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Relevant bibliographies by topics / Plasmid copy number reduction A

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Academic literature on the topic 'Plasmid copy number reduction A'

Author: Grafiati

Published: 4 June 2021

Last updated: 15 February 2022

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Journal articles on the topic "Plasmid copy number reduction A"

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Cook, L. C., and G. M. Dunny. "Effects of Biofilm Growth on Plasmid Copy Number and Expression of Antibiotic Resistance Genes in Enterococcus faecalis." Antimicrobial Agents and Chemotherapy 57, no. 4 (February 4, 2013): 1850–56. http://dx.doi.org/10.1128/aac.02010-12.

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ABSTRACTBiofilm growth causes increased average plasmid copy number as well as increased copy number heterogeneity inEnterococcus faecaliscells carrying plasmid pCF10. In this study, we examined whether biofilm growth affected the copy number and expression of antibiotic resistance determinants for several plasmids with diverse replication systems. Four differentE. faecalisplasmids, unrelated to pCF10, demonstrated increased copy number in biofilm cells. In biofilm cells, we also observed increased transcription of antibiotic resistance genes present on these plasmids. The increase in plasmid copy number correlated with increased plating efficiency on high concentrations of antibiotics. Single-cell analysis of strains carrying two different plasmids suggested that the increase in plasmid copy number associated with biofilm growth was restricted to a subpopulation of biofilm cells. Regrowth of harvested biofilm cells in liquid culture resulted in a rapid reduction of plasmid copy number to that observed in the planktonic state. These results suggest a possible mechanism by which biofilm growth could reduce susceptibility to antibiotics in clinical settings.

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Smith, M. Alex, and Michael J. Bidochka. "Bacterial fitness and plasmid loss: the importance of culture conditions and plasmid size." Canadian Journal of Microbiology 44, no. 4 (April 1, 1998): 351–55. http://dx.doi.org/10.1139/w98-020.

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Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective environments are important to the understanding of the evolution of the bacteria-plasmid associations and the appreciation of the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.Key words: pBluescript, plasmid, stress, fitness, starvation.

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Slavcev, Roderick A., and Barbara E. Funnell. "Identification and Characterization of a Novel Allele of Escherichia coli dnaB Helicase That Compromises the Stability of Plasmid P1." Journal of Bacteriology 187, no. 4 (February 15, 2005): 1227–37. http://dx.doi.org/10.1128/jb.187.4.1227-1237.2005.

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ABSTRACT Bacteriophage P1 lysogenizes Escherichia coli cells as a plasmid with approximately the same copy number as the copy number of the host chromosome. Faithful inheritance of the plasmids relies upon proper DNA replication, as well as a partition system that actively segregates plasmids to new daughter cells. We genetically screened for E. coli chromosomal mutations that influenced P1 stability and identified a novel temperature-sensitive allele of the dnaB helicase gene (dnaB277) that replaces serine 277 with a leucine residue (DnaB S277L). This allele conferred a severe temperature-sensitive phenotype to the host; dnaB277 cells were not viable at temperatures above 34°C. Shifting dnaB277 cells to 42°C resulted in an immediate reduction in the rate of DNA synthesis and extensive cell filamentation. The dnaB277 allele destabilized P1 plasmids but had no significant influence on the stability of the F low-copy-number plasmid. This observation suggests that there is a specific requirement for DnaB in P1 plasmid maintenance in addition to the general requirement for DnaB as the replicative helicase during elongation.

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Percival-Smith, A., and J. Segall. "Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 7 (July 1987): 2484–90. http://dx.doi.org/10.1128/mcb.7.7.2484.

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We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.

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Percival-Smith, A., and J. Segall. "Increased copy number of the 5' end of the SPS2 gene inhibits sporulation of Saccharomyces cerevisiae." Molecular and Cellular Biology 7, no. 7 (July 1987): 2484–90. http://dx.doi.org/10.1128/mcb.7.7.2484-2490.1987.

