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.. ...
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 ...
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 ...
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 ...
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...
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 ...
Easy Yeast Plasmid Isolation Kit: rescue plasmids from yeast using spin columns. Simple and highly efficient method. Zymolyase included.
Easy Yeast Plasmid Isolation Kit: rescue plasmids from yeast using spin columns. Simple and highly efficient method. Zymolyase included.
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 ...
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...
Plasmids are found across bacteria, archaea, and eukaryotes and play an important role in evolution. Plasmids exist at different copy numbers, the number of copies of the plasmid per cell, ranging from a single plasmid per cell to hundreds of plasmids per cell. This feature of a copy number greater than one can lead to a population of plasmids within a single cell that are not identical clones of one another, but rather have individual mutations that make a given plasmid unique. During cell division, this population of plasmids is partitioned into the two daughter cells, resulting in a random distribution of different plasmid variants in each daughter. In this study, we use stochastic simulations to investigate how random plasmid partitioning compares to a perfect partitioning model. Our simulation results demonstrate that random plasmid partitioning accelerates mutant allele fixation when the allele is beneficial and the selection is in an additive or recessive regime where increasing the copy ...
Duncan Clark wrote: , , I was always curious about the replicons used in some clinical E.colis , that carry umpteen different plasmids. I remember many many years ago , that one such E.coli was used for plasmid markers and had may 10 , different plasmids in it. They didnt appear to lose those plasmids , despite no selection. May have been published in Plasmid back in the , late 70s or early 80s. It all depends on the replication control mechanism of the replicons I suppose. ColE1 replicon is the standard one taught to students and I havent really look into others, so Im speaking in complete ignorance here, but there is no reason why a plasmid with different replicon would necessary result in plasmid incompatibility (because ColE1 has an unusual replication control mechanism?), although I understand that many would. , Duncan ...
View Notes - BMI_Lab8DNAPurification from PHS 2301 at St. Johns Duplicate. Laboratory VIII Topic: Purification of DNA, plasmid isolation using alkaline method Introduction: Genetic transformation
SimPlot analysis.Similarity plots with plasmids R751, pBP136 and pB3 as reference plasmids. Each coloured plot corresponds to a specific plasmid depicted in the
The isolation method is optimized for cultures grown in LB media; other rich media may require increased volumes of Suspension-, Lysis-, and Neutralization Buffer, and an additional wash step. The isolation procedure is suitable for all plasmid sizes; lysates of larger constructs (up to 100 kb) should be cleared by filtration to avoid shearing.. The yield of plasmid DNA preparations is dependent on several parameters, e.g., quality of the bacterial culture growth, amount of used culture suspension for the preparation, plasmid type used etc. As a rule of thumb the typical yield of a high copy number plasmid is about 3 - 5 µg of DNA per ml of original bacterial culture (pUC, pTZ, pGEM in common host strains like XL-1 blue, HB101, JM 109). The typical yield of low copy number plasmids is about 0.2 - 1 µg of DNA per ml of original bacterial culture.. The Genopure kits are supplied with folded filters to eliminate the time-consuming centrifugation step after the alkaline lysis. In approximately 2 ...
Plasmids, strains and primers used in this study are listed in Additional file 7: Table S1, Additional file 8: Table S2, Additional file 9: S3 1. Oligonucleotides and gBlocks were ordered from IDT and Eurofins. All fragments obtained by PCR were gel- or column purified (Nucleospin® Gel and PCR Clean-up columns) before cloning, and resulting plasmids were verified by sequencing (Eurofins). Yeast transformations were done using lithium acetate and PEG3350, and genomic integrations were performed with various helper plasmids and pre-expressed iCas9 from plasmid pCT (Addgene #60620) and plated on Sc-Leu+cloNAT. Strains were cured for pCT and helper plasmids after genome engineering and before proceeding to transcriptional regulation using dCas9.. EasyClone-MarkerFree vectors pCfB2909, pCfB3035, pCfB3037 and helper plasmids pCfB3042, pCfB3046, and pCfB3050 as well as genomic integration verification primers were adapted as previously described [46]. Yeast strains were plated according to ...
