Antibiotic resistance represents a significant public health problem. When resistance genes are mobile, being carried on plasmids or phages, their spread can be greatly accelerated. Plasmids in particular have been implicated in the spread of antibiotic resistance genes. However, the selective pressures which favour plasmid-carried resistance genes have not been fully established. Here we address this issue with mathematical models of plasmid dynamics in response to different antibiotic treatment regimes. We show that transmission of plasmids is a key factor influencing plasmid-borne antibiotic resistance, but the dosage and interval between treatments is also important. Our results also hold when plasmids carrying the resistance gene are in competition with other plasmids that do not carry the resistance gene. By altering the interval between antibiotic treatments, and the dosage of antibiotic, we show that different treatment regimes can select for either plasmid-carried, or chromosome-carried,
Individual bacterial cells may contain several different types of plasmids and in some cases more than 10 at a time. Plasmids are generally isolated from the bacterial cells in the supercoiled configuration. So far, thousands of different types of plasmids have been isolated. More than 300 different types of naturally occurring plasmids have been isolated from E.coli alone. Though, plasmids are not considered as part of the cells genome, when a bacterial cell divides each daughter cells receives a copy of each plasmid. Plasmids can also be transferred from one bacterial cell to another by the process called conjugation. Plasmids that govern their own transfer by conjugation are called conjugative plasmids but not all plasmids are conjugative.. ...
Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum beta-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2A1B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25bH4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2A1B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence
Götting C, Thierbach G, Pühler A, Kalinowski J. Versatile low-copy-number plasmids for temperature-inducible overexpression of bacterial genes in Escherichia coli. BIOTECHNIQUES. 1998;24(3):362 ...
The prevalence of 20-30 kb plasmids, almost half of which belong to only three restriction types by RFLP analysis, in staphylococci isolated from sources very distant in time and space suggests that these nonconjugative plasmids are surprisingly widespread for non-self-mobile plasmids. Plasmids in this size range can potentially be transferred by transducing phages (Lindsay and Holden 2006; Malachowa and Deleo 2010; Smillie et al. 2010); most phage genomes identified in staphylococci are ,40 kb, and transduction is thought to be restricted by phage genome size (Smillie et al. 2010). More of these 20-30 kb plasmids may be mobilizable than is apparent with current genome data if they contain mob genes not yet identified as such. However, the scarcity of conjugative plasmids (only 12 in total) implies that mobilization is rare and that staphylococcal plasmid transfer occurs mainly by transduction (Lindsay and Holden 2004; Lindsay 2010). The now larger dataset makes the mechanism of intercellular ...
The presence of the amplified product PR is oraut by electrophoresis in 1% agarose gel.. Example 2. Construction of recombinant plasmid DNA pFastBac-G2R-IgG.. 5-10 μg of plasmid pFastBac [28] hydrolyzing the restriction endonucleases BamHI and HindIII; 1-5 μg of the amplified product corresponding gene G2R UPE without termination codon, hydrolyzing the restriction endonucleases BamHI and XbaI; the plasmid pBluescript-IgG1 hydrolyzing the restriction endonucleases XbaI and HindIII in standard conditions. The resulting fragments are electrophoresis in 1% agarose gel, followed by elution. 0.2 μg of the vector and 0.6 µg of fragments are ligated under standard conditions and received ligase mixture transform competent cells of E. coli strain XL-blue. Cellular Apr-clones grown at 37°C in LB medium containing 100 μg/ml ampicillin, until the stationary phase. Recombinant plasmid DNA secrete according to standard methods and analyzed by restriction endonucleases BamHI, XbaI and HindIII. Clones ...
Although IncP-1 plasmids are important for horizontal gene transfer among bacteria, in particular antibiotic resistance spread, so far only three plasmids from the subgroup IncP-1 alpha have been completely sequenced. In this study we doubled this number. The three IncP-1 alpha plasmids pB5, pB11 and pSP21 were isolated from bacteria of two different sewage treatment plants and sequenced by a combination of next-generation and capillary sequencing technologies. A comparative analysis including the previously analysed IncP-1 alpha plasmids RK2, pTB11 and pBS228 revealed a highly conserved plasmid backbone (at least 99.9% DNA sequence identity) comprising 54 core genes. The accessory elements of the plasmid pB5 constitute a class 1 integron interrupting the parC gene and an IS6100 copy inserted into the integron. In addition, the tetracycline resistance genes tetAR and the ISTB11-like element are located between the klc operon and the trfA-ssb operon. Plasmid pB11 is loaded with a Tn5053-like ...
OBJECTIVES: To investigate the diversity of plasmids that carry blaTEM-52 genes among Escherichia coli and Salmonella enterica originating from animals, meat products and humans. METHODS: A collection of 22 blaTEM-52-encoding plasmids was characterized by restriction fragment length polymorphism (RFLP), replicon typing (by PCR or replicon sequencing), susceptibility testing, assessment of plasmid ability to self-transfer by conjugation and typing of the genetic environment of the blaTEM-52 gene. Detected IncI1 plasmids underwent further plasmid multilocus sequence typing. RESULTS: RFLP profiles demonstrated dissemination of blaTEM-52 in Denmark (imported meat from Germany), France, Belgium and the Netherlands from 2000 to 2006 by mainly two different plasmids, one encoding blaTEM-52b (IncX1A, 45 kb) and the other blaTEM-52c (IncI1, 80 kb). In addition, blaTEM-52b was also found to be located on various other plasmids belonging to IncA/C and IncL/M, while blaTEM-52c was found on IncN-like as well ...
In this semirural community, we found that numerically dominant commensal E. coli strains (showing similar antimicrobial resistance and same antibiotic resistance genes) colonizing children and domestic animals in the same period of time and in the same community are genotypically diverse. We also found that plasmids carrying the same antibiotic resistance genes were distinct, which is consistent with recent reports showing that AMR genes move frequently among different plasmids (28, 29). Our research suggests that a common pool of AMR genes could be cocirculating on different plasmids among different E. coli clones in a community (Table 2)-probably through dissemination mediated by transposons, integrons, or gene cassettes (28, 30). Even when the same resistance gene alleles and same plasmid replicon types were identified across isolates, the plasmids harboring these traits were still distinct. We also found potential evidence of Tn2 participation in mobility of the gene blaTEM-1B, as we found ...
Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale
Plasmids are important members of the bacterial mobile gene pool, and are among the most important contributors to horizontal gene transfer between bacteria. They typically harbour a wide spectrum of host beneficial traits, such as antibiotic resistance, inserted into their backbones. Although these inserted elements have drawn considerable interest, evolutionary information about the plasmid backbones, which encode plasmid related traits, is sparse. Here we analyse 25 complete backbone genomes from the broad-host-range IncP-1 plasmid family. Phylogenetic analysis reveals seven clades, in which two plasmids that we isolated from a marine biofilm represent a novel clade. We also found that homologous recombination is a prominent feature of the plasmid backbone evolution. Analysis of genomic signatures indicates that the plasmids have adapted to different host bacterial species. Globally circulating IncP-1 plasmids hence contain mosaic structures of segments derived from several parental plasmids that
A plasmid partition system is a mechanism that assures the stable transmission of plasmids during bacterial cell division. Each plasmid has its independent replication system which controls the number of copies of the plasmid in a cell. The higher the copy number is, the more likely the two daughter cells will contain the plasmid. Generally, each molecule of plasmid diffuses randomly, so the probability of having a plasmid-less daughter cell is 21−N, where N is the number of copies. For instance, if there are 2 copies of a plasmid in a cell, there is a 50% chance of having one plasmid-less daughter cell. However, high-copy number plasmids have a cost for the hosting cell. This metabolic burden is lower for low-copy plasmids, but those have a higher probability of plasmid loss after a few generations. To control vertical transmission of plasmids, in addition to controlled-replication systems, bacterial plasmids use different maintenance strategies, such as multimer resolution systems, ...
