Stability Of Recombinant Plasmids Research Articles

Stability of recombinant plasmids on the continuous culture of Bifidobacterium animalis ATCC 27536.

Bifidobacterium animalis ATCC 27536 represents among bifidobacteria a host-model for cloning experiments. The segregational and structural stabilities of a family of cloning vectors with different molecular weights but sharing a common core were studied in continuous fermentation of the hosting B. animalis without selective pressure. The rate of plasmid loss (R) and the specific growth rate difference (delta mu) between plasmid-free and plasmid-carrying cells were calculated for each plasmid and their relationship with plasmid size was studied. It was observed that both R and the numerical value of delta mu increased exponentially with plasmid size. The exponential functions correlating the specific growth rate difference and the rate of plasmid loss with the plasmid molecular weight were determined. Furthermore, the smallest of the plasmids studied, pLAV (4.3-kb) was thoroughly characterized by means of its complete nucleotide sequence. It was found that it contained an extra DNA fragment, the first bifidobacterial insertion sequence characterised, named IS 1999.

Stability of Recombinant Plasmids in Transgenic Microorganisms under Different Environmental Conditions

The copy number of R plasmids weakly depends on the selective pressure of the respective antibiotic but does depend on the physiology of the host species and the type of plasmids and cloned genes, whose expression leads to a further load on the biosynthetic apparatus of cells. The last factor is critical in the maintenance of recombinant plasmids in transgenic microorganisms.

Study of stability of recombinant plasmids during the continuous culture ofBacillus stearothermophilus NUB3621 in nonselective medium

The optimal culture conditions for Bacillus stearothermophilus NUB3621 (BGSC 9A5) in chemostat were studied. The results obtained showed that the optimal culture conditions in terms of biomass concentration and maximum growth rate were 65 degrees C, pH 6.8 to 7.2. Dissolved oxygen became growth limiting at pO(2) levels below 10%. Furthermore, this strain was transformed with three new hybrid vectors (pPAM2, pPCH2, or pPLY2) constructed by cloning in pRP9, a plasmid based on the thermophilic replicon, pBC1, and three heterologous genes: the alpha-amylase gene from Bacillus licheniformis, the cholesterol oxidase gene from Streptomyces sp., and the lipase gene from Pseudomonas fluorescens. The influence of several fermentative conditions on segregational and structural stability of the recombinant B. stearothermophilus NUB3621 transformants was studied.The parameters of plasmid loss, that is, rate of plasmid loss (R) and specific growth rate difference (deltamu), were calculated. B. stearothermophilus NUB3621 carrying pRP9 showed great segregational stability in all the assayed conditions, exceeding more than 300 generations without significant plasmid loss, whereas NUB3621 carrying pPAM2, pPCH2, or pPLY2 exhibited relatively low plasmid stability. The segregational instability of the recombinant constructs increased by increasing the fermentation temperature, decreased by increasing the dilution rate, and was not affected by the level of dissolved oxygen. On the other hand, plasmid maintenance decreased in minimal medium if compared with the results obtained in complex medium. Restriction analyses carried out on cultures of NUB3621 carrying pRP9, pPAM2, pPCH2, or pPLY2, grown for 200 generations on nonselective media, revealed that all the clones tested contained the parental plasmids. These results indicate that the heterologous inserts did not affect the structural stability of the recombinant plasmids. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 507-514, 1997.

Cloning of sucrase operon with mini-Mu and plasmid-mediated metabolism of sucrose.

The in vivo cloning system based on mini-Mu derivatives was used for cloning the sucrase operon. We constructed several recombinant plasmids pJT21, pMM2324, pMM2325 with a complete sucrase operon. Two strains of Escherichia coli containing this plasmid replicon were able to grow on different concentrations of sucrose. The stability of recombinant plasmids was determined after cultivation under nonselective conditions. The stability after 5-d cultivation was higher than 75%.

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture

Effects of growth environment on recombinant plasmid stability in Saccharomyces cerevisiae grown in continuous culture.

A recombinant strain of Saccharomyces cerevisiae, containing a 2-microns-fragment-based plasmid (pYE alpha a4) was grown under non-selective conditions in continuous culture. The decrease in the population carrying the plasmid-encoded auxotrophic marker, LEU2, was examined under different physiological conditions. The difference in growth rate (delta mu) between plasmid-free and plasmid-containing cells and the rate of plasmid segregation (R) were determined using a non-linear regression technique. Loss rates were greater in defined glucose-limited cultures than in complex glucose-limited cultures. Plasmid loss was delta mu-dominated in cultures grown on defined media whereas delta mu and R were co-dominant in cultures grown on complex medium. Loss rates increased with increasing dilution rate in complex glucose-limited cultures. The reverse was found in defined glucose-limited cultures. Plasmid retention and loss kinetic determined from defined magnesium-limited cultures were not significantly different from those observed in defined glucose-limited cultures. Although plasmid retention in defined phosphate-limited culture was not significantly different from that in defined glucose-limited culture, reduced R and increased delta mu indicated an alternative physiological effect of phosphate limitation on plasmid stability.

