known to impact long-term growth dynamics of...
Plasmid conjugation is a major mechanism responsible for the spread of antibiotic resistance. Plasmid fitness costs are known to impact long-term growth dynamics of microbial populations by providing plasmid-carrying cells a relative (dis)advantage compared to plasmid-free counterparts. Separately, plasmid acquisition introduces an immediate, but transient, metabolic perturbation. However, the impact of these short-term effects on subsequent growth dynamics has not previously been established. Here, we observed that de novo transconjugants grew significantly slower and/or with overall prolonged lag times, compared to lineages that had been replicating for several generations, indicating the presence of a plasmid acquisition cost. These effects were general to diverse incompatibility groups, well-characterized and clinically captured plasmids, Gram-negative recipient strains and species, and experimental conditions. Modeling revealed that both fitness and acquisition costs modulate overall conjugation dynamics, validated with previously published data. These results suggest that the hours immediately following conjugation may play a critical role in both short- and long-term plasmid prevalence. This time frame is particularly relevant to microbiomes with high plasmid/strain diversity considered to be hot spots for conjugation.
Topics: Bacteria; Conjugation, Genetic; Models, Biological; Plasmids; Time Factors
PubMed: 33646643
DOI: 10.15252/msb.20209913 -
Thoracic Cancer Nov 2020Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular...
BACKGROUND
Plasmid construction of small fragments of interest (such as insertion of small fragment marker genes, expression of shRNA, siRNA, etc) is the basis of many biomolecular experiments. Here, we describe a method to clone short DNA into vectors by polymerase chain reaction (PCR), named one-step PCR cloning. Our method uses PCR to amplify the entire circular plasmid. The PCR was performed by the primers containing the gene of short DNA with overlapping sequences between 10-15 bp. The PCR products were then transformed into E. coli and cyclized by homologous recombination in vivo.
METHODS
The pEGFP-N1-HA plasmid was constructed by one-step PCR and transformation. Cells were transfected with pEGFP-N1-HA and pEGFP-N1 plasmid using TurboFect transfection reagent. Protein expression was detected by western blotting and the HA-GFP fusion protein was detected by confocal microscopy.
RESULTS
The pEGFP-N1-HA plasmid was successfully constructed and HA expression in cells.
CONCLUSIONS
Free from the limitations of restriction enzyme sites and omitting the ligation process, our method offers a flexible and economical option of plasmid construction.
KEY POINTS
Significant findings of the study A method to clone short DNA into plasmids was found. What this study adds Our study provides a flexible and economical option to clone short DNA into plasmids.
Topics: Cloning, Molecular; DNA; Humans; Plasmids; Polymerase Chain Reaction
PubMed: 33015950
DOI: 10.1111/1759-7714.13660