Plasmid-mediated resistance is the transfer of antibiotic resistance genes which are carried on plasmids. The plasmids can be transferred between bacteria within the same species or between different species via conjugation. Plasmids often carry multiple antibiotic resistance genes, contributing to the spread of multidrug-resistance (MDR). Antibiotic resistance mediated by MDR plasmids severely limits the treatment options for the infections caused by Gram-negative bacteria, especially Enterobacteriaceae family. The global spread of MDR plasmids has been enhanced by selective pressure from antibiotic usage in human and veterinary medicine. Resistance plasmids by definition carry one or more antibiotic resistance genes. They are frequently accompanied by the genes encoding virulence determinants, specific enzymes or resistance to toxic heavy metals. Multiple resistance genes are commonly arranged in the resistance cassettes. The antibiotic resistance genes found on the plasmids confer resistance ...
As I understand it, plasmids, like mitochondria, have their own genetic material and are capable of self-replication. According to Wikipedia: Plasmids are considered replicons, units of DNA capable of replicating autonomously within a suitable host. However, like viruses, they are not classified as life. Plasmids are transmitted from one bacterium to another through conjugation. Unlike viruses, plasmids are naked DNA. However, some classes of plasmids encode the conjugative sex pilus necessary for their own transfer. My understanding of that is that a bacteria gets their plasmids not because of the replication of their circular chromosome, nor because that chromosome have genes to code for the plasmid (I dont really know if thats possible), but because of the self-replication of their own plasmids. So, my question is how the first plasmid got into the first bacteria, if they are not in their chromosomes? Were they a virus other prokaryotic cell that had circular DNA, and got phagocytosed ...
Fragments ofCandida boidinii chromosomal DNA were inserted into the integrative vector YIp-kanr and examined for the presence of sequences promoting autonomous replication of plasmids inSaccharomyces cerevisiae. Restriction maps of two plasmids, designated S6/4 and S6/5, originating from the sameS. cerevisiae transformant, were constructed. Southern hybridization data confirmed that the plasmids carry sequences from theC. boidinii chromosome. Both plasmids transformS. cerevisiae strains at 4-5-fold higher frequency than cloning vectors based on the replication origin of the 2μm plasmid. Mitotic stability of the constructed plasmids is similar to that of the 2μ-based vector pNF2 inS. cerevisiae.
To study thermal adaptations in the cyanobacterium, Synechococcus sp. PCC 7002, we screened about 3,000 mutants for their tolerance to high temperature, and found one, SHT1, that is sensitive to high-temperature stress. The mutant had a modified gene construct in the endogenous plasmid, pAQ1. One of the four ORFs, ORF93, was duplicated, and its mRNA level was higher than in the wild type. At 38°C, the growth of SHT1 was retarded as compared with the wild type, and above 38°C, almost all the cells of SHT1 died. This temperature is much lower than that required for induction of heat shock proteins. Interestingly, in both the wild type and SHT1, the thermal stability of oxygen-evolving machinery increased upon acclimation to high temperatures. These findings indicate that the lack of thermal tolerance in the SHT1 strain is likely independent of the adaptation of the PSII complex and heat shock responses, whereas there are essential contributions of genes in the endogenous plasmid to the ...
SUMMARY: Phylogenetic and epidemiological relatedness among transferable plasmids belonging to the IncC, IncM and IncH incompatibility groups has been studied by DNA-DNA filter hybridization. Hybridization was carried out on nitrocellulose microfilters, at low temperature, in formamide and under paraffin oil. The degree of hybridization among plasmids belonging to the IncC and IncM groups supported the conclusions drawn from genetic classification. Studies on relatedness among plasmids belonging to the IncH group allowed their classification into three phylogenetic sub-groups. Comparison of DNA sequences of three plasmids sharing the same genetic properties and isolated from different bacterial species suggested an epidemiological spread of the same plasmid.
Recreate original plasmid by cutting out insert - posted in Molecular Cloning: Maybe this is a stupid question, but I am going to ask it anyway: I would like to cut out an insert from my plasmid (pET28c) between restriction sites Nhe1 and Not1 and ligate the plasmid back to its orginial state without the insert (no I dont have the original plasmid anymore). However, obviously these restriction sites dont generate compatible sticky ends. Does anyone know how to recreate this...
Objectives: To investigate the genetic features of three plasmids recovered from an MCR-1 and ESBL-producing Escherichia coli strain, HYEC7, and characterize the transmission mechanism of mcr-1. Methods: The genetic profiles of three plasmids were determined by PCR, S1-PFGE, Southern hybridization and WGS analysis. The ability of the mcr-1-bearing plasmid to undergo conjugation was also assessed. The mcr-1-bearing transposon Tn6330 was characterized by PCR and DNA sequencing. Results: Complete sequences of three plasmids were obtained. A non-conjugative phage P7-like plasmid, pHYEC7-mcr1, was found to harbour the mcr-1-bearing transposon Tn6330, which could be excised from the plasmid by generating a circular intermediate harbouring mcr-1 and the ISApl1 element. The insertion of the circular intermediate into another plasmid, pHYEC7-IncHI2, could form pHNSHP45-2, the original IncHI2-type mcr-1-carrying plasmid that was reported. The third plasmid, pHYEC7-110, harboured two replicons, IncX1 and ...
Despite the near-ubiquity of plasmids in bacterial populations and the profound contribution of plasmid-borne genes and infectious gene transfer to the adaptation and evolution of bacteria, the mechanisms responsible for the maintenance of plasmids in bacterial populations are poorly understood. In this report, we address the question of how plasmids manage to persist over evolutionary time. Previous explanations have typically relied upon the ability of plasmids to deliver occasionally-useful genes to the right place at the right time. In contrast, we present a general mathematical proof that if (as suggested by several empirical studies) plasmids are not infectiously transmitted at a rate high enough to be maintained as genetic parasites in single populations, they will not be able to persist indefinately in these populations by carrying genes that are beneficial or sometimes beneficial to their host bacteria. Using more specific mathematical models, along with computer simulations, we ...
uncut plasmid dna vs linearized plasmid gel - posted in Molecular Cloning: Hello, I am going to run a gel comparing my uncut plasmid dna vs linearized plasmid. My insert is 135 bps and my vector is 3Kb. What can I expect to see on my gel, and how many bands can I expect to see. I am assuming the uncut plasmid will have several bands at different sizes and the linearized will have only one, is that right ? I am assuming the total size of my product will now be 3135 bps. Pls advise.