Anion exchange monolithic chromatography is increasingly becoming a prominent tool for plasmid DNA purification but no generic protocol is available to purify all types of plasmid DNA. In this work, we established a simple framework and used it to specifically purify a plasmid DNA model from a clarified alkaline-lysed plasmid-containing cell lysate. The framework involved optimising ligand functionalisation temperature (30-80°C), mobile phase flow rate (0.1-1.8 mL/min), monolith pore size (done by changing the porogen content in the polymerisation reaction by 50-80%), buffer pH (6-10), ionic strength of binding buffer (0.3-0.7. M) and buffer gradient elution slope (1-10% buffer B/min). We concluded that preferential pcDNA3F adsorption and optimum resolution could be achieved within the tested conditions by loading the clarified cell lysate into 400. nm pore size of monolith in 0.7. M NaCl (pH 6) of binding buffer followed by increasing the NaCl concentration to 1.0. M at 3%B/min. © 2010 ...
Genetic reagents for generating plasmids containing multiples of a DNA segment are prepared by modification of the restriction sites of plasmid rings. The modified plasmids permit cloning of DNA segments which can be recovered with sticky ends that are complementary but not rotationally equivalent. Such segments will polymerize in a head-to-tail conformation. The plasmid rings with modified restriction sites are also used in linear form to obtain head-to-tail joining of multiple DNA segments into plasmid rings for further cloning and/or expression of the DNA segment-directed protein. The critical restriction site sequences of the modified plasmid rings can be prepared as a reagent which permits the sequence to be introduced into any plasmid. The reagents have utility in preparing multiples of protein-forming genes, and in preparing large amounts of homogeneous DNAs which can themselves be used as reagents.
Fig 3. Scheme of construction of 4 isogenic biosensor strains. It was a pyramiding process during which the phenotypes of bacterial reporter developed previously in our project were combined, in order to implement strains with different mercury sensitivity. (E) BBa_J23103, BBa_J23101 and BBa_J23117 belong to a constitutive promoter library from partsregistry, among which BBa_J23103 is a very weak one, BBa_J23117 is medium and BBa_J23101 is the second strongest in the library. (A)Expression of MerR driven by weak constitutive promoter BBa_J23103 on low copy number plasmid backbone pSB3K3 was pyramided with wild type PmerT which is the most mercury-sensitive promoter combined with LacZ alpha fragment as the reporter gene on plasmid backbone pSB1A3. It is an extreme to confer the bioreporter the most sensitivity, based on our previous results. (B) Strong promoter BBa_J23101 drive the high expression intensity of MerR on plasmid backbone pSB1A2, while PmerT 88 on pSB3K3 is the most insensitive ...
Replication of the miniF plasmid pML31 was examined during the division cycle of Escherichia coli growing with doubling times between 40 and 90 min at 37°C and compared to the replication of plasmid pBR322 and the minichromosome pAL70. The replication pattern of pML31 was indistinguishable from that of pBR322 at all growth rates and very different from the cell- cycle-specific replication of the minichromosome. It is concluded that both pML31 and pBR322 plasmids can replicate at all stages of the division cycle, with a probability of replication that increases gradually, but perhaps nut exponentially, during the cycle. In contrast, the modes of segregation of pML31 and pBR322 plasmids into daughter cells at division appeared to differ, raising the possibility that pML31 may segregate in a nonrandom fashion similar to that of chromosomes and minichromosomes ...
Our method for disrupting E. coli chromosomal genes is analogous to one that has been used for many years in yeast (8). It is based on results of K. Murphy (18) who provided us with his materials before publication. Because multicopy plasmids might interfere with recombination by acting as competitive inhibitors (18), we cloned the Red genes (γ, β, and exo) into a low copy number plasmid. We used a vector which shows temperature-sensitive replication (37) to permit its easy curing from the resultant mutants. The plasmids pKD20 and pKD46 (Fig. 2) express the Red system under control of a well-regulated promoter to avoid unwanted recombinational events under noninducing conditions. They differ in that the latter has the native tL3 terminator downstream of exo. Although all recombinants described here were made by using pKD20, we now use pKD46 instead because we recently discovered that pKD46 yields a greatly enhanced number of recombinants. The reason is unknown. Curiously, tL3 encodes a small ...