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We found that the introduction into a yeast cell of a high-copy-number plasmid containing the 5' end of the SPS2 gene, a sporulation-specific gene of Saccharomyces cerevisiae, led to a reduction in the efficiency of spore formation. The plasmid pAP290, which contains the sequence from -138 to +152 of the SPS2 gene, caused a fivefold reduction in spore formation; the presence of the plasmid had no effect on transcription of the chromosomal SPS2 gene. A plasmid containing only the sequence upstream of the TATA box of the SPS2 gene (-350 to -68) was unable to inhibit the completion of sporulation, whereas the downstream sequence, from -70 to +404, although unable by itself to inhibit sporulation, could do so when provided with an upstream fragment containing the CYC1 upstream activation sequence. Deletion of 22 base pairs from pAP290, which introduced a frameshift after codon 17 of the SPS2 gene and reduced the open reading frame to 26 amino acids, generated a plasmid (pAP290 delta Pst) which could no longer inhibit sporulation. The SPS2 inserts of pAP290 and pAP290 delta Pst were found to direct equivalent levels of sporulation-specific transcription. We conclude from these results that the presence of both the SPS2 promoter (or a substitute promoter) and the initial coding sequence of the SPS2 gene is required in the high-copy-number plasmid to generate the asporogenous phenotype. We speculate that the accumulation of a protein containing the amino-terminal portion of the SPS2 gene product, synthesized from the transcripts of the truncated plasmid-borne copies of the SPS2 gene, prevents ascus formation.

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Vila, Pau, Jose L. Corchero, Antoni Benito, and Antonio Villaverde. "Ammonium-Mediated Reduction of Plasmid Copy Number and Recombinant Gene Expression in Escherichia coli." Biotechnology Progress 10, no. 6 (November 1994): 648–51. http://dx.doi.org/10.1021/bp00030a010.

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Robertson, Gregory T., Ann Reisenauer, Rachel Wright, Rasmus B. Jensen, Allen Jensen, Lucille Shapiro, and R. Martin Roop. "The Brucella abortus CcrM DNA Methyltransferase Is Essential for Viability, and Its Overexpression Attenuates Intracellular Replication in Murine Macrophages." Journal of Bacteriology 182, no. 12 (June 15, 2000): 3482–89. http://dx.doi.org/10.1128/jb.182.12.3482-3489.2000.

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ABSTRACT The CcrM DNA methyltransferase of the α-proteobacteria catalyzes the methylation of the adenine in the sequence GAnTC. Like Dam in the enterobacteria, CcrM plays a regulatory role in Caulobacter crescentus and Rhizobium meliloti. CcrM is essential for viability in both of these organisms, and we show here that it is also essential in Brucella abortus. Further, increased copy number of the ccrM gene results in striking changes inB. abortus morphology, DNA replication, and growth in murine macrophages. We generated strains that carryccrM either on a low-copy-number plasmid (strain GR131) or on a moderate-copy-number plasmid (strain GR132). Strain GR131 has wild-type morphology and chromosome number, as assessed by flow cytometry. In contrast, strain GR132 has abnormal branched morphology, suggesting aberrant cell division, and increased chromosome number. Although these strains exhibit different morphologies and DNA content, the replication of both strains in macrophages is attenuated. These data imply that the reduction in survival in host cells is not due solely to a cell division defect but is due to additional functions of CcrM. Because CcrM is essential in B. abortus and increased ccrMcopy number attenuates survival in host cells, we propose that CcrM is an appropriate target for new antibiotics.

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Kazic, T., and D. E. Berg. "Context effects in the formation of deletions in Escherichia coli." Genetics 126, no. 1 (September 1, 1990): 17–24. http://dx.doi.org/10.1093/genetics/126.1.17.

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Abstract We have examined the frequency with which identical deletions are formed in different chromosomal contexts. A panel of six mutant bla genes containing palindrome/direct repeat structures were moved from pBR322 to three locations: at lambda att, at chromosomal lac, and at F'lac. Deletion of the palindromes and one of the direct repeats results in reversion to Ampr. The frequency of deletion for all alleles declines beyond the reduction in copy number when they are moved from the multicopy plasmid environment to a single-copy chromosome. The magnitude of the declines varies in an allele-specific and location-specific manner. Our data support the hypothesis that context can influence the frequency of mutation independent of the immediate DNA sequence.

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McGrath, Stephen, Jos F. M. L. Seegers, Gerald F. Fitzgerald, and Douwe van Sinderen. "Molecular Characterization of a Phage-Encoded Resistance System in Lactococcus lactis." Applied and Environmental Microbiology 65, no. 5 (May 1, 1999): 1891–99. http://dx.doi.org/10.1128/aem.65.5.1891-1899.1999.