Figure 20. The pBR322 plasmid. Two genes of pBR322 confer resistance to antibiotics to any cell that contains the plasmid. AmpR confers resistance to amplicillin and tetR confers resistance to tetracycline to cells containing the plasmids. AmpR is a gene that codes for the periplasmic enzyme beta-lactamase that cleaves the ring structure found in amphicillin, which is a penicillin antibiotic. TetR is a gene that codes for a protein that modifies the bacterial cell wall and prevents tetracycline from entering the cell.. Multiple restriction endonuclease sites are present where foreign DNA fragments may be inserted.. Relaxed plasmid DNA replication continues in the presence of chloramphenicol. An interesting feature of this plasmid is that relaxed plasmid DNA replication continues even in the presence of an inhibitor of protein synthesis such as chloramphenicol. This feature allows increased yields of plasmid/cell of up to 100-fold ...
One of the disadvantages of circular plasmids and chromosomes is their high sensitivity to rearrangements caused by homologous recombination. Odd numbers of crossing-over occurring during or after replication of a circular replicon result in the formation of a dimeric molecule in which the two copies of the replicon are fused. If they are not converted back to monomers, the dimers of replicons may fail to correctly segregate at the time of cell division. Resolution of multimeric forms of circular plasmids and chromosomes is mediated by site-specific recombination, and the enzymes that catalyze this type of reaction fall into two families of proteins: the serine and tyrosine recombinase families. Here we give an overview of the variety of site-specific resolution systems found on circular plasmids and chromosomes.
Santa Cruz Biotechnology now offers target-specific CRISPR/Cas9 Knockout (KO) Plasmids, CRISPR Double Nickase Plasmids and CRISPR/dCas9 Activation Plasmids for over 18,910 human and 18,340 mouse protein encoding genes. CRISPR/Cas9 KO Plasmids and CRISPR Double Nickase Plasmids enable the identification and cleavage of specific genes encoding a protein of interest thereby eliminating production of that gene product (protein). CRISPR/dCas9 Activation Plasmids activate endogenous gene transcription using a robust synergistic activation mediator (SAM) system. This exciting new technology is a useful tool for studying protein function and signalling pathways ...
Studying the transfer of specific mobile genetic elements in complex environmental matrices remains difficult because suitable molecular tools are not yet available to back up classical culture-dependent approaches. In this report, we show that quantitative PCR could be used to monitor the dissemination of the broad-host-range plasmid pB10 in sediment microcosms. This approach lies in the differential measurement of the host and plasmid DNAs used to inoculate the microcosms, using a particular design of quantitative PCR primers/probes where we took advantage of the mosaic aspect of the bacterial genomes to achieve a highly specific quantitative PCR detection system.
The segregational stability of bacterial, low-copy-number plasmids is promoted primarily by active partition. The plasmid-specified components of the prototypical P1 plasmid partition system consist of two proteins, ParA (44.3 kDa) and ParB (38.5 kDa), which, in conjunction with integration host fac …
In spite of the importance of plasmids in bacterial adaptation we have a poor understanding of their dynamics. It is not known if or how plasmids persist in and spread through (invade) a bacterial population when there is no selection for plasmid-encoded traits. Moreover, the differences in dynamics between spatially structured and mixed populations are poorly understood. Through a joint experimental/theoretical approach we tested the hypothesis that self-transmissible IncP-1 plasmids can invade a bacterial population in the absence of selection when initially very rare, but only in spatially structured habitats and when nutrients are regularly replenished. Using protocols that differed in degree of spatial structure and nutrient levels, the invasiveness of plasmid pB10 in E. coli was monitored during at least 15 days, with an initial fraction of plasmid-bearing (p+) cells as low as 1E-7. To further explore the mechanisms underlying plasmid dynamics we developed a spatially explicit mathematical ...
Mono- and Stereopictres of 5.0 Angstrom coordination sphere of Manganese atom in PDB 3dkx: Crystal Structure of the Replication Initiator Protein Encoded on Plasmid PMV158 (Repb), Trigonal Form, to 2.7 Ang Resolution
Difficult to Express Proteins: Novel Plasmid Technology to Significantly Increase Product Yield in CHO Cells, by Marco Cacciutolo
Mcr-1-harboring Enterobacteriaceae are reported worldwide since their first discovery in 2015. However, a limited number of studies are available that compared full-length plasmid sequences of human and animal origins. In this study, mcr-1-bearing plasmids from seven Escherichia coli isolates recovered from patients (n = 3), poultry meat (n = 2) and turkey meat (n = 2) in Switzerland were further analyzed and compared. Isolates were characterized by multilocus sequence typing (MLST). The mcr-1-bearing plasmids were transferred by transformation into reference strain E. coli DH5α and MCR-1-producing transformants were selected on LB-agar supplemented with 2 mg/L colistin. Purified plasmids were then sequenced and compared. MLST revealed six distinct STs, illustrating the high clonal diversity among mcr-1-positive E. coli isolates of different origins. Two different mcr-1-positive plasmids were identified from a single E. coli ST48 human isolate. All other isolates possessed a
The reason that we stock plasmids that only contain one terminator is because sometimes, despite the increased versatility, researchers may not want unnecessary terminators in their plasmids, for example when size constraints are an issue. The button below takes you to the plasmids with single terminators. If you prefer to use a plasmid with triple terminator, we suggest you search using the Plasmid Seach tool since nearly all our plasmids have triple terminators. The Plasmid Search tool will give you access to our full plasmid catalogue.. Finally, please try the Design Your Own Plasmid Online button below, to see what our cloning system can do for you. And remember that, while our plasmids are designed for easy cloning modifications, we are happy to do it for you if you prefer to outsource the cloning work.. ...
Prokaryotic transcriptomes change not only in response to physiological parameters but also to genetic rearrangements mediated by mobile elements. Plasmids are extrachromosomal genetic elements that replicate autonomously, and many can be transmitted between different strains through conjugation. Plasmids provide benefits to their hosts, such as resistance to antibiotics or degradation of recalcitrant aromatic compounds [1]; however, in several cases, the carriage of a large plasmid results in changes in the transcriptome of the host chromosome [2-4]. Similar to the effects of plasmid carriage on the transcriptional network of the host chromosome, differences in host background can alter the transcription patterns of backbone and accessory genes on a plasmid. Many plasmid backbone genes essential for conjugative transfer, replication initiation, and active partitioning are regulated both autogenously and by host factors [5]. Additionally, a number of plasmid-encoded degradative accessory genes ...
Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel
ID YEP367 preliminary; circular DNA; SYN; 8400 BP. XX AC ATCC37735; XX DT 01-JUL-1993 (Rel. 7, Created) DT 01-JUL-1995 (Rel. 12, Last updated, Version 1) XX DE Saccharomyces/E.coli plasmid vector YEp367 - incomplete. XX KW cloning vector. XX OS Cloning vector OC Artificial sequences; Cloning vehicles. XX RN [1] RC YEp352E from YEp352 & linker RC YEp363A from pNM480 & YEp351 RC YEp353A from pNM480 & YEp352 RC YEp353 from YEp353A & YEp352E RC YEp354A from pNM481 & YEp352 RC YEp354 from YEp354A & YEp352E RC YEp355A from pNM482 & YEp352 RC YEp355 from YEp355A & YEp352E RC YEp356, YEp356R from YEp353 & pUC18 RC YEp357, YEp357R from YEp354 & pUC18 RC YEp358, YEp358R from YEp355 & pUC18 RC YEp363 from YEp363A & YEp353 RC YEp364 from YEp363A & YEp354 RC YEp365 from YEp363A & YEp355 RC YEp366 from YEp363A & YEp356 RC YEp367 from YEp363A & YEp357 RC YEp368 from YEp363A & YEp358 RC YEp366R from YEp363A & YEp356R RC YEp367R from YEp363A & YEp357R RC YEp368R from YEp363A & YEp358R RC YIp353 from YEp353 & ...