Like the above examples, pSC101s replication is positively regulated by RepA binding the origin. RepA is also used to control copy number, by two mechanisms. Firstly, RepA negatively regulates its own transcription, thus the RepA protein levels (and its ability to promote replication) is confined to narrow limits. Secondly, The plasmid contains several (3-7) repeats of a 17-22bp sequence called iteron sequences. RepA binds the iterons, and at higher plasmid conncentration, this can lead to "handcuffing" of two plasmids. Interestingly, adding extra iteron sequences on other plasmids can reduce the copy number by this handcuffing mechanism. F, RK6, P1, RK2, and RP4 also use iterons, but the regulating protein and origins differ. pETcoco is an interesting plasmid, made by Novagen. It can be maintained as a single copy plasmid using the origin and positive regulator from the F plasmid (oriS and RepE). It can be swiched to a medium copy plasmid using the machinery from the RK2 plasmid (oviV and ...
Plasmid replication control is usually controlled by balancing the levels of a positive and a negative regulator of replication. For some plasmids (pMB1/colE1 replicons) the positive regulator is an RNA and in others (e.g. pSC101) it is a protein. Plasmids with a protein positive regulator will not replicate in the abscence of protein production - stringent control (although not the same as the stringent response due to a shortage of loaded tRNAs). Plasmids with an RNA positive regulator will continue to replicate in the abscence of protein production. This is termed relaxed control. High yields of plasmid may be obtained by halting protein production (via chloroamphenicol) when the culture reaches a high density and then continuing incubation for a number of hours. This might be of practical relevance when prepping the 1 and 3 series of Synthetic Biology plasmids.--BC 19:05, 3 Sep 2005 (EDT) ...
Plasmid Construction. The pGL3-promoter and pGL3-basic plasmids were purchased from Promega. The pL6 plasmid was generated by ligation of the 1.3-kb KpnI and XhoI (-1326 to +1; the translation start site was designated as +1) DNA fragment of mouse MOR into the polylinker sites of a promoterless luciferase vector, pGL3-basic (Promega, Madison, WI). The sequence of the insertion was confirmed by sequencing. The DNA fragment flanking the poly (A) site was generated by PCR from mouse genomic DNA with a pair of primers (sense, 5′-AATAGGCCGGCCGCATTAGGAGCATTGCTGAG-3′; antisense, 5′-ACGCGTCGACCCTAACTCTGGGATGGCAAG-3′; the underlined nucleotides indicate the overhanging restriction enzyme sites for FseI and SalI, respectively). The pL6PA and pGL3pPA plasmids were constructed by subcloning this DNA fragment after digestion with FseI and SalI into pL6 and pGL3-promoter plasmid, respectively. The pL6 and pGL3-promoter plasmids also have been digested by FseI and SalI, to replace the original SV40 ...
This two part exercise provides state-of-the-art information and practical experience with a variety of techniques that form the foundation of the biotechnology industry. In part A, students create a strain of E. coli that is resistant to the antibiotic ampicillin by introducing a plasmid that contains an ampicillin-resistance gene. The success of the transformation is monitored by growing the bacteria on an ampicillin-containing media. This experiment provides sufficient sterile materials for sixteen platings. In Part B, students isolate the amplified plasmid from the bacteria without the use of toxic chemicals. Then they digest the isolated DNA with EcoR1 and compare the electrophoretic properties of linear and circular plasmid DNA molecules. By inserting desired DNA segments into plasmids, this procedure has enabled scientists to amplify more than 1000 specific genes including those for human interferon, insulin, and growth hormone. Part B of the exercise requires a centrifuge that can be ...
No, RNase A should not be omitted from buffer P1. It is required to prevent RNA contamination of the purified plasmid DNA. RNase A will not interfere with downstream in-vitro transcription experiments, since it will be efficiently removed during the plasmid purification procedures using QIAGEN Plasmid Kits ...
In article ,[email protected][142.20.8.21], jlight at RESUNIX.RI.SICKKIDS.ON.CA writes: ,From: jlight at RESUNIX.RI.SICKKIDS.ON.CA ,Subject: Denaturation of plasmid DNA ,Date: 30 Nov 1995 16:52:45 -0800 ,Heres a basic question for which I should know the answer. Basically, Im ,working with a mixed population of cDNAs cloned into a plasmid and I want ,to anneal a primer (with an affinity tag) to the specific constructs in ,solution to try and recover the homologous constructs intact. When you ,want to separate the strands of plasmid DNA and you have mostly ccc DNA ,(say, from a Qiagen prep), whats the best way to separate the strands only ,partially so that when you reanneal, one strand from any particular plasmid ,will reanneal with the other strand from the same plasmid molecule, rather ,than those of other homologous plasmid molecules? Please dont flame me if ,you think this is stupid - Im trying to be serious. Thanks in advance for ,your help. I really do not think that it is ...
