Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P in continuous culture.
Plasmid stability of recombinant Pseudomonas sp. B13 FR1 pFRC20P, a strain capable of mineralizing 3- and 4-chlorobenzoate and 4-methylbenzoate, was investigated in continuous culture. The hybrid cosmid pFRC20P enables the strain to mineralize 4-methylbenzoate. Rapid plasmid loss was observed under nonselective conditions using 3-chlorobenzoate as the substrate. Plasmid stability decreased with increasing dilution rate. Despite the growth advantage of the generated plasmid free cells a total depletion of plasmid bearing cells was not observed. After approximately 50 generations the fraction of plasmid bearing cells reached a constant level of 10%, which was stably maintained during the next 25 generations. Cells from this stage were used to inoculate a new culture that resulted in a stable level of 50% plasmid bearing cells. By a temporary substrate change to selective conditions (4-methylbenzoate), this level could be further increased to 70%. Literature models on plasmid stability could not be applied to describe the experimental data. Therefore, a new but unstructured model was developed to describe the experimental results. The model is based on the existence of three subpopulations: a plasmid free one, an original plasmid bearing one with a growth disadvantage compared to plasmid free cells, and a second plasmid bearing subpopulation with increased stability that is generated from the original one and has a growth rate comparable to the plasmid free cells.
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Biochemical Engineering Division, GBF-Gesellschaft für Biotechnologische Forschung mbH, Mascheroder Weg 1, 38124 Braunschweig, Germany.
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Deckwer
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Hecht
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D001565:Benzoates / Q000378:metabolism; D001673:Biodegradation, Environmental; D019149:Bioreactors; D001709:Biotechnology; D002723:Chlorobenzoates / Q000378:metabolism; D008954:Models, Biological; D010957:Plasmids / Q000235:genetics*; D011549:Pseudomonas / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic
D001565:Benzoates; D002723:Chlorobenzoates; C036427:3-chlorobenzoic acid; C009504:4-toluic acid; C040768:4-chlorobenzoic acid