Background Prokaryotic plasmids have played out significant roles in the evolution of bacterial genomes and have a great impact on the metabolic functions of the host cell. variable genes (distributed genes and unique genes) than to the chromosomal core genes. Although all the functional categories of the chromosomal genes were exhibited by the plasmid genes, the proportions of each category differed between these two gene sets. The 598 gene families shared between chromosomes and plasmids displayed a uniform distribution between the two groups. A phylogenetic analysis of the shared genes, including the chromosomal core gene set, indicated that gene exchange events between plasmids and chromosomes occurred frequently during the evolutionary histories of the strains and species in this group. Moreover, the shared genes between plasmids and chromosomes usually had different promoter and terminator sequences, suggesting that they are regulated by different elements at the transcriptional level. ...
www.MOLUNA.de Plasmids in Bacteria [4191995] - Structure and Evolution.- Plasmids as Organisms.- Report on a Workshop: Structure and Function.- Evolutionary Relevance of Genetic Rearrangements Involving Plasmids.- Mechanisms of Transposition in Bacteria.- Insertion of Transcriptional Elements Outside the Replication Region Can Interfere with Replication, Maintenance, and Stability of ColE1-Derived Plasmids.- Studies on the Transposition of IS1.- On
ID YEP353 preliminary; circular DNA; SYN; 7944 BP. XX AC U03500; ATCC37725; XX DT 01-JUL-1993 (Rel. 7, Created) DT 01-JUL-1995 (Rel. 12, Last updated, Version 1) XX DE Saccharomyces/E.coli plasmid vector YEp353 - complete. XX KW cloning vector. XX OS Cloning vector OC Artificial sequences; Cloning vehicles. XX RN [1] RP 1-7944 RC YEp352E from YEp352 & linker RC YEp363A from pNM480 & YEp351 RC YEp353A from pNM480 & YEp352 RC YEp353 from YEp353A & YEp352E RC YEp354A from pNM481 & YEp352 RC YEp354 from YEp354A & YEp352E RC YEp355A from pNM482 & YEp352 RC YEp355 from YEp355A & YEp352E RC YEp356, YEp356R from YEp353 & pUC18 RC YEp357, YEp357R from YEp354 & pUC18 RC YEp358, YEp358R from YEp355 & pUC18 RC YEp363 from YEp363A & YEp353 RC YEp364 from YEp363A & YEp354 RC YEp365 from YEp363A & YEp355 RC YEp366 from YEp363A & YEp356 RC YEp367 from YEp363A & YEp357 RC YEp368 from YEp363A & YEp358 RC YEp366R from YEp363A & YEp356R RC YEp367R from YEp363A & YEp357R RC YEp368R from YEp363A & YEp358R RC YIp353 ...
DNA plasmids. Coloured atomic force micrograph of numerous pGL3 plasmids of DNA (deoxyribonucleic acid). A plasmid is a loop of DNA that can exist in a cell independently of the cells chromosomal DNA. Plasmids are important as they can replicate and express genes despite being outside the organisms chromosomes. This means that they can be used to introduce genes to organisms, a process used in gene therapy and genetic engineering. Genes, specific lengths of DNA, determine the development and characteristics of an organism. Atomic force microscopes make an image by moving a sensitive probe over a surface. Magnification: x24,000 at 6x7cm size. - Stock Image G110/0752
The present results concern the recombinant bacteria Escherichia coliHB101(GAPDH) which produces glyceraldehyde 3-phosphate dehydrogenase. An unusual phenomenon was noticed concerning the plasmid...
Many bacteria carry extra DNA molecules beyond their chromosome, so-called plasmids. While plasmids are apriori costly to the cell, they ...
The combination of a p15A and a colE1 origin of replication plasmid is the most common two-plasmid system for use in E. coli. The pSB1A* series of plasmids contain the colE1 plasmid and cannot be co-maintained at stable copy numbers in combination due to plasmid incompatibility. Here, two plasmids in the same cell with origins from the same incompatibility group compete for replication machinery, resulting in unpredictable copy number and often the exclusion of one of the plasmids. This is circumvented by cotransforming plasmids with origins from distinct incompatibility groups. Since no p15A origin plasmid was present in the Registry, we developed part J23001, or pSB3C6 for our studies. This plasmid was used for all our riboregulator experiments for the locked-RFP reporter. Shown here is the variant of the plasmid containing part J01022. Plasmids for constructing basic Biobrick parts ...
TY - JOUR. T1 - Comparative analysis of conjugative plasmids mediating gentamicin resistance in Staphylococcus aureus. AU - Goering, R. V.. AU - Ruff, E. A.. PY - 1983/1/1. Y1 - 1983/1/1. N2 - Five gentamicin-resistant clinical isolates of Staphylococcus aureus were found to contain self-transmissible plasmids of 32 to 37 megadaltons in size. Restriction endonuclease digests of the plasmids were markedly similar to those of reference plasmids of unrelated geographical origin, thus suggesting the significant contribution of common conjugal plasmids to the emergence of gentamicin resistance in S. aureus populations.. AB - Five gentamicin-resistant clinical isolates of Staphylococcus aureus were found to contain self-transmissible plasmids of 32 to 37 megadaltons in size. Restriction endonuclease digests of the plasmids were markedly similar to those of reference plasmids of unrelated geographical origin, thus suggesting the significant contribution of common conjugal plasmids to the emergence of ...
Objectives: To investigate the genetic features of five plasmids recovered from an NDM-5-producing clinical Escherichia coli strain, BJ114, and to characterize the plasmid recombination event that occurred during the conjugation process. Methods: The genetic profiles of the five plasmids were determined by PCR, conjugation, S1-PFGE, Southern hybridization and WGS analysis. Plasmid sequences were analysed with various bioinformatic tools. Results: Complete sequences of five plasmids were obtained. Two small plasmids, pBJ114-141 and pBJ114-46, were speculated to have recombined into a large fusion plasmid, pBJ114T-190. When conjugated to other E. coli strains, some of the fusion plasmids were able to be resolved into the original two single plasmids. A nonconjugative plasmid, pBJ114-96, exhibited a high degree of sequence identity with the phage P7-like plasmid as well as an mcr-1-bearing plasmid. Another plasmid, pBJ114-78, was found to contain multidrug resistance genes and various mobile ...
© 2018 Macmillan Publishers Ltd., part of Springer Nature. Plasmids have a major role in the development of disease caused by enteric bacterial pathogens. Virulence plasmids are usually large (|40 kb) low copy elements and encode genes that promote host-pathogen interactions. Although virulence plasmids provide advantages to bacteria in specific conditions, they often impose fitness costs on their host. In this Review, we discuss virulence plasmids in Enterobacteriaceae that are important causes of diarrhoea in humans, Shigella spp., Salmonella spp., Yersinia spp and pathovars of Escherichia coli. We contrast these plasmids with those that are routinely used in the laboratory and outline the mechanisms by which virulence plasmids are maintained in bacterial populations. We highlight examples of virulence plasmids that encode multiple mechanisms for their maintenance (for example, toxin-antitoxin and partitioning systems) and speculate on how these might contribute to their propagation and success.