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ABSTRACT A specific fragment of the genome of Tuc2009, a temperate lactococcal bacteriophage, was shown to contain several open reading frames, whose deduced protein products exhibited similarities to proteins known to be involved in DNA replication and modification. In this way, a putative single-stranded binding protein, replisome organizer protein, topoisomerase I, and a methylase were identified. When the genetic information coding for the putative replisome organizer protein of Tuc2009, Rep2009, was supplied on a high-copy-number plasmid vector, it was shown to confer a phage-encoded resistance (Per) phenotype on its lactococcal host UC509.9. The presence of this recombinant plasmid was shown to cause a marked reduction in Tuc2009 DNA replication, suggesting that the observed phage resistance was due to titration of a factor, or factors, required for Tuc2009 DNA replication. Further experiments delineated the phage resistance-conferring region to a 160-bp fragment rich in direct repeats. Gel retardation experiments, which indicated a protein-DNA interaction between this 160-bp fragment and the Rep2009protein, were performed. UC509.9 strains harboring plasmids with randomly mutated versions of this fragment were shown to display a variable phage resistance phenotype, depending on the position of the mutations.

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Kwong, Stephen M., Ronald A. Skurray, and Neville Firth. "Replication Control of Staphylococcal Multiresistance Plasmid pSK41: an Antisense RNA Mediates Dual-Level Regulation of Rep Expression." Journal of Bacteriology 188, no. 12 (June 15, 2006): 4404–12. http://dx.doi.org/10.1128/jb.00030-06.

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ABSTRACT Replication of staphylococcal multiresistance plasmid pSK41 is negatively regulated by the antisense transcript RNAI. pSK41 minireplicons bearing rnaI promoter (P rnaI ) mutations exhibited dramatic increases in copy number, approximately 40-fold higher than the copy number for the wild-type replicon. The effects of RNAI mutations on expression of the replication initiator protein (Rep) were evaluated using transcriptional and translational fusions between the rep control region and the cat reporter gene. The results suggested that when P rnaI is disrupted, the amount of rep mRNA increases and it becomes derepressed for translation. These effects were reversed when RNAI was provided in trans, demonstrating that it is responsible for significant negative regulation at two levels, with the greatest repression exerted on rep translation initiation. Mutagenesis provided no evidence for RNAI-mediated transcriptional attenuation as a basis for the observed reduction in rep message associated with expression of RNAI. However, RNA secondary-structure predictions and supporting mutagenesis data suggest a novel mechanism for RNAI-mediated repression of rep translation initiation, where RNAI binding promotes a steric transition in the rep mRNA leader to an alternative thermodynamically stable stem-loop structure that sequesters the rep translation initiation region, thereby preventing translation.

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Dissertations / Theses on the topic "Plasmid copy number reduction A"

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Dillingham, Mark Simon. "Biochemical studies on DNA helicases." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312245.

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Zealey, Gavin Ross. "Plasmid copy number in Saccharomyces cerevisiae." Thesis, University of Bath, 1985. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333232.

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Studies were made of 2 mum based chimaeric plasmid copy number in Saccharomyces cerevisiae. A plasmid (pAYE56) containing three selectable genes in yeast (yeast LEU2, bacterial CAT and HSV-1 - TK) was constructed to reflect changes in copy number. Yeast transformants could be grown under three selection regimes and plasmid copy number estimated. During selective growth for the LEU2 gene there are about 20 plasmids per cell. This increases to about 100 during selective growth for the TK gene and furthermore the copy number can be controlled by the stringency of selection. Simultaneous selection for the TK and CAT genes may lead to a further increase (160 copies). Two models are proposed to account for these increases. The amplification model proposes plasmid replication without cell growth whilst the selection model suggests that plasmid copy number varies greatly in a population of transformants and cells with a high copy number are selected for growth under the TK/CAT selection conditions. Whilst the mechanism of copy number increase is unclear, an attempt was made to relate the expression of a heterologous gene (Human alpha2-IFN) to gene dosage using the promotion and secretion signals of the alpha-factor gene. Production of intracellular alpha2-IFN was unaffected by copy number whilst secreted material showed a 100 fold increase over a ten fold increase in gene dosage. Attempts were made to isolate plasmid copy number mutants. After mutagenesis (of cells or plasmid) transformants were selected under conditions for simultaneous over-expression of the TK and CAT genes. Mutants capable of growth under these conditions were obtained. In one group the mutant phenotype was lost upon curing but did not return upon retransformation. In a second group a chromosomal mutation was isolated. Plasmid copy number estimates indicated that this was unchanged however. Alternative strategies are discussed for the isolation of mutants.

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Erdmann, Natalie. "Identification of high-copy-number inhibitors of P1 plasmid stability." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp01/MQ34055.pdf.

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Eldek, Ahmed. "Antibiotic-Regulated Plasmid Copy Number Variation: A Driver of Antibiotic Resistance?" Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-388523.