Plasmid pCLIPf-NK1R Control Plasmid from Dr. Ana Eganas lab contains the insert Neurokinin-1 Receptor, Truncated and is published in Unpublished This plasmid is available through Addgene.
In order to measure RPU, we must use two types of E.coli transformed with the test promoter circuit and the standard promoter circuit respectively to compare absolute activity of the test promoter with that of the standard. BBa_K358000 has the lactose promoter (R0011) immediately upstream of GFP, and BBa_K358001 has the standard promoter (J23101). We constructed both parts with a low copy number plasmid, pSB4K5, because lacI cannot repress lactose promoter completely even in the absence of IPTG if a high copy number plasmid is used. The E.coli transformed with pSB4K5 was also used for correcting the autofluorescence signal. We picked up three colonies from each plate, and cultivated them in M9 medium supplemented with 50 µg/mL of kanamycine at 37x2103; for about 16h. The precultures were diluted to 1:100 in pre-warmed fresh M9 medium supplemented with 50 µg/mL of kanamycine and incubated with shake at 37℃. After 3h or 3.5h, we measured OD600 followed by GFP fluorescence of the culture. We ...
This video explain plasmid cloning. I am also adding here background information: Plasmids are circular, double-stranded DNA (dsDNA) molecules that are separate from a cells chromosomal DNA. These...
Plasmid constructions.The plasmids used for mapping assays were constructed as described below. All of the plasmids were sequenced to verify the expected genotypes.. Full-length UL31 was cloned into the pCDNA3 vector such that UL31 expression was driven by the human cytomegalovirus immediate early promoter-enhancer. The construct was designated pJB261.. The full-length UL34 sequence (encoding amino acids [aa] 1 to 275) was PCR amplified from HSV-1(F) viral DNA by use of the primers 5′ GTA GTC GAC ATA TGG CGG GAC TGG GCA AG 3′ and 5′ GCG GTC GAC AGG GCT GTG TGG GGC GAA GGC GTC 3′. The PCR product was then removed with SalI and ligated into the pGEX4T-1 SalI restriction site. This plasmid was designated pJB253. The UL34 fragment was then cut from pJB253 with the SalI enzyme and ligated into the XhoI site of pCDNA3, and the construct was designated pJB280a. To include sequences sufficient to promote translation of the gene, we PCR amplified a full-length UL34 fragment from pJB253 by using ...
Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.
This exercise was designed to provide an exciting introduction to specific gene structure and function. In the experiment, students are given two plasmids (A and B) which are identified in the instructors guide. One plasmid (A) has a functional gene for the enzyme ß-galactosidase. The ß-galactosidase gene in the other plasmid (B) is inactive because it contains a segment of foreign DNA. In the first part of the exercise, students analyze restriction digests of both plasmids in order to determine which plasmid should have a functional ß-galactosidase gene. In the second part of the exercise, the plasmids are introduced into E. coli by transformation and the color of the resulting colonies (blue or white) is then used to assess the functional status of the ß-galactosidase gene. This exercise can be completed within a single 3-hour laboratory session or two 2-hour laboratory periods.. ...
GENETIC BASIS OF RESISTANCE:. Most drug resistance is due to a genetic change in the organism, either as a chromosomal mutation or the acquisition of a plasmid ortransposon. a) CHROMOSOME-MEDIATED-RESISTANCE:. Chromosomal resistance is due to a mutation in the gene that codes for either the target of the drug or the transport system in the membrane that controls the uptake of the drug. The frequency of spontaneous mutations usually ranges from 10~ 7 to 10 ~ 9 which is much lower then the frequency of acquisition of resistance plasmids. Therefore, chromosomal resilience is less of a clinical problem than plasmid. Mediated resistance. b) PLASMID-MED1ATED RESISTANCE:. Plasmid-mediated resistance is very important from clinical point of view for 3 reasons: 1) It occurs in many different species, especially gram negative rods. 2) Plasmida frequently madiate resistance to multiple drugs. 3) Plasmids have a high rate of trans. for from one cell to another, usually by conjugation. Resistance plasmids ...
Study Flashcards On restriction enzymes/plasmids at Cram.com. Quickly memorize the terms, phrases and much more. Cram.com makes it easy to get the grade you want!