Biotechnical production processes often operate with plasmid-based expression systems in well-established prokaryotic and eukaryotic hosts such as Escherichia coli or Saccharomyces cerevisiae, respectively. Genetically engineered organisms produce important chemicals, biopolymers, biofuels and high-value proteins like insulin. In those bioprocesses plasmids in recombinant hosts have an essential impact on productivity. Plasmid-free cells lead to losses in the entire product recovery and decrease the profitability of the whole process. Use of antibiotics in industrial fermentations is not an applicable option to maintain plasmid stability. Especially in pharmaceutical or GMP-based fermentation processes, deployed antibiotics must be inactivated and removed. Several plasmid addiction systems (PAS) were described in the literature. However, not every system has reached a full applicable state. This review compares most known addiction systems and is focusing on biotechnical applications.. ...
TY - JOUR. T1 - Restriction endonuclease analysis of Staphylococcus aureus plasmid DNA from three continents. AU - Doebbeling, Bradley. AU - Pfaller, M. A.. AU - Hollis, R. J.. AU - Boyken, L. D.. AU - Pignatari, A. C.. AU - Herwaldt, L. A.. AU - Wenzel, R. P.. PY - 1992/1. Y1 - 1992/1. N2 - Staphylococcus aureus isolates (n=1201) from 20 centers in Europe, the USA and Brazil were evaluated for the presence of epidemiologic markers. Plasmid typing and restriction endonuclease analysis of plasmid DNA confirmed the presence of an apparently identical plasmid in 13 % of clinical isolates. The plasmid was recovered from all 20 hospitals studied, with an overall frequency of ,10 % on each of the three continents. Since relatively few staphylococcal plasmids may be shared by epidemiologically unrelated strains, there are inherent limitations to this otherwise useful technique. Additionally, these data demonstrate the importance of including unrelated strains of Staphylococcus aureus from the local ...
To further characterize the fosB-carrying plasmids of 19 vancomycin-resistant enterococci, the complete sequences of the fosB- and vanA-containing plasmids of E. faecium (pEMA120) and E. avium (pEA19081) were obtained by single-molecule, real-time sequencing. We found that these two plasmids are essentially identical (99.99% nucleotide sequence identity), which proved the possibility of interspecies transmission. Comparative analysis of the plasmids revealed that the backbone of pEMA120 is 99% similar to a conjugative fosB-negative E. faecium plasmid, pZB18. There is a traE disrupted in the transfer region of pEMA120, in comparison to pZB18 with an intact traE. The difference of their transfer frequencies between pEMA120 and pZB18 suggests this interruption of traE might affect conjugative transfer. Two copies of the fosB gene linked to a tnpA gene, forming an ISL3-like transposon, were found at separate locations within pEMA120, which had not been reported previously. These two fosB-carrying
The sequence analysis of the 7383 bp plasmid pCIZ2 from |i|Enterococcus faecium|/i| L50 enabled the identification of a DNA region involved in its replication. The structural organization of the pCIZ2 replication region is highly similar to those of well-known theta-replicating plasmids. It contains an untranslated region, the putative replication origin (|i|ori|/i|), constituted by two sets of direct repeats of 12 and 22 bp (iterons), and followed by three open-reading frames (|i|orf8|/i| to |i|orf10|/i|). |i|orf8|/i| encodes the replication initiation protein (RepE). The transcriptional start site of the replication locus was identified 13 nucleotides upstream of the |i|repE|/i| start codon. A two-dimensional agarose gel electrophoresis analysis revealed pCIZ2 intermediates profile typical of the theta-type replication mechanism. Subcloning of different DNA fragments of the pCIZ2 replication region in |i|Escherichia coli|/i| and, subsequently, in the plasmidless |i|E. faecium|/i| L50/14-2 allowed the
Gene transfer mediated by plasmids plays an important role in the spread of antibiotic resistance genes. Nowadays, high molecular weight plasmids carrying resistance genes to antibiotics and growth inhibitors are commonly found in pathogenic bacteria. Moreover, conjugative plasmids coding for antibiotic resistance may interact with virulence plasmids thus resulting in plasmid transferring both antibiotic resistance and virulence genes. In this thesis I therefore pursued genomics and proteomics studies on plasmids of two incompatibility group. In the first part of the thesis we determined the complete nucleotide sequences of multidrug resistance plasmid p9134 and its two variants, p9134dT which spontaneously lost tetracycline resistance gene, and p9134dAT which spontaneously lost ampicillin and tetracycline resistance genes. Plasmids were 133 802 bp, 127 291 bp a 109 512 bp in size, respectively, and their basic backbone was similar to that of IncI plasmids. In plasmid p9134 genes coding for ...
ii) Evolution of the blaNDM-1 locus.The mer operon of pAR060302 is part of a complex hybrid transposon inserted into the IncA/C backbone flanked by direct repeats, and related structures are found in other IncA/C plasmids. The strA-strB genes in pYR1 are part of a Tn5393 that was recently described (3, 4). The synteny among the compared plasmids restarted at the DNA primase gene (Fig. 1). IncA/C plasmid pP91278 from P. damselae likely represents the IncA/C scaffold before the acquisition of the accessory genes, since it does not show any insertion between the phage-integrase, rhs, and DNA primase genes (Fig. 1).. An analysis of the pNDM-KN sequence revealed that the rhs gene was deleted at its 3′ and 5′ extremities and was adjacent to the heat shock chaperone groEL-groES cluster (28). In pAR060302, this cluster flanked a class 1 integron that contained the aac(3)-IVa and aadA1 gene cassettes, but it was absent in plasmids pYR1 and pP91278 (Fig. 1). Analysis of the sequences located at the ...
Integrative and conjugative elements (ICEs, also known as conjugative transposons) are mobile elements that are found integrated in a host genome and can excise and transfer to recipient cells via conjugation. ICEs and conjugative plasmids are found in many bacteria and are important agents of horizontal gene transfer and microbial evolution. Conjugative elements are capable of self-transfer and also capable of mobilizing other DNA elements that are not able to self-transfer. Plasmids that can be mobilized by conjugative elements are generally thought to contain an origin of transfer (oriT), from which mobilization initiates, and to encode a mobilization protein (Mob, a relaxase) that nicks a site in oriT and covalently attaches to the DNA to be transferred. Plasmids that do not have both an oriT and a cognate mob are thought to be nonmobilizable. We found that Bacillus subtilis carrying the integrative and conjugative element ICEBs1 can transfer three different plasmids to recipient bacteria at ...
