RESUMO
Methods for transfer of exogenous DNA into cells are essential for genetics and molecular biology, and the lack of effective methods hampers research on many different species of bacteria which have shown to be particularly recalcitrant to transformation. This review presents the progress on the development of methods for artificial transformation of bacteria with emphasis on different methodologies and the range of bacteria that can be transformed. The methods' strengths and weaknesses are described.
Assuntos
Bactérias/genética , Transformação Bacteriana , Bactérias/classificação , Proteínas de Bactérias/genética , DNA Bacteriano/genética , Genes Bacterianos , Engenharia Genética , Mutação , FenótipoRESUMO
The inducible Pm-xylS promoter system has proven useful for production of recombinant proteins in several gram-negative species and in high-cell-density cultivations of Escherichia coli. In this study we subjected a 24-bp region of Pm (including the -10 element) to random mutagenesis, leading to large mutant libraries in E. coli. Low-frequency-occurring Pm mutants displaying strongly increased promoter activity (up-mutants) could be efficiently identified by using beta-lactamase as a reporter. The up-mutants typically carried multiple point mutations positioned throughout the mutagenized region, combined with deletions around the transcription start site. Mutants displaying up to about a 14-fold increase in beta-lactamase expression (relative to wild-type Pm) were identified without loss of the inducible phenotype. The mutants also strongly stimulated the expression of two other reporter genes, luc (encoding firefly luciferase) and celB (encoding phosphoglucomutase), and were found to significantly improve (twofold) a previously optimized process for high-level recombinant production of the medically important granulocyte-macrophage colony-stimulating factor in E. coli under high-cell-density conditions. These results demonstrate the potential of using random mutagenesis of promoters to improve protein expression at industrial levels and indicate that targeted modifications of individual functional elements are not sufficient to obtain optimized promoter sequences.
Assuntos
Proteínas de Escherichia coli/biossíntese , Escherichia coli/genética , Expressão Gênica , Mutagênese , Regiões Promotoras Genéticas , beta-Lactamases/biossíntese , Sequência de Bases , Análise Mutacional de DNA , Proteínas de Escherichia coli/genética , Genes Reporter , Fator Estimulador de Colônias de Granulócitos e Macrófagos/biossíntese , Fator Estimulador de Colônias de Granulócitos e Macrófagos/genética , Luciferases/biossíntese , Luciferases/genética , Dados de Sequência Molecular , Fosfoglucomutase/biossíntese , Fosfoglucomutase/genética , Mutação Puntual , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Deleção de Sequência , Regulação para Cima , beta-Lactamases/genéticaRESUMO
Methodologies for introduction of DNA into cells are essential in molecular genetics and vital for applications such as genetic engineering and gene therapy. The use of cyclodextrins (CyDs) for increased efficiency of introducing DNA into eukaryotic cells (transfection) has been reported, but CyDs' effect on the introduction of DNA into bacterial cells (transformation) is unknown. Here, we have investigated the potential of using CyDs in the transformation of chemically competent in-house, commercially available, and, on non-competent bacterial cells, with plasmid DNA of two different sizes. Possible interactions between CyDs and DNA were studied with nuclear magnetic resonance (NMR) spectroscopy. The presence of CyDs resulted in an up to fourfold increment of the transformation rate for in-house cells, with beta-CyD and derivates giving the strongest effect. For commercial cells and transformation with megaplasmids, a more moderate effect around 1.4-fold was obtained. However, CyDs have little or no effect on DNA uptake by noncompetent cells. Results obtained from NMR spectroscopy show no interactions between CyDs and DNA-like molecules, which indicated that the CyDs' effect is related to the bacterial cell wall.