Plasmid最新文献

IncI1 has become one of the most common plasmid families in contemporary Enterobacteriaceae from both human and animal sources. In clinical epidemiology, this plasmid type ranks first as the confirmed vehicle of transmission of extended spectrum beta-lactamase and plasmid AmpC genes in isolates from food-producing animals. In this review, we describe the epidemiology and evolution of IncI1 plasmids and closely related IncIγ plasmids. We highlight the emergence of epidemic plasmids circulating among different bacterial hosts in geographically distant countries, and we address the phylogeny of the IncI1 and IncIγ family based on plasmid Multilocus Sequence Typing.

Promoter engineering has been employed as a strategy to enhance and optimize the production of bio-products. Availability of promoters with predictable activities is needed for downstream application. However, whether promoter activity remains the same in different gene contexts remains unknown. Six consecutive promoters that have previously been determined to have different activity levels were used to construct six different versions of plasmid backbone pTH1227, followed by inserted genes encoding two polymer-producing enzymes. In some cases, promoter activity in the presence of inserted genes did not correspond to the reported activity levels in a previous study. After removing the inserted genes, the activity of these promoters returned to their previously reported level. These changes were further confirmed to occur at the transcriptional level. Polymer production using our newly constructed plasmids showed polymer accumulation levels corresponding to the promoter activity reported in our study. Our study demonstrated the importance of re-assessing promoter activity levels with regard to gene context, which could influence promoter activity, leading to different outcomes in downstream applications.

Advances in neuroscience have relied on the development of techniques that examine neuronal cell activities. One major challenge involves the limitations in labeling and controlling neuronal activities relating to the cell's activation state. In this study, the modified human codon-optimized channelrhodopsin-2 photoreceptor hChR2(C128S) was integrated into function with inducible gene expression methods and materials: the Tet system and the highly efficient minimum promoter of Arc/Arg3.1. The system successfully expressed the target fusion gene exclusively in activated SH-SY5Y human neuroblastoma cells while maintaining the essential characteristics of ChR2. The expression of the channelrhodopsin construct was observed, while the expression duration was refined by treatment with doxycycline. The optogenetic construct here tested the application of the minimum Arc/Arg3.1 promoter, an advanced immediate-early gene promoter, for the expression of the channelrhodopsin gene. Along with its noninvasive nature, this expression system promises to serve dual functions as a cell activity indicator and cell actuator, creating the possibility for researchers to precisely label cells according to their activation state and control the activities of specific neuronal cell populations.

Gluconobacter oxydans is an obligate Gram-negative bacterium that belongs to the family Acetobacteraceae. It is one of the most frequently used microorganisms in industrial biotechnology to produce chemicals related to incomplete oxidation. However, the fine-tuning of G. oxydans is hampered by the lack of efficient genetic tools to enable sophisticated metabolic manipulations. Thus, a series of shuttle vectors for G. oxydans inspired by a series of wild-type plasmids in different G. oxydans strains were constructed. Fifteen shuttle vectors were employed to express mCherry in G. oxydans WSH-003 using the replication origin of these wild-type plasmids. Among them, the intensity of fluorescent proteins expressed by p15-K-mCherry was about 10 times that of fluorescent proteins expressed by p5-K-mCherry. Quantitative real-time polymerase chain reaction showed that the relative copy number of p15-K-mCherry reached 19 and had high stability. In contrast, some of the plasmids had a relative copy number of less than 10. The co-expression of multiple shuttle vectors revealed five shuttle vectors that could be transformed into G. oxydans WSH-003 and could express five different fluorescent proteins. The shuttle vectors will facilitate genetic operations for Gluconobacter strains to produce useful compounds more efficiently.

Proteins from food-grade expression systems can be used in food products and medical applications. Herein, we describe a one-step method of constructing an expression vector in Kluyveromyces lactis by combining a URA3-deficient strain and a plasmid vector with no drug-resistant selection. Adjacent DNA elements of the vector were assembled in a targeted manner through a reaction with a special recombinase to form a plasmid vector using a one-step reaction. The unnecessary fragments containing the pUC origin and the ampicillin resistance gene were removed, and the vector was isolated and purified before transformation. A single transformation of the vector can produce a URA3-deficient strain. PCR assay, sequencing, and western blot analysis all indicated that the method of vector construction and target protein expression (mCherry and human serum albumin) were successful. This method may potentially be applied to any species containing the URA3 gene; this system has the potential to become a safe and powerful tool for promoting protein expression in food-safe species.