When you want sustained transgene expression without introducing any foreign DNA-such as for model animal and gene therapy development-Minicircle Technology is a great gene expression option. Produced as small excised, circular DNA fragments from a parental plasmid, the non-viral, episomal Minicircle expression cassette is free of any bacterial plasmid DNA sequences, and comes with a variety of promoter and reporter combinations. Their small size facilitates more efficient transfection than whats possible with standard-sized plasmids, and, while Minicircles do not replicate with the host cell, expression lasts for 14 days or longer in dividing cells, and can continue for months in non-dividing cells.. Product Note:. Parental minicircle plasmids and the ZYCY10P3S2T Producer Bacterial Strain are available for purchase by not-for-profit researchers only. Commercial users may purchase pre-made, ready-to-transfect minicircle DNA only. SBI also offers custom parental plasmid cloning and minicircle ...
|jats:title|ABSTRACT|/jats:title||jats:p|OXA-48-like enzymes have emerged as important extended-spectrum β-lactamases/carbapenemases in|jats:named-content xmlns:xlink=http://www.w3.org/1999/xlink content-type=genus-species xlink:type=simple|Escherichia coli|/jats:named-content|sequence type 131 (ST131). We report the structures of the first fully sequenced|jats:italic|bla|/jats:italic||jats:sub|OXA-163|/jats:sub|plasmid and of two other|jats:italic|bla|/jats:italic||jats:sub|OXA-48|/jats:sub|plasmids in this lineage.|jats:italic|bla|/jats:italic||jats:sub|OXA-163|/jats:sub|was located on a 71-kb IncN plasmid with other resistance genes.|jats:italic|bla|/jats:italic||jats:sub|OXA-48|/jats:sub|was present on IncL/M plasmids, genetically similar to other|jats:italic|bla|/jats:italic||jats:sub|OXA-48|/jats:sub|plasmid sequences, and consistent with interspecies/interlineage spread. The presence of|jats:italic|bla|/jats:italic||jats:sub|OXA-48-like|/jats:sub|genes on epidemic plasmids in ST131 is of
This chapter provides a broad overview of many applications of plasmids for genetic analysis, primarily in bacteria. Ever since DNA sequencing became accessible to most research laboratories, reverse genetic analysis has become a standard experimental approach to study bacterial gene function. Similar suicide vectors have also been used for nontargeted insertional mutagenesis by cloning random chromosomal DNA fragments into the plasmid. The use of suicide vectors also allows for easy identification of the insertion mutations. Plasmids that utilize different combinations of double-counter selective markers have been used for diverse applications, including the search for extremely rare suppressor mutations of essential Escherichia coli genes, and to improve the efficiency of allelic exchange on bacterial artificial chromosomes (BACs). Although temperature-sensitive vectors represent the majority of conditionally replicating plasmids, other plasmids that exhibit conditional replication have been described
Non-viral gene therapy is being considered as a treatment for cystic fibrosis. In clinical studies and in studies using the mouse airways as a model, current formulations result in only transient transgene expression. A number of reasons for this have been proposed including the loss of plasmid DNA from cells. The aim of these studies was to investigate why transgene expression from non-viral vectors is transient in the mouse lung. Plasmid DNA encoding the luciferase reporter gene was complexed with the cationic lipid GL67 and delivered to the mouse airways. The persistence of plasmid DNA in the mouse lungs was investigated using quantitative PCR and Southern hybridization. Results showed that intact plasmid DNA persisted in the mouse lung in the absence of any detectable luciferase activity. The de novo methylation of plasmid DNA in vivo was investigated as a potential cause of this transient gene expression but results suggested that plasmid DNA does not become de novo methylated in the mouse lung.
The conditions promoting the persistence of a plasmid carrying a trait that may be mutually beneficial to other cells in its vicinity were studied in structured and unstructured environments. A large plasmid encoding mercury resistance in Pseudomonas fluorescens was used, and the mercury concentration allowing invasion from rare for both plasmid-bearing and plasmid-free cells was determined for different initial inoculum densities in batch-culture structured (filter surface) and unstructured (mixed broth) environments. A range of mercury concentrations were found where both cell types could coexist, the regions being relatively similar in the two types of environment although density-dependent in the unstructured environment. The coexistence is explained in terms of frequency-dependent selection of the mutually beneficial mercury resistance trait, and the dynamics of bacterial growth under batch culture conditions. However, the region of coexistence was complicated by conjugation which increased plasmid
The International Society for Plasmid Biology and other Mobile Genetic Elements (ISPB) was established in 2004. The purpose of the ISPB is to promote the study of plasmids and other mobile genetic elements.. The Society will engage in collective activities that increase the awareness and understanding of the importance of these genetic elements both in the scientific community and in the public arena.. The Society will highlight important discoveries in the plasmid field as well as new and novel applications of plasmids and other mobile genetic elements, and will act as an advocate for increased research support of this field.. ISPB will also promote the importance of retaining the study of plasmids and other mobile genetic elements in undergraduate teaching curriculum.. The Society will provide a forum for the plasmid biology community to work with sequence databases to ensure the highest quality of annotation.. The constitution of ISPB can be found here.. Information about previous conferences ...
SUMMARY: A PUB 110-derived plasmid encoding chloramphenicol resistance, kanamycin resistance and high-temperature α-amylase showed a high degree of segregational instability when inserted into Bacillus subtilis. In an attempt to obtain stable derivatives, the organism was grown in chemostat culture in the presence of chloramphenicol. It was periodically found necessary to increase the concentration of chloramphenicol in the medium feed in order to avoid plasmid loss. Strains were isolated after 19 and 160 generations, which showed high levels of plasmid stability. This characteristic appeared to be genotypic. No detectable difference in plasmid copy number was found between the original and the improved strains. The stability characteristics resided in the host, rather than in the plasmid. Stable isolates possessed elevated MICs for both chloramphenicol and kanamycin. Their maximum specific growth rates were higher than that of the original strain, and similar to that of the plasmid-free parent strain.
BACKGROUND: Electroporation is an established technique for enhancing plasmid delivery to many tissues in vivo, including the skin. We have previously demonstrated efficient delivery of plasmid DNA to the skin utilizing a custom-built four-plate electrode. The experiments described here further evaluate cutaneous plasmid delivery using in vivo electroporation. Plasmid expression levels are compared to those after liposome mediated delivery. METHODS: Enhanced electrically-mediated delivery, and less extensively, liposome complexed delivery, of a plasmid encoding the reporter luciferase was tested in rodent skin. Expression kinetics and tissue damage were explored as well as testing in a second rodent model. RESULTS: Experiments confirm that electroporation alone is more effective in enhancing reporter gene expression than plasmid injection alone, plasmid conjugation with liposomes followed by injection, or than the combination of liposomes and electroporation. However, with two time courses of multiple
Extended-spectrum cephalosporins have been classified by the World Health Organization as critically important antibiotics in human medicine (19). Ceftiofur is an extended-spectrum cephalosporin licensed for the treatment of respiratory infections in pigs and cattle. Various authors have hypothesized that the veterinary use of extended-spectrum cephalosporins may select for extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae in animals, resulting in an increased risk of the zoonotic transmission of ESBL-carrying bacteria and plasmids (2, 10, 11, 17, 18).. A statistical association between the prophylactic use of ceftiofur and the occurrence of cefotaxime-resistant E. coli in healthy pigs was recently demonstrated at two Danish pig farms, and most of the isolates were shown to be CTX-M-1 producers (11). In this study, we revisited the farms 1 year after the previous study to investigate the distribution, persistence, and transmission of blaCTX-M-1 between pigs, farm workers, and ...
산타크루즈바이오테크놀러지는 광범위한 유전자편집 제품을 제공하고 있으며 유전자침묵에 쓰이는 CRISPR/Cas9 Knockout 와 CRISPR Double Nickase plasmids를 제공합니다. Ribosomal Protein L39유전자침묵에는 Ribosomal Protein L39 CRISPR/Cas9 Knockout plasmids 와 Ribosomal Protein L39 Double Nickase Plasmids를 제공합니다. 또한 유전자활성화에 쓰이는 Ribosomal Protein L39 CRISPR/dCas9 Activation Plasmids와 CRISPR Lenti Activation Systems도 제공합니다. 유전자침묵과 활성화는 유전자연구에 유용하게 쓰이며 이는 항체와 결합하여 단백질의 검출에 유용하게 쓰입니다.