QIAGEN has established a partnership with Aldevron, a company that specializes in custom plasmid DNA preparation services. Through this partnership, Aldevron is offering customers the possibility to order standard plasmid DNA preparation services performed using high-quality QIAGEN plasmid purification products. This service offers three different scales of endotoxin-free* plasmid DNA. This standardized service is only available via the Web site |span style=text-decoration: underline;>www.plasmid.com|/span>. |br /> |br /> Learn more:
February 8, 2011 at 2:28 pm , nick , cool results, everyday science Few experiments in science are conclusive. So, it is very exciting when an experiment provides completely unambiguous results. Today that happened.. Molecular biologists use bacteria-specifically strains of E. coli that dont make people sick (i.e., non-pathogenic)-to make lots of copies of circular DNA, called plasmids; one can think of E. coli as a photocopier for plasmids. Bacteria love to take up plasmids, copy them, and express them to make proteins with unique functions, and it is this ability that makes them so evolutionarily successful. Bacteria can transfer plasmids containing antibiotic resistance genes to each other, for example, and in this manner, can become "superbugs." Staph is one pathogen that has done this so successfully in hospitals that we will soon run out of antibiotics to treat it.. Interestingly, molecular biologists give E. coli antibiotic resistance genes on purpose. Its not because were ...
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InvivoGen provides two different E. coli competent strains : GT115 cells are specifically designed for cloning and propagation of plasmids containing hairpin structures and the R6K gamma origin of replication, such as pCpG-siRNA plasmids, GT116 cells are specifically designed for cloning and propagation of shRNA-expressing plasmids which contain hairpin structures, such as psiRNA plasmids.
Hi, in this agarose gel photo (70 volts, 3.5uL Gel Red in 30ml of TBE, 1% Agarose, 1 hour run), Ive checked the digestion of a large plasmid (15Kbp, one cut site). The theory says that the digested plasmid (linearized) should migrate slower than the supercoiled form of the uncut one (bigger band of the Undigested Plasmid). In my case the digested plasmid is faster than the undigested one. Ive found that my digested plasmid could be single-stranded (http://bitesizebio.com/13524/how-to-identify-supercoils-nicks-and-circles-in-plasmid-preps/). Do you think that could be the case?. ...
OriGene offers the largest collection of cDNA expression plasmids, ready for transfecting into mammalian cells. Genome-wide coverage of tagged ORF vectors, non-tagged genes and lentiviral vectors.
This could be a few things: 1. Bad antibiotic - check to see if bacteria that dont harbor the same resistance will grow/not grow in the media or on the plate. 2. Something wrong with the method of DNA prep - try to prep other plasmids using the same kit/reagents. Try to prep plasmids with the same antibiotic resistance. 3. No plasmid to prep - Use a method of colony cracking where you take some of the culture, pop the bacteria and run it straight on a gel without prepping for clean plasmid. This will let you know if anything is there to prep. In general, clones should not be able to grow on a particular antibiotic if there is no plasmid expressing resistance ...
The mKate2 Optimization plasmid is similar to the Edit-R Cas9 nuclease expression plasmid with the Blasticidin resistance marker, where the mKate2 fluorescent gene sequence has replaced the Cas9 nuclease gene. The mKate2 Optimization plasmid can be used to perform transfection optimization in a similar manner as described in the ��Transfection Optimization�� section of the technical manual. However, if you are working with a SMARTCas9 nuclease expression plasmid, then we recommend performing transfection optimization with a corresponding SMARTCas9-mKate2 expression plasmid under the control of a promoter you know to be active in your cells ...
Chapter 19) (a) Minimal defective prophage created by removal of the N through kil genes in the P L operon and replacement of rexA and rexB with a drug resistance cassette, either ampicillin (bla) or chloramphenicol (cat). With this system, raising the temperature induces the operon directly without N-mediated antitermination. (b) Minimal prophage moved onto a high-copy-number vector. The pBR322 origin of DNA replication between nucleotide coordinates 2348 and 3296 was amplified.This linear PCR product was used to clone the minimal prophage in a gap repair reaction. This linear pBR322 fragment contains ori but lacks an antibiotic resistance gene, so only those plasmid clones that have undergone successful recombineering will contain an antibiotic resistance marker inherited from the prophage.The high-copy-number plasmids thus generated, pSIM2 and pSIM4 (Cmr and Ampr, respectively),were used as targets in subsequent recombineering reactions.The pBR322 segment was replaced precisely with a linear ...