IncI1 plasmids are known disseminators of the extended-spectrum cephalosporin resistance (ESC) gene bla_CTX-M-1, among species of the Enterobacteriaceae family. In several IncI1 plasmids, this gene was found incorporated into the transposition unit, ISEcp1-bla_CTX-M-1-orf477, interrupting a shufflon region, a hallmark of IncI1 conjugative plasmids. The shufflon diversifies pilV gene that encodes the adhesine-type protein found on the tip of the conjugative pilus. To further elucidate the shufflon rearrangement, we examined to what extent the shufflon rearrangement was affected by the transposition-unit insertion. As expected, the interrupted shufflons generated a lower number of shufflon variants and exhibited an altered segment-deletion pattern compared to the non-interrupted shufflon. Interestingly, segment-loss frequency of the interrupted shufflons was distinctive in different plasmid hosts. Finally, the analysis of the 3′ end of the pilV gene revealed that shufflon rearrangement favoured segment A to complete pilV partial open reading frame regardless of the shufflon. Thereby, it could be assumed that the A-segment has greater importance during conjugation, however, this remained a hypothesis. Further exploration of shufflon rearrangement and its importance in the plasmid-recipient selection during conjugation would be beneficial as the knowledge could be applied in developing a strategy to limit IncI1 mediated antimicrobial resistance dissemination.

Francisella tularensis is a Gram-negative intracellular pathogen causing tularemia. A number of its potential virulence factors have been identified, but their biology and functions are not precisely known. Understanding the biological and immunological functions of these proteins requires adequate genetic tools for homologous and heterologous expression of cloned genes, maintaining both original structure and post-translational modifications. Here, we report the construction of a new multipurpose shuttle plasmid – pEVbr – which can be used for high-level expression in F. tularensis. The pEVbr plasmid has been constructed by modifying the TetR-regulated expression vector pEDL17 (LoVullo, 2012) that includes (i) a strong F. tularensis bfr promoter, and (ii) two tet operator sequences cloned into the promoter. The cloned green fluorescent protein (GFP), used as a reporter, demonstrated almost undetectable basal expression level under uninduced conditions and a highly dynamic dose-dependent response to the inducer. The utility of the system was further confirmed by cloning the gapA and FTT_1676 genes into the pEVbr vector and quantifying proteins expression in F. tularensis LVS, as well as by studying post-translational modification of the cloned genes. This study demonstrates that high levels of recombinant native-like Francisella proteins can be produced in Francisella cells. Hence, this system may be beneficial for the analysis of protein function and the development of new treatments and vaccines.

There are currently 47 characterized species in the Naegleria genus of free-living amoebae. Each amoeba has thousands of extrachromosomal elements that are closed circular structures comprised of a single ribosomal DNA (rDNA) copy and a large non-rDNA sequence. Despite the presence of putative open reading frames and introns, ribosomal RNA is the only established transcript. A single origin of DNA replication (ori) has been mapped within the non-rDNA sequence for one species (N. gruberi), a finding that strongly indicates that these episomes replicate independently of the cell's chromosomal DNA component. This article reviews that which has been published about these interesting DNA elements and by analyzing available sequence data, discusses the possibility that different phylogenetically related clusters of Naegleria species individually conserve ori structures and suggests where the rRNA promoter and termination sites may be located.

Mobile genetic elements (MGEs) are instrumental in natural prokaryotic genome editing, permitting genome plasticity and allowing microbes to accumulate genetic diversity. MGEs serve as a vast communal gene pool and include DNA elements such as plasmids and bacteriophages (phages) among others. These mobile DNA elements represent a human health risk as they can introduce new traits, such as antibiotic resistance or virulence, to a bacterial strain. Sequencing libraries targeting environmental circular MGEs, referred to as metamobilomes, may broaden our current understanding of the mechanisms behind the mobility, prevalence and content of these elements. However, metamobilomics is affected by a severe bias towards small circular elements, introduced by multiple displacement amplification (MDA). MDA is typically used to overcome limiting DNA quantities after the removal of non-circular DNA during library preparations. By examining the relationship between sequencing coverage and the size of circular MGEs in paired metamobilome datasets with and without MDA, we show that larger circular elements are lost when using MDA. This study is the first to systematically demonstrate that MDA is detrimental to detecting larger-sized plasmids if small plasmids are present. It is also the first to show that MDA can be omitted when using enzyme-based DNA fragmentation and PCR in library preparation kits such as Nextera XT® from Illumina.

The fission yeast, Schizosaccharomyces pombe, is an excellent model for basic research but is not useful for commercial scale protein expression due to lack of strong expression vectors. Earlier, we showed that the lsd90 promoter elicited significantly greater GFP expression level than the adh1 and nmt1 promoters, albeit in different vector backbones. Here, we have systematically investigated the contribution of selectable markers, LEU2 and URA3m to GFP expression: while LEU2 elicited very low expression, the URA3m gene, with truncated promoter, elicited much greater GFP expression level with all promoters. Paradoxically, an inverse correlation was observed between the GFP transcription and translation efficiency. This system can be useful for understanding the factors governing recombinant gene expression and optimization of protein production.