산타크루즈바이오테크놀러지는 광범위한 유전자편집 제품을 제공하고 있으며 유전자침묵에 쓰이는 CRISPR/Cas9 Knockout 와 CRISPR Double Nickase plasmids를 제공합니다. Ribosomal Protein L39L유전자침묵에는 Ribosomal Protein L39L CRISPR/Cas9 Knockout plasmids 와 Ribosomal Protein L39L Double Nickase Plasmids를 제공합니다. 또한 유전자활성화에 쓰이는 Ribosomal Protein L39L CRISPR/dCas9 Activation Plasmids와 CRISPR Lenti Activation Systems도 제공합니다. 유전자침묵과 활성화는 유전자연구에 유용하게 쓰이며 이는 항체와 결합하여 단백질의 검출에 유용하게 쓰입니다.
Vol 9: Complete Nucleotide Sequence and Analysis of Two Conjugative Broad Host Range Plasmids from a Marine Microbial Biofilm.. This article is from PLoS ONE, volume 9.AbstractThe complete nucleotide sequence of plasmids pMCBF1 and pMCBF6 was deter. Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.
TM 10 6670-277 13&P APPENDIX B MAINTENANCE ALLOCATION CHART Section I. INTRODUCTION B 1. General. a. This section provides a general explanation of all maintenance and repair functions authorized at various maintenance categories. b. The Maintenance Allocation Chart (MAC) in Section II designates overall authority and responsibility for the performance of maintenance functions on the identified end item or component. The application of the maintenance functions to the end item or component will be consistent with the capacities and capabilities of the designated maintenance categories. c. Section Ill lists the tools and test equipment (both special tools and common tool sets) required for each maintenance function as referenced from Section Il. d. Section IV contains supplemental instructions and explanatory notes for a particular maintenance function. B 2. Maintenance Functions. Maintenance functions will be limited to and defined as follows: a. Inspect. To determine the serviceability of an ...
We have previously shown that the intramuscular injection of naked plasmid DNA enables foreign gene expression in muscle. Further studies showed that the intravascular delivery of naked plasmid DNA enables high levels of expression not only in muscle but also in hepatocytes. For the liver, this tech …
Bacterial circular chromosomes replicate bidirectionally by the theta mechanism. Circular plasmids replicate by three general mechanisms: theta type, strand displacement, and rolling circle (43). Different machineries are involved in different plasmid replication mechanisms (43). For example, initiation of theta-type plasmid replication generally requires a plasmid-encoded initiator protein. In contrast, initiation of strand displacement replication requires the plasmid-encoded helicase, primase, and initiator protein, and initiation of rolling-circle replication requires the plasmid-encoded protein with DNA strand transferase enzymatic activity. The absence of the above essential proteins for the latter two mechanisms indicated that Pseudoalteromonas chromids replicate by the theta mechanism. Consistent with this, no such proteins in chromids from other bacteria have been reported. Considering the different replication factors involved in different mechanisms, a parsimony assumption is that ...
Objective: Carbapenem-producing organisms have spread worldwide and cause significant morbidity. Horizontal gene transfer of carbapenemases may play a role in this spread. Given that conjugation is influenced by a number of factors, we sought to perform a systematic analysis of blaKPC encoding plasmid transfer into multiple species using hospital isolates. Methods: Plasmids were isolated from patient donor strains from the NIH and University of Virginia and a subset were tagged with GFP and electroporated into a K. pneumoniae patient isolate cured of its blaKPC plasmid. Broth and filter matings were performed, and transconjugants were isolated on selective media. Plasmids tested included those found in multiple species during hospital surveillance. Results: Transfer frequency was dependent on the recipient, temperature, substratum, and the specific plasmid. pKPC-47e was extremely attenuated in conjugation efficiency across all conditions tested compared to pKpQIL. In vitro studies showed a low ...
Interpretive Summary: A novel plasmid (pHoss1) for Listeria monocytogenes was constructed. pHoss1 is very efficient for the generation of in-frame deletion mutants. The L. monocytogenes ispG and ispH genes were in-frame mutated. The L. monocytogenes ispG and ispH genes are not involved in cell adhesion. Technical Abstract: Although several plasmids have been used in Listeria monocytogenes for generating mutants by allelic exchange, construction of L. monocytogenes mutants has been inefficient due to lack of effective selection markers for first and second recombination events. To address this problem, we have developed a new suicide plasmid, pHoss1, by using the pMAD plasmid backbone and anhydrotetracycline selection marker (secY antisense RNA) driven by an inducible Pxyl/tetO promoter. Expression of the secY antisense RNA eliminates merodiploids and selects for the loss of plasmid via a second allelic exchange, which enriches the number of mutants with deleted genes. To assess the effectiveness ...
In the PBE lab we study the role of plasmids as catalysts of bacterial evolution, with a special focus on the evolution of plasmid-mediated antibiotic resistance.. Currently, we have two ongoing projects:. - In vivo evolution of plasmid-mediated resistance.. Conjugative plasmids play a key role in the horizontal spread of antibiotic resistance mechanisms among bacteria. One of the key factors undermining the successful spread of a conjugative plasmid is the initial fitness cost produced by the plasmid in the recipient bacteria. The factors involved in this cost and its potential compensation remain largely unknown. In our lab we are trying to understand the evolutionary and genetic determinants that promote the emergence and establishment of successful associations between bacterial clones and resistance plasmids in vivo.. To do so we study conjugation events between different enterobacteria occurring in the gut of hospitalized patients. We study the cost produced by the plasmids when they first ...
Tagging proteins is a standard method facilitating protein detection, purification or targeting. When tagging a certain protein of interest, it is challenging to predict which tag will give optimal results and will not interfere with protein folding, activity or yields. Ideally, multiple tags and positions are tested which however complicates molecular cloning and expression vector generation. In conventional cloning, tags are either added on PCR primers (requiring a distinct primer and PCR product per tag) or provided on the vector (typically leaving a restriction site scar). Here we report a vector family of 40 plasmids allowing simple, seamless fusions of a single PCR product with various N- and C-terminal tags, signal sequences and promoters. The restriction site free cloning (RSFC) strategy presented in this paper relies on seamless cloning using type IIS restriction endonucleases. After cutting out a stuffer (placeholder) fragment from the vectors, a single PCR product can be directly inserted in
The F plasmid origin needs to be designed. The complete F plasmid with partitioning genes in ~10kb in length. It contains several BioBricks restriction sites in both coding and noncoding regions. Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the vector scaffold to generate a new single copy BioBricks plasmid. Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site). The R6K origin is a conditional origin. It only works in the presence of the trans-acting protein Π (encoded by pir) for replication. R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts. ...
The F plasmid origin needs to be designed. The complete F plasmid with partitioning genes in ~10kb in length. It contains several BioBricks restriction sites in both coding and noncoding regions. Once designed, the F plasmid origin can be assembled with an antibiotic resistance marker and cloned into the vector scaffold to generate a new single copy BioBricks plasmid. Chris Anderson suggested inclusion of the R6K origin in these plasmids (rather than inclusion of a pUC19 origin in the multiple cloning site). The R6K origin is a conditional origin. It only works in the presence of the trans-acting protein Π (encoded by pir) for replication. R6K replicates at a medium copy (15 per cell) in pir+ strains and high copy (250 per cell) in pir-116 (high-copy-number mutant) E. coli hosts. ...