5121 Telomeres, centromeres, and other highly repetitive genomic regions are typically under-represented in shotgun libraries, and they are often absent from partial-digest BAC libraries. Accurate cloning of isolated fragments containing short tandem repeats, AT-rich DNA, or regions with strong secondary structure likewise can be difficult or impossible using conventional supercoiled plasmids. We have developed vectors and methods to alleviate these types of cloning bias. A transcription-free, linear plasmid was derived from the linear coliphage N15 for cloning into E.coli. The ends of the vector are free to rotate during replication, minimizing formation of secondary structures that are substrates for deletion or rearrangement. The "pJAZZ" linear vectors are shown to provide unprecedented ability to maintain regions of up to 30 kb that are unclonable in circular plasmids. Examples include regions of di-, tri-, and tetra-nucleotide repeats up to several kb, as well as highly AT-rich fragments of ...
Wein, Tanita, Hülter, Nils F., Mizrahi, Itzhak and Dagan, Tal (2019) Emergence of plasmid stability under non-selective conditions maintains antibiotic resistance Nature Communications, 10 (1). DOI 10.1038/s41467-019-10600-7. Full text not available from this repository ...
... : Found or purchased all 11 basic Plasmid types - worth 20 GamerScore. Find guides to this achievement here.
Plasmids can be submitted in either form. For DNA, aliquot 15 µL of DNA into a 1.5 mL microfuge tube (at a concentration of 0.1 -1µg/µL)...
Description: Small toxic membrane polypeptide, Qin prophage; homologous to plasmid-encoded plasmid stabilization toxins regulated by antisense RNA; functional relevance of chromosomal homologs is ...
Basically, plasmid DNA can be isolated from cells on the basis of size and their conformation. As the plasmid DNA is smaller than chromosomal DNA.
Transfections were performed in six-well culture plates (Corning Inc., Corning, NY). Each well has approximately 10-cm2 surface for cell culture growth. Cells in each well were transfected with 2 μg of total plasmid DNA. To test the transactivating activity of wild-type and mutant hPXRs, the cells were transfected with 50 ng of pcDNA3, wild-type or mutant hPXR, and 1950 ng of pGL3-CYP3A4-luc plasmids. To examine the protein expression and localization, the cells were transfected with 2 μg of pcDNA3 and wild-type or mutant hPXR plasmids. For the rapamycin experiments, the cells were transfected with 50 ng of pcDNA3 or hPXR and 1950 ng of pGL3-CYP3A4-luc plasmids. In p70 S6K overexpression experiments, the cells were transfected with 100 ng of FLAG-pcDNA3, pcDNA3-FLAG-hPXR, or pcDNA3-FLAG-hPXRT57A; 500 ng of constitutively active p70 S6K; and 1000 ng of pGL3-CYP3A4-luc and 100 ng of pGL4-hRluc; FLAG-pcDNA3 was used to make up the total plasmid DNA to 2 μg in each transfection. In mammalian ...
A plasmid is a small DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Plasmids are commonly used to multiply (make many copies of) …
Vector ConstructionHuman Akt-Myr cDNA with myristolation signal for greater activity, also known as PKB, was cloned into the multiple cloning site of pAC-CMV-pLpA. The resulting plasmid was cotransfected into HEK293 cells with plasmid pJM17 which contains the Ad5 genome. Homologous recombination between the two plasmids resulted in the replacement of the Ad5 early region 1 with the human Akt cDNA expression cassette, generating a replication deficient recombinant virus. ...
Existing methods for cloning and recombination of DNA enable construction of arbitrary sequences. However, the sequential nature of these techniques makes them time-consuming and expensive. Furthermore, while the transformation of an existing plasmid into a host strain can be reliable when a selection marker is used, there are many current limitations: the number of different plasmids that can be co-transformed is limited by the choice of markers and compatible origins of replication; plasmids are less stable than chromosomal DNA and are difficult to maintain indefinitely without mutation; and cistronic interactions cannot be designed since each new nucleotide sequence added is on an unconnected DNA molecule. To overcome these limitations, we are designing reconfigurable chromosomes consisting of both fixed and variable regions. While the fixed region is carefully optimized and tuned ahead of time, the variable region can be modified in the field, at the point-of-use, leading to rapid and ...