Effect of growth rate and cultivation environments on cloned gene stability and the cloned gene product formation in Streptomyces lividans

The growth rate and environmental effects on the stability of recombinant plasmid, pDML6 containing β-lactamase gene, and the cloned gene product formation in Streptomyces lividans were studied. A maximum production rate of the cloned gene product was obtained at a specific growth rate 0.106 h −1 in glucose-limited chemostat cultivations without genetic selection pressure. Optimum environmental conditions for the recombinant plasmid stability and maximum formation rates of the cloned gene product were determined using continuous cultivations at the optimum specific growth rate. The fractions of plasmid harboring mycelium in prolonged cultivation up to 50 generations were varied from 77 to 95%. The recombinant plasmid was stably maintained in the host cells grown in different temperatures (24 to 36°C) and pH (6.0 to 8.5). The formation of the cloned gene product was optimum at pH 7.0 and 27°C, at which the maximum enzyme production rate was 0.82 kU g −1 h −1. Continuous cultivations varying the dissolved oxygen tension (10 to 80% air saturation) showed that the plasmids were maintained stably and the specific enzyme production rates were increased with increasing dissolved oxygen levels.

Construction of broad‐host‐range plasmids for the expression of heterologous genes in Acetobacter methanolicus B58

AbstractThe construction of different plasmids reported here on the basis of a broad‐host‐range RSF1010 replicon allows an efficient expression of heterologous genes in the acidophilic methanol‐assimilating bacterium Acetobacter methanolicus B58.The promoter‐probe vector pRS201 was used for the identification and isolation of the promoter containing sequences derived from the DNA of the Acetobacter phage Acm1. Further, this plasmid was coupled with the Escherichia coli promoters tac and pr creating the expression vectors pRS201tac and pRS201pr, respectively. After the insertion of the chloramphenicol acetyltransferase (cat) gene of the cloned promoters downstream, the chloramphenicol acetyltransferase (CAT) was determined in a cell‐free extract of both E. coli and A. methanolicus.Using E. coli promoters as well as promoters of the Acetobacter phage Acm1 arranged in tandem with the promoters of the heterologous genes to be expressed, the pectat lyase gene (ptlB) of Erwinia carotovora and the threonine A gene (thrA) of E. coli were successfully expressed in A. methanolicus.The stability of recombinant plasmids under various conditions in A. methanolicus strains was tested using antibiotic‐free media.

Improvement in segregational stability of recombinant plasmids by retransformation of E. coli host cells

Strains of E. coli JM103 harboring recombinant pUC8 plasmids were found to exhibit severe segregational instability in the absence of antibiotic selection. This was reversed by retransformation into fresh JM103. pUC8 was considerably more stable than its recombinant derivatives.

Increased stability of recombinant plasmids by Tn1000 insertion in chemostat cultures of recombinantEscherichia coli GT123

The stability of several pBR322-derived recombinant plasmids, carrying thethr operon fromEscherichia coli, was investigated in sulfur-limited chemostat cultures ofE. coli GT123. A marked increase in the segregational stability of one of these plasmids was observed. It is concluded that the increased stability was due to the spontaneous insertion of Tn1000 from the chromosome of the host into the plasmid.

Stability of recombinant plasmids in yeast

Yeasts represent one class of host for the production of recombinant proteins. Heterologous DNA is usually introduced into yeast strains in the form of multi-copy plasmids. During production, protein expression levels and rates are often limited by the stability of the recombinant organism. In this paper, we review the major factors affecting the stability of yeast strains containing multi-copy recombinant plasmids. Models for predicting plasmid loss are summarised, comparisons are made with relevant bacterial systems and strategies are described for overcoming such problems.

The effect of gene expression on copy number and heritable stability of recombinant plasmids in Bacillus subtilis

The effect of gene expression on copy number and heritable stability of recombinant plasmids in <i>Bacillus subtilis</i>

Effect of immobilization on stability of recombinant plasmids in gene-engineered microorganisms.

Effect of immobilization on stability of recombinant plasmids in gene-engineered microorganisms.

