Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Influence of immobilization on the stability of pTG201 recombinant plasmid in some strains of Escherichia coli.

The stability of pTG201 plasmid was examined by continuous culture in three genetically different Escherichia coli hosts. Two types of experiment were carried out, one with free cells and one with immobilized cells. When cells were cultivated in free continuous culture in the absence of antibiotic selection, the plasmid was maintained with various degrees of stability in the three host organisms. By contrast, in continuous culture with immobilized cells, plasmid pTG201 was stably maintained in the three strains. We showed that the increase in pTG201 stability in immobilized cells is due neither to plasmid transfer between immobilized cells nor to an increase of the plasmid copy number of immobilized cells. We also showed that plasmid-free cells, when coimmobilized and grown in competition with plasmid-containing cells, cannot overrun the culture.     

Alteration of the specificity of PvuII restriction endonuclease.

The restriction endonuclease PvuII which cleaves the sequence CAGCTG, at the position indicated by the arrow, was found to decrease its substrate specificity in the presence of organic solvents. Thirty-three sites, that we have named PvuII sites, were identified on the nucleotide sequence of pBR322 DNA. The new recognition sequences cleaved in pBR322 DNA, at the positions indicated by the arrows, were shown to be AAGCTG, GAGCTG, CNGCTG, CANCTG, CAGNTG, CAGCNG, CAGCTC and CAGCTT. (TAGCTG and the complementary sequence CAGCTA are not present in pBR322 DNA). From these recognition sequences, we deduced that PvuII activity recognizes and cleaves degenerate sequences which differ from the standard PvuII sequence CAGCTG at only one of the recognition site. Any substitution can occur at any one of the six positions in the hexanucleotide sequence. The optimum incubation medium for PvuII activity was found to be: 10-50 mM Tris-HCl, pH 8.5, 12-15 mM MgCl2, 50 mM NaCl, 10% ethanol + 10% dimethylsulfoxide (DMSO).     

Effect of environmental growth conditions on plasmid stability, plasmid copy number, and catechol 2,3-dioxygenase activity in free and immobilized Escherichia coli cells

In order to better understand the high plasmid stability in immobilized recombinant E. coli cells, the effects of dilution rate on the pTG201 plasmid stability, the copy number, and the catechol 2,3-dioxygenase (encoded by XyIE gene) production were, at first, studied in free E. coli W3101 continuous cultures in minimal media. It was found that decreasing specific growth rate increased the plasmid copy number and the catechol 2,3-dioxygenase activity but the stability decreased. In continuous culture with immobilized cells, an increase was shown in plasmid copy number and catechol 2,3-dioxygenase activity probably due to the distribution of growth in the gel beads. Besides mechanical properties of gel beads which may allow limited cell divisions, the increase in plasmid copy number is involved in enhanced plasmid stability in immobilized cells. In the same way, an experiment conducted in LB medium dealing with competition between pTG201-free and pTG201-containing E. coli B cells was described. It was shown that the competition was not more pronounced in gel bead compared to a free system. The effects of nutritional limitations on pTG201 plasmid stability and catechol 2,3-dioxygenase activity during chemostat cultivations in free and immobilized E. coli B cells were also investigated. It was found that immobilization of cells increased the stability of pTG201 even under glucose, nitrogen, or phosphate limited cultures. However in the case of magnesium depleted culture, pTG201 was shown to be relatively instable and a decrease in viable cell number during the immobilized continuous culture was observed. By contrast to the free system, the catechol 2,3-dioxygenase activity increased in immobilized cells under all culture conditions used.     

Effect of growing conditions of recombinantE.coli in carrageenan gel beads upon biomasse production and plasmid stability

Summary The biomass production and the plasmid stability of immobilizedE. coli cells in K-carrageenan gel beads were investigated in continuous cultures. Several factors, such as inoculum size, gel bead volume and gel concentration were examined in order to increase the cell concentration inside the immobilized cell reactor, and therefore to increase the overall productivity.     

Stability fluctuations of plasmid-bearing cells: immobilization effects

The maintenance of the plasmid vectors pTG201 and pTG206 (which both carry the Pseudomonas putida xylE gene) and pB lambda H3 in Escherichia coli hosts was studied in free and immobilized continuous cultures. pTG201, containing the strong lambda PR promoter, was more quickly lost than plasmid pTG206, containing the tetracycline resistance gene promoter. The instability of pTG201 seems to be related to high expression of the cloned xylE genet. Fluctuations in the proportion of pTG201-containing cells were observed in the free system, suggesting the appearance of adaptive descendants (with and without plasmid) from the initial strains. The loss of plasmid vectors from E. coli cells and the fluctuations in the proportion of plasmid-containing cells could be prevented by immobilizing plasmid-containing bacteria in carrageenan gel beads.     

Fatal Invasive Pulmonary Aspergillosis in COVID-19 Patient with Acute Myeloid Leukemia in Iran

Mycopathologia - Although patients with severe immunodeficiency and hematological malignancies has been considered at highest risk for invasive fungal infection, patients with severe pneumonia due...