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Master's Dissertation
DOI
10.11606/D.87.2014.tde-13052014-134327
Document
Author
Full name
Felipe Almeida da Silva
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Biondo, Ronaldo (President)
Correa, Benedito
Huenuman, Nilton Erbet Lincopan
Title in Portuguese
Avaliação da estabilidade genética conferida pelo lócus parB e força de expressão dos promotores deste sistema gênico.
Keywords in Portuguese
Citometria de fluxo
Expressão gênica
Genética bacteriana
Microscopia de fluorescência
Plasmídeos
Abstract in Portuguese
Em aplicações biotecnológicas, a estabilização de informações e a expressão gênica em bactérias Gram-negativas são fundamentais. Neste trabalho, analisou-se o efeito de estabilização plasmidial promovido pelo lócus parB (hok/sok) nas bactérias E. coli, C. metallidurans e C. necator transformadas com os plasmídeos pCM2, pBBR1MCS e seus respectivos derivados contendo o lócus parB, pCM3 e pBBPAR. As bactérias transformadas com pCM2 exibiram perda plamidial acentuada após 50 gerações, e as bactérias transformadas com pCM3 e pBBPAR exibiram estabilidade próxima a 100%, após 100 gerações. Também foi avaliada a força relativa de expressão dos promotores hok e sok em relação ao promotor lac, inseridos no plasmídeo pBBEGFP. Por microscopia de fluorescência, foi observado que regulado pelos promotores sok e hok, os transformantes expressaram EGFP. Utilizando citometria de fluxo, foi observado que o promotor hok apresentou maior força de expressão nas bactérias transformantes de E. coli e C. metalliduras; já o promotor sok apresentou maior força de expressão em C. necator.
Title in English
Evaluation of the genetic stability conferred by the parB locus and the expression strength of the promoters from this genic system.
Keywords in English
Bacterial genetics
Flow cytometry
Fluorescence microscopy
Gene expression
Plasmids
Abstract in English
In biotechnological applications, stabilization of information and gene expression in Gram-negative bacteria are important. In this work, we analyzed the effect of plasmid stabilization promoted by locus parB (hok/sok) in the bacteria E. coli, C. metallidurans and C. necator transformed with the plasmids pCM2, pBBR1MCS, and their derivatives containing the parB locus, pCM3 and pBBPAR. The Bacteria transformed with pCM2 showed a pronounced plasmidial loss after 50 generations, and bacteria transformed with pCM3 and pBBPAR exhibited stability close to 100 % after 100 generations. We also evaluated the relative expression strength of hok and sok promoters relative to the lac promoter, inserted into plasmid pBBEGFP. For fluorescence microscopy, it was observed that regulated by hok and sok promoter, transformants expressed EGFP. Using flow cytometry, it was observed that the hok promoter showed higher expression levels in E. coli and C. metallidurans transformants; as sok promoter showed higher expression level in C. necator.
 
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Publishing Date
2014-05-20
 
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