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Zawartość zarchiwizowana w dniu 2024-04-15

ANALYSIS OF IN SITU BEHAVIOUR OF GENETICALLY ENGINEERED BACTERIA IN STANDARDISED MICROCOSMS

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Activated sludge microcosms simulated the level of aeration. Nutrient makeup and microbial community structure associated with activated sludge reactors. Soil microcosms were initially sterilised, but maintained many of the physicochemical characteristics of soil. The fate and behaviour of the genetically engineered microorganisms (GEMs) were consistent within microcosms and allowed for comparisons to be made between microcosm types.

Polyclonal and monoclonal antibodies were specific for the GEMs when tested against closely related Pseudomonads and bacteria isolated from the microcosms. Nucleic acid probes were specific for recombinant deoxyribonucleic acid (DNA), did not hybridise to closely related microorganisms and isolated bacteria and were used in colony hybridisation procedures to detect plasmid DNA in putative transconjugant bacteria.

GEMs maintained population levels of 10{4} bacteria/ml sludge. In soil microcosms, the density of GEMs varied depending on physicochemical properties. The GEMs degraded substituted aromatic compounds present in both microcosms.

Recombinant DNA was stable in GEMs added to the microcosms both in the absence and presence of the aromatic compounds which they specifically degrade P. sp. strain FR1(pFRC20P) contains recombinant DNA necessary for the degradation of alkylbenzoates on both the chromosome and plasmid, pFRC20P. Transfer of this DNA was not detected in any microcosm. The plasmid was mobilised in vitro by helper plasmids at frequencies of 10{-5} transconjugants/recipient. P. putida (pWWO-EB62) has the modified catabolic pathway for ethylbenzoate present on the plasmid. Transconjugants arising by transfer of plasmid, pWWO-EB62, to recipient bacteria were observed in activated sludge microcosms at densities of 1000 bacteria/ml and in soil microcosms. The frequency of in vitro conjugative transfer of pWW0-EB62 to recipient bacteria was 1 to 0.1 transconjugants per donor cell.

Conjugative transfer of pWWO-EB62 was a nalysed on solid medium and continuous culture. On agar, the plasmid transfers to and is expressed in Pseudomonads group I and Escherichia coli. Other bacteria including Pseudomonads belonging to group II, III and IV did not act as recipients, either because the plasmid was not transferred or stably maintained. In continuous culture, the transfer rate of pWWO-EB62 from P. putida KT2440 (pWWO-EB62) to P. putida UWC1, was dependent on cell doubling time, the temperature, and degree of agitation.

Temat(-y)

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Zaproszenie do składania wniosków

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CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
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