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Plasmids are small circular DNA molecules within bacterial cells that are separated from the bacterial chromosome and replicate independently. Also, they play a crucial role in the dissemination of antibiotic resistance genes among bacteria through horizontal gene transfer. They can be present in many copies within host cell, which is known as plasmid copy number. Plasmids can regulate their own copy number by different mechanisms. Additionally, the selective pressure can also play a pivotal role in determining plasmid copy number. The presence of antibiotics in the surrounding environment can drive variations of plasmid copy number. In this study, we examined plasmid copy number variations of multidrug resistance plasmids in presence of antibiotics by using EvaGreen® - based multiplexed digital droplet PCR. We could observe that cultures of Klebsiella pneumoniae and Escherichia coli harboring multidrug resistance plasmids grown in presence of sub-MIC concentrations of the antibiotics did not show high variations in plasmid copy numbers. On the other hand, mutants of K. pneumoniae selected for increased antibiotic resistance showed high increases in copy number of a multidrug-resistance plasmid.

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Perry, Clarice Lorraine. "Specialized Replication Operons Control Rhizobial Plasmid Copy Number in Developing Symbiotic Cells." BYU ScholarsArchive, 2015. https://scholarsarchive.byu.edu/etd/6167.

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The rhizobium – legume symbiosis is a complex process that involves genetic cooperation from both bacteria and plants. Previously, our lab described naturally occurring accessory plasmids in rhizobia that inhibit this cooperation. A transposon mutagenesis was performed on the plasmids to detect the genetic factor that blocked nitrogen fixation. Several of the plasmids were found to possess a replication operon that when disrupted by transposon insertion, restored symbiotic function. This study describes an in-depth investigation into one of those plasmids, pHRC377, and into its replication operon. The operon, which we have called repA2C2, comes from the repABC family of replication and partitioning systems commonly found in alphaproteobacteria. In this study we show that this operon is not necessary for pHRC377 replication in LB culture or free living cells, but is necessary for plasmid amplification in the plant, specifically during rhizobial differentiation into nitrogen fixing bacteroids. We also show how the other repABC type operons on pHRC377 function in relation to plasmid maintenance and copy number during endoreduplication and how they do not have the same phenotypic effect as repA2C2.

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Ahn, Yong-tae. "Studies on the 2 micron plasmid encoded components for plasmid stability and copy number control in Sacchromyces [i.e. Saccharomyces] cerevisiae /." Digital version accessible at:, 1998. http://wwwlib.umi.com/cr/utexas/main.

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Jansen, Yvette. "Characterisation of a high copy number mutant pAL5000 origin of replication." Thesis, Stellenbosch : Stellenbosch University, 2001. http://hdl.handle.net/10019.1/52159.

