RESUMO
Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.
Assuntos
Bactérias/genética , Resistência Microbiana a Medicamentos/genética , Escherichia coli/genética , Genoma Bacteriano , Proteínas Virais , Acetiltransferases/genética , Bactérias/efeitos dos fármacos , Cloranfenicol/farmacologia , Cloranfenicol O-Acetiltransferase/genética , DNA Recombinante , Resistência a Múltiplos Medicamentos/genética , Escherichia coli/efeitos dos fármacos , Deleção de Genes , Marcadores Genéticos , Vetores Genéticos , Gentamicinas/farmacologia , Integrases/genética , Canamicina/farmacologia , Canamicina Quinase/genética , Óperon Lac/genética , Mutagênese Insercional , Plasmídeos/genética , UDPglucose 4-Epimerase/genéticaRESUMO
A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.
Assuntos
Cromossomos Bacterianos , Escherichia coli/genética , Genética Microbiana/métodos , Plasmídeos/genética , beta-Galactosidase/genética , Clonagem Molecular/métodos , Estabilidade Enzimática , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Reação em Cadeia da Polimerase , Temperatura , Transdução Genética , alfa-Amilases/genética , beta-Galactosidase/metabolismoRESUMO
Lipases are esterases able to hydrolyze water-insoluble esters such as long-chain triglycerides. These enzymes also catalyze the formation of esters (esterification) and the exchange of ester bonds (transesterification) when present in nonaqueous media. Lipases display a high degree of specificity and enantioselectivity for esterification and transesterification reactions, and thus their potential uses in industry are very wide. These potential industrial applications have been an important driving force for lipase research during the last several years, and in particular for the study of lipases produced by microorganisms. Pseudomonas lipases are very interesting because they display special biochemical characteristics not common among the lipases produced by other microorganisms, such as their thermoresistance and activity at alkaline pHs. Recently, several Pseudomonas genes have been cloned and sequenced, and the regulation of their expression is beginning to be understood. The molecular genetic approach to the study of Pseudomonas lipases will permit the construction of recombinant strains with increased lipase productivity and will provide the opportunity to modify these enzymes to suit particular industrial applications.