Results: Our goal was to develop a high-throughput recombineering system, primarily for the coupling of genes to epitope tags, which could also be used for deletion of genes in both pathogenic and K-12 E. coli strains. To that end we have designed a series of donor plasmids for use with the lambda-Red recombination system, which when cleaved in vivo by the I-SceI meganuclease generate a discrete linear DNA fragment, allowing for C-terminal tagging of chromosomal genes with a 6xHis, 3xFLAG, 4xProteinA or GFP tag or for the deletion of chromosomal regions. We have enhanced existing protocols and technologies by inclusion of a cassette conferring kanamycin resistance and, crucially, by including the sacB gene on the donor plasmid, so that all but true recombinants are counter-selected on kanamycin and sucrose containing media, thus eliminating the need for extensive screening. This method has the added advantage of limiting the exposure of cells to the potential damaging effects of the lambda-Red ...
This topic contains 2 study abstracts on Vaccination: Plasmid DNA Vaccines indicating they may negatively impact Infertility, Vaccination: Abortion, and Vaccine-induced Toxicity
The pCMV-GLuc 2 Control Plasmid is a mammalian expression vector that encodes the secreted luciferase from the copepod Gaussia princeps as a reporter, under the control of the constitutive CMV (cytomegalovirus) promoter. Gaussia luciferase (GLuc) is a 19 kDa protein encoded by a humanized sequence, and it contains a native signal peptide at the N-terminus that allows it to be secreted from mammalian cells into the cell culture medium. A neomycin resistance gene under the control of an SV40 promoter allows selection for stable integration of the plasmid into the mammalian cell genome using G418.
The mitochondrial gene oxi1, carried on a bacterial plasmid, has been used to transform the mitochondria of a yeast strain lacking mtDNA (rho0). The plasmid DNA behaved in a manner entirely consistent with the known properties of normal yeast rho- mtDNA after its introduction by high-velocity microprojectile bombardment. Like the mtDNA sequences retained in natural rho- strains, the plasmid DNA in the transformants was reiterated into concatemers whose size was indistinguishable from that of wild-type mtDNA. The oxi1 sequences in the transformants were surrounded by restriction sites derived from the plasmid that were not present in wild-type mtDNA. oxi1 genetic information in these synthetic rho- strains could be expressed in diploids either after marker rescue by recombination with rho+ mtDNA carrying an appropriate oxi1 point mutation or in trans during the growth of diploids heteroplasmic for both the plasmid-derived oxi1 sequences and rho+ mtDNA with oxi1 deleted. The ability to ...
I was hoping for suggestions as I am having difficulty getting two plasmids into E.coli at library efficiences. I am hoping to add a M13 derived vector to cells containing a pUC/pBR3222 plasmid, or vice versa. Electroporating the M13 derived vector has been at low efficiency so far, making double electroporations very inefficient. The cells containing these plasmids were less electrocompetent, less than 10^6 col/ ug of the second plasmid. Therefore, this approach would require many electroporations to get a large library. I next looked to make the M13 derived vector infectious by growing with helper phage, but had too much cross over with the helper phage. I am now trying this in a Rec minus strain. I was hoping to electroporate in one plasmid and then add the other plasmid through lambda infection but was unable to locate a non lysing system. Please let me know if you have had similar experiences or any suggestions. Thanks tj t j cradick ucsf dept of immunology please reply to:tjc at ...
The procedure has been used successfully for isolation of high- and low-copy-number plasmids from various Bacillus subtilis strains. Yield of plasmid DNA was typically 10-20 µg plasmid DNA from 100 ml culture ...
In the PBE lab we are interested in the evolutionary forces that drive plasmid dynamics in bacterial populations. Plasmids play a crucial role in bacterial ecology and evolution because they can transfer genes horizontally between different bacteria. The most striking example of how plasmids drive bacterial evolution is the global spread of plasmid-mediated antibiotic resistance over the last few decades. Plasmids are arguably the main vehicle for the spread of antibiotic resistance genes among clinically relevant bacteria, contributing to the overwhelming antibiotic resistance crisis we are currently facing.. In our group we try to understand the population genetics of antibiotic resistance plasmids using advanced molecular and evolutionary techniques. Ultimately, we intend to apply the concepts that we learn from the study of the evolution of plasmid-mediated antibiotic resistance to develop more rational intervention strategies to control infectious diseases.. ...
The same plasmid carrying blaCTX-M-14b was identified from an Escherichia coli isolate and an Enterobacter cloacae isolate collected from cattle in the United Kingdom by complete plasmid sequencing. This 35,341-bp plasmid, pSAM7, had an IncX4 backbone that is 99% identical to that of pJIE143 from a human isolate in Australia. PCR screening identified pSAM7-like plasmids in three other E. coli isolates of different multilocus sequence types isolated from cattle on different farms in the United Kingdom.. ...
pRV500 is, to our knowledge, the first plasmid of L. sakei to be entirely sequenced. Careful sequence analysis showed that pRV500 belongs to the pUCL287 family, itself related to class A theta-type plasmids of the pUCL22 and pSC101 families. Experimental evidence of the theta replication mode was obtained for pUCL287 itself (2). Although the minimum length of the pRV500 oriA region remains to be determined, it is likely to involve the same two kinds of DNA repeats as other pUCL287-type plasmids. The involvement of repetitive sequences of 22 bp (iterons) as a target for binding of the Rep protein was demonstrated for pSBO1 (40), and the upstream 11-bp repeated sequences were observed to be essential for pUCL287 replication (3).. Like other plasmids (14, 49), pRV500 appears to be a composite structure containing DNA segments from different sources. Some ORFs are shared by plasmids from both subfamilies pUCL22 and pUCL287; this applies to orf6 in pRV500 and ORFs of unknown function around the gene ...
Members of the family Enterobacteriaceae have been isolated from raw wastewater, identified, and characterized with respect to their plasmid content and antibiotic resistance. Several strains possessing both antibiotic resistance and high-molecular-weight plasmid(s) transferred their resistance characteristics to recipient cells during a 25 h coincubation. Eight were characterized (six Escherichia coli and two Klebsiella pneumoniae); each produced 10(2) to 10(7) transconjugants per ml by the end of the incubation period. They were also able to mobilize pBR325 from a laboratory E. coli strain into plasmid-free recipients to yield 10(2) to 10(7) transconjugants per ml. These transconjugants possessed phenotypic characteristics specified by pBR325, the R plasmid, and the chromosome of the recipient. Many transconjugants exhibited recombinational rearrangements of the acquired plasmid material. ...
Polymerase chain reaction-based replicon typing represents a novel method to describe the dissemination and follow the evolution of resistance plasmids. We used this approach to study 26 epidemiologically unrelated Enterobacteriaceae and demonstrate the dominance of incompatibility (Inc) A/C or Inc N-related plasmids carrying some emerging resistance determinants to extended-spectrum cephalosporins and carbapenems.
Bioz Stars score, Techniques, Protocol Conditions and more for Expression Plasmids, supplied by . Data for Expression Plasmids gathered from related PubMed articles.
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