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Thesis (MScMedSc)--Stellenbosch University, 2001.
ENGLISH ABSTRACT: The plasmid pAL5000 is a mycobacterial plasmid isolated from Mycobacterium fortuitum. It is a low copy number plasmid, which replicates in both fast growing (e.g. M. smegmatis) and slow growing (e.g. M. bovis BCG) mycobacteria. Most mycobacterial-E. coli shuttle vectors utilise the pAL5000 origin of replication. The minimum replicon consists of ORF1 (RepA), ORF2 (RepB) and the origin of replication. Dr W.R. Bourn created an E. coli-mycobacterial vector based on the pAL5000 origin of replication (pORI) and then subjected it to semi-random mutagenesis. A high copy number mutant was identified (pHIGH) and the causative mutation was tentatively identified as a 3bp deletion situated just upstream of repB. This work describes the further characterisation of the mutant plasmid. Firstly, it was shown by retransforming M. smegmatis with both the original and mutant plasmids (pORI and pHIGH), that the mutation causing the increased copy number was plasmid-encoded and not on the chromosome. Following this, it was demonstrated by simple subcloning of the region that carries the 3 bp deletion, that other pAL5000-based vectors could be converted to high copy number. In addition to this, the subcloned region was sequenced and the nature of the mutations was confirmed. The subcloning experiment confirmed that the 3bp deletion caused the high copy number phenotype. Following this, the exact copy number of pHIGH and the relative increase in copy number was determined. From this, the copy number of pORI could also be determined. The plasmid pHIGH has a copy number of approximately 54, compared to the 8 of pORI (a relative increase by a factor of 7). Because it is important for researchers to know the characteristics of the vectors that they use, especially the influence it will have on its host, stability tests and growth curves were also performed. It was seen that the higher copy number did not markedly increase the stability, however, this is because pORI is already extremely, and unexpectedly, stable in the host M. smegmatis. According to the growth curves, the increased copy number has little effect on the growth of the host M. smegmatis. Possible mechanisms for the increased copy number were then investigated. By using a promoter probe vector, the possible existence of a promoter situated between the two open reading frames of pAL5000 (repA and repB) was investigated. It was thought that the mutation might have created, or changed an existing promoter, situated between repA and repB. The results showed, however, that in both pORI and pHIGH there might be a very weak promoter upstream of repB, but the mutation did not cause any change that was measurable by the method that was used. A further possibility was that the mutation caused a change in the RNA secondary structure, which might then have an effect on the translational efficiency of RepB. It was found that the 3bp deletion in pHIGH causes a change in the local RNA secondary structure around the ribosomal binding site and the start codon, when compared to pORI (wild type). This change may cause the translation initiation rate of RepB to be different between pHIGH and pORI. Ultimately it would lead to a different ratio of RepA and RepB in the cell.
AFRIKAANSE OPSOMMING: Die plasmied pAL5000 is ‘n mikobakteriele plasmied wat vanuit Mycobacterium fortuitum gei'soleer is. Dit is ‘n lae kopie-getal plasmied wat in beide vinnig groeiende (bv. M. smegmatis) en stadig groeiende (bv. M. bovis BCG) mikobakteriee kan repliseer. Die meeste mikobakteriele-E. coli shuttle vektore gebruik die pAL5000 oorsprong van replisering. Die minimum replikon bestaan uit ORF1 (RepA), ORF2 (RepB) en die oorsprong van replisering. Dr. W.R. Bourn het ‘n E. coli-mikobakteriele vektor gemaak wat gebaseer is op die pAL5000 oorsprong van replisering (pORI), en dit onderwerp aan semi-random mutagenese. ‘n Hoë kopie-getal mutant is gei'dentifiseer (pHIGH) en die mutasie hiervoor verantwoordelik was tentatief gei'dentifiseer as ‘n 3bp delesie, net stroomop van repB. Die projek beskryf die verdere karakterisering van die mutante plasmied. Eerstens, deur M. smegmatis te hertransformeer met die plasmied DNA (pORI en pHIGH), is dit bewys dat dit mutasie wat die toename in kopie-getal veroorsaak, deur die plasmied gekodeer word, en dat dit nie ‘n mutasie op die chromosoom is nie. Hierna is dit deur eenvoudige subklonering bewys dat die gedeelte wat die 3bp delesie dra, ander pAL5000-gebaseerde vektore ook kan verander in ‘n hoër kopie-getal. Die sub-klonerings eksperiment het ook bewys dat die 3 bp delesie die oorsaak is vir die hoë kopie-getal fenotipe. Volgende is die presiese kopie-getal van pHIGH en die relatiewe toename in kopiegetal bepaal. Die kopie-getal van pORI kon vanaf hierdie data bepaal word. Die plasmied pHIGH het ‘n kopie-getal van ongeveer 54 in M. smegmatis, in vergelyking met die 8 van pORI (‘n relatiewe toename met ‘n faktor van 7). Aangesien dit vir navorsers belangrik is om die eienskappe van die vektore wat hulle gebruik, te ken, en veral die invloed wat dit op die gasheer sal hê, is stabiliteits toetse, en groeikurwes gedoen. Die hoër kopie-getal het nie die stabiliteit werklik verbeter nie, maar dit is omdat pORI alreeds uiters stabiel is in die gasheer M. smegmatis. Volgens die groeikurwes het die toename in kopie-getal ‘n minimale effek op die groei van die gasheer M. smegmatis. Moontlike meganismes vir die hoër kopie-getal is ook ondersoek. Die moontlike bestaan van ‘n promoter tussen die twee oop-leesrame van pAL5000 (repA en repB) is ondersoek deur gebruik te maak van ‘n “promoter probe” vektor. Die mutasie kon moontlik ‘n promoter geskep het, of ‘n bestaande een tussen repA en repB verander het. Die resultate het gewys dat daar in beide pORI en pHIGH moontlik ‘n baie swak promoter stroomop van repB is, maar die mutasie het nie enige veranderinge veroorsaak wat meetbaar was met die metode wat gebruik is nie. ‘n Verdere moontlikheid was dat die mutasie ‘n verandering in die RNA sekondere struktuur kon veroorsaak het, en dit mag ‘n effek hê op die translasie effektiwiteit van RepB. Daar is gevind dat, in vergelyking met pORI, het die 3bp delesie in pHIGH ‘n verandering in die lokale RNA sekondere struktuur rondom die ribosomale bindings posisie en die begin-kodon veroorsaak. Die verandering mag veroorsaak dat die translasie inisiasie tempo van RepB verskillend is vir pORI en pHIGH. Uiteindelik sal dit lei tot ‘n heeltemal ander verhouding van RepA en RepB in die sel.

