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Doctoral Thesis
DOI
https://doi.org/10.11606/T.87.2012.tde-06062012-111417
Document
Author
Full name
Oeber de Freitas Quadros
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2012
Supervisor
Committee
Vicente, Elisabete Jose (President)
Huenuman, Nilton Erbet Lincopan
Paula, Márcia Oliveira de
Perpetuo, Elen Aquino
Rivera, Irma Nelly Gutierrez
Title in Portuguese
Construção de sistemas bacterianos para a detecção de metais pesados em amostras ambientais.
Keywords in Portuguese
Bactérias
Bioluminescência
Biotecnologia
Meio ambiente
Metais pesados
Regulação gênica
Abstract in Portuguese
Visando a construção de três biossensores bacterianos para os metais mercúrio, arsênio e chumbo, Cupriavidus metallidurans CH34 foi escolhida para obtenção dos fragmentos de DNA (por PCR) correspondentes aos operons mer, ars e pbr. Os fragmentos foram inseridos à montante do gene EGFP, no plasmídeo pBB-EGFP, obtendo três novos plasmídeos: pGHg, pGAs e pGPb. Estes foram clonados em C. metallidurans CH34 e Escherichia coli DH5α. As linhagens recombinantes foram submetidas a várias situações de cultivo e diferentes intensidades de fluorescência, detectadas em microscopia e quantificadas por citometria de fluxo. As linhagens recombinantes C. metallidurans CH34 / pGHg, E. coli DH5α / pGHg, C. metallidurans CH34 / pGAs e C. metallidurans CH34 / pGPb mostraram-se eficazes e, consequentemente, poderão ser utilizadas em rápidos diagnósticos de amostras contaminadas por mercúrio, arsênio e chumbo.
Title in English
Construction of bacterial systems for the detection of heavy metals in environmental samples.
Keywords in English
Bacteria
Bioluminescence
Biotechnology
Environment
Gene regulation
Heavy metals
Abstract in English
Aiming at the construction of three bacterial biosensors for metals mercury, arsenic and lead, Cupriavidus metallidurans CH34 was chosen to obtain the DNA fragments (by PCR) corresponding to operons mer, ars and pbr. The fragments were inserted upstream of the EGFP gene, in plasmid pBB-EGFP, getting three new plasmids: pGHg, pGAs and pGPb. They were cloned in C. metallidurans CH34 and Escherichia coli DH5α. The recombinant strains were subjected to various conditions of cultivation. Different intensities of fluorescence were detected microscopy and quantified by flow cytometry. The recombinant strains C. metallidurans CH34 / pGHg, E. coli DH5α / pGHg, C. metallidurans CH34 / pGAs and C. metallidurans CH34 / pGPb were effective and therefore may be used in rapid diagnosis of samples contaminated by mercury, arsenic and lead.
 
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Publishing Date
2012-07-06
 
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