Existing methods for cloning and recombination of DNA enable construction of arbitrary sequences. However, the sequential nature of these techniques makes them time-consuming and expensive. Furthermore, while the transformation of an existing plasmid into a host strain can be reliable when a selection marker is used, there are many current limitations: the number of different plasmids that can be co-transformed is limited by the choice of markers and compatible origins of replication; plasmids are less stable than chromosomal DNA and are difficult to maintain indefinitely without mutation; and cistronic interactions cannot be designed since each new nucleotide sequence added is on an unconnected DNA molecule. To overcome these limitations, we are designing reconfigurable chromosomes consisting of both fixed and variable regions. While the fixed region is carefully optimized and tuned ahead of time, the variable region can be modified in the field, at the point-of-use, leading to rapid and ...
Vector ConstructionHuman cDNA for apoptosis signaling kinase 1 with a mutation in catalytic lysine (presumed dominant negative). It was cloned into the multiple cloning site of pAC-CMV-pLpA. The resulting plasmid was cotransfected into HEK293 cells with plasmid pJM17 which contains the Ad5 genome. Homologous recombination between the two plasmids resulted in the replacement of the Ad5 early region 1 with the mouse cDNA for ASK1-KM, generating a replication deficient recombinant virus. ...
Transgenic mice. To make the 7X-tetOp A-FOS transgene, the plasmid pUHD 10-3 ( 19) was linearized with SacII and BamHI and ligated to a 62-bp polylinker: top strand, 5′-GGCCACCATGGCGTATCCCTACGACGTGCCCGATTATGCCCGATTATGCCCATATGCAGGAATTCAAGCTTG-3′ that encodes a Kozak consensus and a Hemagglutinin tag (YPYDVPDYA). An SV40 poly(A) fragment containing the small T-antigen intron that preceded the poly(A) site was obtained from the plasmid pRSVNeo as a 1,026-bp BamHI-SmaI fragment. This fragment, when ligated to the BamHI-NaeI-digested 2.7-kb vector (pTRE 850/85-ds) backbone, produced a plasmid with the SV40 poly(A) in reverse orientation (pTRE850/851 ds reversed). To obtain a plasmid with the SV40 poly(A) in the correct orientation, "pTRE850/851-ds-reversed" was digested with HindIII. The 2,704-bp vector and the 1,067-bp insert were then religated, and recombinants were selected in which the insert fragment [representing the SV40 poly(A)] is reversed when compared with the parental plasmid. This ...
To test whether ORF14 is cotranscribed with gumM, we employed a method consisting of the application of lacZtranscriptional fusions combined with plasmid integration (13). A promoterless lacZ-aacC1 interposon (4) was inserted into the unique BamHI site of the SmaI-ClaI fragment (nucleotides 14056 [GenBank accession no. U22511] and 2071 [GenBank accession no.U70053], respectively), which was previously subcloned into pK19mobGII. The hybrid plasmid was transferred from the broad-host-range mobilizing strain E. coli S17-1 (21) to wild-type X. campestris FC2 (14), as described previously (19). Exconjugants were selected in agar medium containing gentamicin, rifampin, and X-gluc. Yellow colonies appeared with a frequency of 10−4 and were sensitive to kanamycin. Correct gene replacement was verified by Southern hybridization. The strain mutated in ORF14 (XcORF14) produced normal amounts of xanthan, as judged by precipitation of the polymer from the broth as previously described (6) (not shown). ...
GenScripts industrial-grade Plasmid DNA can help your experiments achieve highly efficient cell transfection, helping to improve experimental outcomes in research areas such as protein expression, antibody production, and other research projects.
There are numerous methods for detecting protein-protein associations, each with advantages and disadvantages. Advantages of the FLS platform technology presented here are its versatility and simplicity with the capacity to evaluate proteins from many different sources yet without the need for purified proteins, unusual technical expertise, or highly specialized equipment. The most demanding requirement is for a fluorescence microscope with standard imaging capabilities. Regarding ease of use, it is also significant that this technology is designed to work upon transient expression of proteins from transfected plasmids and does not require the generation of stably expressing cell lines. Another advantage is that associations are visualized inside cells in a setting that approximates the native environment of the proteins. In addition to CV-1 and COS-7 cells, we have shown the technology to work as well in other cell types (BHK-21 and NIH 3T3) (data not shown) so that cell type restrictions seem ...