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Lyle, Keenan Harris. "Comparison of plasmids from clinical Lactobacillus strains." University of the Western Cape, 2018. http://hdl.handle.net/11394/6397.

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Magister Scientiae - MSc (Biotechnology)
The vaginal mucosa is dominated by Gram positive, rod shaped lactobacilli which serve as a natural barrier against infection. In both healthy and BV infected women Lactobacillus crispatus and Lactobacillus jensennii has been found to be the predominant Lactobacillus species. Many studies have been conducted to assess factors influencing lactobacilli dominance in the vaginal microbiome. However, no study has evaluated the impact of plasmids on the vaginal lactobacilli. In the present study two plasmids, pLc17 and pLc4, isolated from vaginal Lactobacillus species of both healthy and BV infected women were characterized. pLc4 was present in both Lactobacillus crispatus and Lactobacillus jensennii while pLc17 was only present in Lactobacillus crispatus. pLc17 (16663 bp in size) encoded a ribonucleotide diphosphate reductase (RNR), a filamentation induced by cAMP-like (FIC-like) protein and numerous mobile elements. The FIC-like protein may assist pLc17 to persist within the bacterial population, while RNR is commonly associated with phages and may indicate phage infection. pLc4 (4224 bp in size) encodes for a replication initiator protein and a plasmid partitioning protein. The replication protein on pLc4 shows 44% identity with the replication initiation protein of pSMQ173b_03. On further phylogenetic and sequence analysis with other Rolling Circle Replication (RCR) plasmids, pLc4 appears to be novel as the plasmid shows a low degree of similarity to these RCR plasmids. pLc17 appears to carry both a RCR replicon as well as a theta replicon, similar to pIP501, the broad-host-range plasmid from Bacillus subtilis. The relative Plasmid Copy Number (PCN) for pLc4 and pLc17 was analysed using quantitative polymerase chain reaction (qPCR) for the healthy state relative to the disease state from twentyeight vaginal swab samples obtained from the National Institute for Communicable Diseases (NICD). The relative PCN for pLc4 and pLc17 had a fold increase of ~2.803 and ~1.693, respectively in the healthy patient samples relative to BV infected patient samples. However, there were not found to be significant differences when taking the standard error into account Due to the novelty of these plasmids further analysis and characterisation is required for both plasmids, to establish what role they may play in the health of the vaginal milieu.

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Menil, Sidiky. "Cascade bi-enzymatique autosuffisante in vivo : le jeu des plasmides." Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0040.

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Une attention croissante est portée aux cascades multi enzymatiques pour l’élaboration de procédés de synthèse plus efficaces. Cependant, le contrôle de l’expression hétérologue de plusieurs gènes dans un même hôte s’avère difficile et peut mener à un déséquilibre du flux réactionnel. Pour exploiter au mieux les avantages d’une cascade in vivo, il est nécessaire d’ajuster les activités de chaque étape, et de construire des catalyseurs cellulaires capables de programmer la stœchiométrie des enzymes. Nous avons développé dans ce projet une approche originale pour moduler le ratio de deux enzymes in cellulo en jouant sur le nombre de copies de plasmides par cellule (PCN). Nous avons choisi comme modèle un système autosuffisant associant une Alcool Déshydrogénase (ADH) et une Baeyer-Villiger MonoOxygenase (BVMO), NADP(H)-dépendantes. Plusieurs plasmides recombinants portant les deux gènes ont été conçus et combinés dans E. coli. Les souches de co-expression construites ont été comparées en termes de PCN, de production d’enzymes et d’activité. Nous avons montré l’importance d’un choix judicieux de la combinaison de plasmides ainsi que l’existence d’une corrélation entre ratios d’enzymes et activité. Nos biocatalyseurs s’étendent sur une gamme allant du système inactif à un système affichant un TTN d’environ 6000. Ce système a permis la synthèse de lactones d’intérêt industriel, la dihydrocoumarine et la caprolactone, à partir d’indanol et de cyclohexanol. Enfin, sur ce modèle de combinaison de plasmides, trois nouveaux biocatalyseurs cellulaires, associant l’ADH à diverses BVMOs, ont été créés afin d’élargir la gamme d’esters et de lactones synthétisables à partir d’alcools
Growing attention is paid to multienzymatic cascades to develop more efficient synthetic processes. However, in in cellulo process, the control of the simultaneous heterologous expression of several genes in the same host is often difficult and can lead to imbalances in the reaction flow. To exploit the benefits of cascades, activities of each step have to be adjusted and thus, cellular biocatalysts capable of programming enzymes stoichiometry have to be constructed. In this work, to modulate the stoichiometry of two enzymes in vivo, we developed an original approach based on the copy number per cell of plasmids (PCN) used as vectors. The PCN is regulated in bacteria by three main mechanisms leading, according to the replicon, to low, medium or high PCN. As proof of concept, we chose a self-sufficient system combining an Alcohol Dehydrogenase (ADH) and a Baeyer-Villiger MonoOxygenase (BVMO), both NADP(H)-dependent. Several recombinant plasmids harboring both genes were designed and combined in E. coli. Coexpression strains constructed were compared in terms of PCN, enzyme production and activity. We showed the importance of a judicious choice of plasmids combination and the existence of a correlation between enzymes ratios and activity. Our biocatalysts ranged from an inactive system to a system with a TTN of about 6000. This system allowed the synthesis of lactones of industrial interest, dihydrocoumarin and caprolactone, via double oxidation of indanol and cyclohexanol. Finally, based on this plasmids combination model, three new cellular biocatalysts combining ADH with various BVMOs were designed to broaden the range of esters and lactones synthesizable from alcohols