The effect of the growth environment on the stability of recombinant plasmids in Bacillus subtilis

Conference Article| April 01 1987 The effect of the growth environment on the stability of recombinant plasmids in Bacillus subtilis BRIAN LICKEN; BRIAN LICKEN 1Department of Industrial Microbiology, University College Dublin, Belfield, Dublin 4, Ireland Search for other works by this author on: This Site PubMed Google Scholar AIDEN. J. McLOUGHLIN; AIDEN. J. McLOUGHLIN 1Department of Industrial Microbiology, University College Dublin, Belfield, Dublin 4, Ireland Search for other works by this author on: This Site PubMed Google Scholar CAROLINE HUSSEY CAROLINE HUSSEY 1Department of Industrial Microbiology, University College Dublin, Belfield, Dublin 4, Ireland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1987) 15 (2): 282–283. https://doi.org/10.1042/bst0150282 Article history Received: September 26 1986 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation BRIAN LICKEN, AIDEN. J. McLOUGHLIN, CAROLINE HUSSEY; The effect of the growth environment on the stability of recombinant plasmids in Bacillus subtilis. Biochem Soc Trans 1 April 1987; 15 (2): 282–283. doi: https://doi.org/10.1042/bst0150282 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1987 Biochemical Society1987 Article PDF first page preview Close Modal You do not currently have access to this content.

The relationship between copy number and stability of recombinant plasmids in Bacillus subtilis

The relationship between copy number and stability of recombinant plasmids in <i>Bacillus subtilis</i>

Enhanced stability of a 2?-based recombinant plasmid in diploid yeast

The stability of a 2μ-based recombinant plasmid, pJDB219, has been compared in glucose-limited chemostat cultures of two haploid strains ofSaccharomycescerevisiae and a diploid derived from them. The stability of the recombinant plasmid differed in the two haploid hosts but was greatest in the diploid. Enhanced stability in the diploid is probably a function of both the increased copy number and reduced selective burden of the plasmid.

Application of recombinant DNA technology to the production of useful biomaterials.

It is now possible to clone and express a gene of interest in widely differing hosts. Among many the steps necessary for the production of useful biomaterials in industry, gene cloning, scale-up of the process, and isolation and purification of products are the most important. For the efficient cloning of specific genes, suitable host-vector systems should be selected. For the successful cultivation of recombinant organisms in a large fermentor, both expression of cloned genes and stability of recombinant plasmid are essential. For the isolation of products, protein secretion systems must be improved.

Stability of Recombinant Plasmids in Industrial Microorganisms

(1986). Stability of Recombinant Plasmids in Industrial Microorganisms. Critical Reviews in Biotechnology: Vol. 4, No. 3, pp. 263-277.

Stability of recombinant plasmids containing the ars sequence of yeast extrachromosomal rDNA in several strains of Saccharomyces cerevisiae

The mitotic stabilities of hybrid plasmid Rcp21/11, which contains the replicator of yeast rDNA, have been compared for four yeast host strains of different origins. In two related strains, Saccharomyces cerevisiae A62-1G-P188 and 1A-P3812 from the Peterhof genetic stocks, the plasmid was much more stable than in strains DC5 and GRF18 from the USA stocks. The enhanced mitotic stability of Rcp21/11 in these two yeast strains is obviously attributable to a higher rate of integration of the plasmid into the chromosomal rDNA repeats of the hosts. The centromeric locus CEN3 was inserted into Rcp21/11 because it provides high mitotic and meiotic stability of plasmids with yeast replicators, due to an ordered distribution of plasmids throughout cell division. Using the new centromeric plasmid Rcp CEN3, transformation of the four above-described yeast strains was carried out. It was found that, similarly to centromeric plasmids with other chromosomal replicators, Rcp CEN3 remains in the cell as a single copy. In strains DC5, GRF18 and A62-1G-P188 the mitotic stability of Rcp CEN3 was 20–50%, i.e., less than half that of plasmids containing locus CEN3 and other yeast repliiators, ars1, ars2 and the 2μ DNA replicator. The mitotic stability of Rcp CEN3 in strains 1A-P3812 (from the Peterhof genetic stocks) for individual clones reached 85%, i.e. close to that of the other plasmids. Genetic analysis showed that the capacity of strain 1A-P3812 to stably retain Rcp CEN3 has a recessive polygenic character. We suggest that the observed differences in mitotic stability of centromeric plasmid Rcp CEN3 between various yeast strains reflects the differences in activity of rDNA replicator in these strains. The nature of extrachromosomal rDNA circles, found in some strains of S. cerevisiae, is discussed from the point of view of the data.

A perspective on the application of genetic engineering: stability of recombinant plasmid.

A perspective on the application of genetic engineering: stability of recombinant plasmid.