Read about how mammalian plasmids differ from their bacterial counterparts, including how replication occurs and whether selection is necessary for transfected cells.
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Miniprep plasmid DNA extraction kit is used when the starting E. coli culture volume is 1~5 ml of LB broth and the expected DNA yield is 20~30 μg. Other extraction and purification plasmid DNA Kits are available from varying manufacturers, named by size of bacterial culture, includes gigaprep, megaprep, and midiprep - Plasmid DNA Extraction (Miniprep) - AbVideo™ - Support - Abnova
The default terms for deposit agreements allows Addgene to transfer plasmids to academic and nonprofit institutions only. Unless...
Those sneaky file searchers over at the 2K Games forums have uncovered a few text strings embedded in the PC version of BioShocks game code which hint at a new set of plasmids coming our way. After looking through the games install files for a reference to a PC game editor, forum member Zemlor stumbled across a reference to a piece of content called PlasmidPack1. ...
The resultant PCR solution was cloned into a TA vector and remodeled into E. coli, which underwent ampicillin variety (a hundred mg/mL) on luria broth plates
Creative Biogene offers a wide range of plasmid preparation services for many applications, including research, preclinical, clinical, and diagnostic applications.
Bacterial plasmids are nucleotide sequences floating in the cytoplasm of bacteria. These molecules replicate independently from the main chromosomal DNA and are not essential to the survival or replication of their host. Plasmids are thought to be part of the bacterial domains mobilome (for overview, see Siefert, 2009), a sort of genetic commonwealth which most,…
The VTC4 gene (At3g02870) was amplified by PCR with primers 5′-ATGGCGGACAATGATTCTCT-3′ (forward) and 5′-TCATGCCCCTGTAAGCCGCA-3′ (reverse). The template was generated by RT (Omniscript RT kit) of RNA extracted from wild-type plants using the RNAeasy Plant Mini kit (both kits from Qiagen) according to the manufacturers instructions. The resulting PCR product was cloned into plasmid pCRT7/NT-TOPO using the pCR T7 TOPO TA Expression kit (Invitrogen). The newly created plasmid pAtIMPH contains the amplified At3g02870 gene (816 nucleotides) from the start ATG to the stop TGA along with upstream plasmid regions including a T7 promoter, ribosome binding site, ATG start site, 6xHIS region, Xpress epitope, and EK cleavage site. Because of the extra upstream plasmid sequences, the theoretical size of the protein is 36 amino acids longer than the 271 amino acids of IMP. pAtIMPH was used to transform TOP10F′ Escherichia coli cells, and the gene sequence was verified. Similarly, plasmids containing ...
MOESM1 of Construction and characterization of broad-host-range reporter plasmid suitable for on-line analysis of bacterial host responses related to recombinant protein production
RNase, Purification, Plasmid DNA, CIM® Columns, Plasmids, episomes, eukaryotic, prokaryotic, genetic vectors, gene manipulation, ribonuclease A, hydrophobic chromatography, ion-exchange, size exclusion, affinity
Bars = estimated 16S copies based on nicked-circular plasmid standard. Black and white striped bars = estimated 16S copies based on linearized plasmid standard.
Domain architecture and assignment details (superfamily, family, region, evalue) for gi|146276053|ref|YP_001166213.1|NC_009427 from NCBI plasmid sequences. Plus protein sequence and external database links.
C-terminal 6His and EGFP dual affinity and reporter tag yeast expression plasmid. This vector allows the creation of fusion proteins with an EKT cleavage tag.
Clone #1 was selected from each of the screened clones.. A sterile pipette tip was used to inoculate 5mL of 1x LBAmp100 in a 15mL conical tube. The tubes were incubated O/N @ 37C on a rocker.. 3mL of liquid culture was used as input for plasmid isolation with the QIAprep Spin Mini Kit (Qiagen) according to the manufacturers protocol.. 1mL of each culture was combined with 1mL of 50% sterile glycerol (25% glycerol final concentration) and stored @ -80C with existing bacterial stocks.. Plasmid DNA was eluted with 50μL of Buffer EB and quantified on the Roberts Lab NanoDrop1000 (ThermoFisher) in order to have a rough idea of concentrations to submit for Sanger sequencing. A dye-based quantification will be performed after sequencing results are back in order to obtain a more accurate assessment for use in ISH and/or qPCR standard curve creation.. Results:. ...
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