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Benevides, Kristina, Oscar Broström, Kalman Grim Elison, Hugo Swenson, Andrei Vlassov, and Josefin Ågren. "Stabil och antibiotikafri läkemedelsproduktion i rekombinant Escherichia coli." Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-323719.

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Den här rapporten presenterar ett antibiotikafritt, stabilt och kromosombaserat expressionssystem för läkemedelsproduktion i Escherichia coli på beställning av företaget Affibody AB. E. coli-stammen BL21(DE3) valdes som värdorganism för expressionssystemet. Systemet består av en genkassett som innehåller en T7-promotor, en 5′-UTR från genen ompA och en terminatorsekvens från RNA-operonet rrnB. Fyra kopior av genkassetten ska integreras i pseudogenerna caiB, yjjM, hsdS och yjiV. En datormodell som modellerar det egentliga kopietalet i cellerna har skapats i mjukvaran MATLAB, vilket visar att det uppskattas vara maximalt 32 kopior av genkassetten per cell på grund av replikation av kromosomen. Ett högt pH i fermentorn; att använda fed-batch och blandade kolhydratkällor; och att använda stammen BL21(DE3) minskar acetatproduktionen i cellen. En lägre acetatproduktion kan leda till en högre produkthalt. En proteinutbytesmodell för mjukvaran MATLAB har konstruerats för att uppskatta koncentrationen av Affibody®-molekylen i en E. coli cell.

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Books on the topic "Plasmid copy number reduction A"

1

Erdmann, Natalie. Identification of high-copy-number inhibitors of P1 plasmid stability. Ottawa: National Library of Canada, 1998.

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2

Xu, Donghong. Searching for high-copy-number suppressors of a E. coli growth defect caused by the excess of a plasmid partition protein. Ottawa: National Library of Canada, 1996.

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Book chapters on the topic "Plasmid copy number reduction A"

1

Friehs, Karl. "Plasmid Copy Number and Plasmid Stability." In New Trends and Developments in Biochemical Engineering, 47–82. Berlin, Heidelberg: Springer Berlin Heidelberg, 2003. http://dx.doi.org/10.1007/b12440.

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Million-Weaver, Samuel, David L. Alexander, Jennifer M. Allen, and Manel Camps. "Quantifying Plasmid Copy Number to Investigate Plasmid Dosage Effects Associated with Directed Protein Evolution." In Methods in Molecular Biology, 33–48. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-61779-483-4_3.

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Meyer, Richard J., Lung-Shen Lin, Kyunghoon Kim, and Michael A. Brasch. "Broad Host-Range Plasmid R1162: Replication, Incompatibility, and Copy-Number Control." In Plasmids in Bacteria, 173–88. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4613-2447-8_16.

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Miyake, Masato, Hiroshi Nagai, Makoto Shirai, Ryuichiro Kurane, and Yasuo Asada. "A High-Copy-Number Plasmid Capable of Replication in Thermophilic Cyanobacteria." In Twentieth Symposium on Biotechnology for Fuels and Chemicals, 267–75. Totowa, NJ: Humana Press, 1999. http://dx.doi.org/10.1007/978-1-4612-1604-9_25.

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Kelly, Michael D., and Joel E. Mortensen. "A Low-Copy Number Plasmid Mediating β-Lactamase Production by Xanthomonas Maltophilia." In Antimicrobial Resistance, 71–80. Boston, MA: Springer US, 1995. http://dx.doi.org/10.1007/978-1-4757-9203-4_6.

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Futcher, Bruce. "The Copy Number Control System of the 2μm Circle Plasmid of Saccharomyces Cerevisiae." In Genetic Engineering, 33–48. Boston, MA: Springer US, 1989. http://dx.doi.org/10.1007/978-1-4615-7084-4_3.

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Kirinaka, Hideyo, Shinji Iijima, and Takeshi Kobayashi. "A ts-SV40 Based New Mammalian Plasmid Vector and Its Copy Number Control by Temperature Change." In Animal Cell Technology: Basic & Applied Aspects, 79–84. Dordrecht: Springer Netherlands, 1993. http://dx.doi.org/10.1007/978-94-011-2044-9_11.

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Liu, Yen-Ting, Saumitra Sau, Chien-Hui Ma, Aashiq H. Kachroo, Paul A. Rowley, Keng-Ming Chang, Hsiu-Fang Fan, and Makkuni Jayaram. "The Partitioning and Copy Number Control Systems of the Selfish Yeast Plasmid: An Optimized Molecular Design for Stable Persistence in Host Cells." In Plasmids, 325–47. Washington, DC, USA: ASM Press, 2015. http://dx.doi.org/10.1128/9781555818982.ch19.

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Conference papers on the topic "Plasmid copy number reduction A"

1

Mendoza-Chamizo, Belén, Rocίo González-Soltero, and Emilia Botello. "Plasmid copy number quantification by spectrofluorometry." In MICROBES IN APPLIED RESEARCH - Current Advances and Challenges. WORLD SCIENTIFIC, 2012. http://dx.doi.org/10.1142/9789814405041_0087.

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Hajimorad, Meghdad, and Jeffrey A. Gralnick. "Plasmid copy number variation of a modular vector set in Shewanella oneidensis MR-1." In 2020 42nd Annual International Conference of the IEEE Engineering in Medicine and Biology Society (EMBC) in conjunction with the 43rd Annual Conference of the Canadian Medical and Biological Engineering Society. IEEE, 2020. http://dx.doi.org/10.1109/embc44109.2020.9176029.

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Kong, Jinhwa, Jaemoon Shin, Jungim Won, Jeehee Yoon, and Unjoo Lee. "An efficient noise reduction method for copy number variations detection from whole exome sequencing data." In 2016 International Conference on High Performance Computing & Simulation (HPCS). IEEE, 2016. http://dx.doi.org/10.1109/hpcsim.2016.7568451.

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Perry, Kristina, Kim Tsui, Clint Baldwin, Antonio de las Morenas, and Carol Rosenberg. "Abstract 2153: Copy number variants in histologically normal breast epithelium from reduction mammoplasty controls and breast cancer cases." In Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-2153.

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Nedyalkov, Ivaylo, Alec Cunningham, and Adam Lovell. "Effects of Cyclist Size and Position Within Formations on Drag and Side Force in the Presence of Cross Winds." In ASME-JSME-KSME 2019 8th Joint Fluids Engineering Conference. American Society of Mechanical Engineers, 2019. http://dx.doi.org/10.1115/ajkfluids2019-5476.

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Abstract In the absence of cross-winds, a cyclist can expend up to 90% of their energy to overcome drag and can save up to 30% of that energy if riding behind another cyclist. The aerodynamic forces acting on cyclists in the presence of cross wind have not been studied in much detail. The effect of the offset distances between cyclists on the aerodynamic forces has been investigated in the literature for configurations of two cyclists. In the present study, 1:11 scale models of two different cyclists were rapid-prototyped and tested in a wind tunnel. The effect of the size of the cyclist was studied by placing the larger cyclist model behind the smaller one; the smaller behind the larger one; and the larger model behind an identical (larger model) copy. The effect of position within the group was studied by measuring the forces on each of the four cyclists placed in a favorable formation. The results suggest that the size of the cyclist matters, particularly when the leading cyclist is smaller than the drafting cyclist, and the effect is more prominent for the side forces. The results also show that in a formation of four cyclists, the leading cyclist experiences minor drag reduction compared to riding alone. The second and third cyclists experience the largest force reductions within the group, and the fourth cyclist experiences force reduction, which is not as significant. The results appear to be dependent on the Reynolds number, but may still be valuable for racing strategies and recreational cycling.

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