RESUMEN
The present study evaluated the stability of Bacillus subtilis strains transformed with a replicative or integrative plasmid (via CRISPR-Cas9) to express a recombinant phytase. Both transformation methods did not affect the growth of B. subtilis, but the stability of the construct and the enzymatic activity was reduced in the strain transformed with the replicative plasmid.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Sistemas CRISPR-Cas , Técnicas Genéticas , Plásmidos , Proteínas Recombinantes , 6-Fitasa/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Replicación del ADN , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Transformación BacterianaRESUMEN
The present study aimed to evaluate the influence of induction conditions (IPTG concentration, temperature, and induction time) on the plasmid pQE-30 stability and 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15. Batch cultures were performed at 37 °C and induced by the addition of different IPTG concentrations (0.01 to 1.5 mM). Subsequently, experiments were carried out at different temperatures (27 to 42 °C), evaluating the influence of induction time (0.5 to 6 h after the start of the culture). The results showed that IPTG toxicity caused a metabolic stress in the cells and, consequently, the microorganism growth reduced. The induction with IPTG may also be associated with the plasmid pQE-30 instability, due to metabolic burden imposed by the recombinant protein expression. The optimal conditions for 503 antigen expression of Leishmania i. chagasi in Escherichia coli M15 were an IPTG concentration of 1.0 mM, temperature of 37 °C, and induction time of 2 h. The maximum antigen concentration obtained was 0.119 ± 0.009 g/L, about seven times higher than the lowest concentration. Therefore, the results showed that 503 antigen can be produced in laboratory; however, it requires more studies to minimize the plasmid instability and improve to industrial scale.
Asunto(s)
Antígenos de Protozoos/biosíntesis , Escherichia coli/metabolismo , Expresión Génica , Leishmania/genética , Proteínas Recombinantes/biosíntesis , Activación Transcripcional , Antígenos de Protozoos/genética , Escherichia coli/genética , Inestabilidad Genómica/efectos de los fármacos , Isopropil Tiogalactósido/metabolismo , Plásmidos , Proteínas Recombinantes/genética , TemperaturaRESUMEN
Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244⯵g/mL of rSm14, 181⯵g/mL and 392⯵g/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147⯵g/mL of rSm14 and 162⯵g/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.
Asunto(s)
Antibacterianos , Escherichia coli/metabolismo , Proteínas de Transporte de Ácidos Grasos/metabolismo , Vectores Genéticos/química , Factor Estimulante de Colonias de Granulocitos/metabolismo , Proteínas del Helminto/metabolismo , Plásmidos/química , Proteínas Recombinantes/metabolismo , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Transporte de Ácidos Grasos/química , Proteínas de Transporte de Ácidos Grasos/genética , Vectores Genéticos/genética , Factor Estimulante de Colonias de Granulocitos/química , Factor Estimulante de Colonias de Granulocitos/genética , Proteínas del Helminto/química , Proteínas del Helminto/genética , Humanos , Plásmidos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genéticaRESUMEN
OBJECTIVE: The genome of Vibrio cholerae has three paralog genes encoding for distinct pyruvate kinases. We were interested in elucidating whether they were expressed, and contributed to the pyruvate kinase activity of V. cholerae. VcIPK and VcIIPK were transformed and expressed in BL21-CodonPlus(DE3)-RIL strain, whereas VcIIIPK could not be transformed. Those studied did contribute to the pyruvate kinase activity of the bacteria. Therefore, our aim was to find an efficient transformation and commonly used over-expression heterologous system for VcIIIPK and develop its purification protocol. RESULTS: vcIpk, vcIIpk and vcIIIpk genes were transformed in six different BL21 expression strains. No transformants were obtained for the vcIIIpk gene using BL21(DE3), BL21(DE3)pLysS and BL21(DE3)CodonPlus-RIL strains. Reduced rates of cell growth were observed for BL21-Gold(DE3)pLysS and Origami B(DE3)pLysS. High efficiency of transformation was obtained for BL21-AI. Using this strain, VcIIIPK was purified but proved to be unstable during its purification and storage. Therefore, the transformation of vcIIIpk gene resulted in a toxic, mildly toxic or nontoxic product for these BL21 strains. Despite VcIIPK and VcIIIPK being phylogenetically related, the preservation of the proteins is drastically different; whereas one is preserved during purification and storage, the other is auto-proteolyzed completely in less than a week.
Asunto(s)
Expresión Génica , Piruvato Quinasa/metabolismo , Vibrio cholerae/enzimología , Genes Bacterianos , IsoenzimasRESUMEN
Expression vectors for industrial production should be stable and allow tight control of protein synthesis. This is necessary to ensure plasmid transmission to daughter cells in order to achieve a stable population capable of synthesizing high amounts of the target protein. A high-copy-number plasmid, pAE, was previously used for laboratory-scale production of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and the Schistosoma mansoni fatty acid binding protein (rSm14), but it was unstable for large-scale production. Therefore, here we evaluated a new expression vector derived from pAE, pAR-KanI, which combines two plasmid replication strategies: a high-copy plasmid pUC origin of replication as pAE, and a par locus sequence derived from pSC101, which is typical of low copy plasmids, for rhG-CSF and rSm14 production in Escherichia coli. Clones bearing these constructs were cultivated in two complex media (2YT and auto-induction) and both yielded higher-than-95% resistant colonies, before and after induction, either with or without antibiotics. In 2YT medium, we obtained 244?µg/mL of rSm14, 181?µg/mL and 392?µg/mL for rhG-CSF, with and without glucose, respectively. In auto-induction medium without antibiotics, 147?µg/mL of rSm14 and 162?µg/mL of rhG-CSF were obtained. The new vector presented high stability for the production of both recombinant proteins in complex media in Escherichia coli, even in the absence of antibiotics, making the pAR-KanI a promising vector for industrial production of recombinant proteins.
RESUMEN
Pseudomonas aeruginosa plasmid pUM505 possesses a pathogenicity island that contains the pumAB genes that encode products with sequence similarity to Toxin-Antitoxin (TA) modules. RT-PCR assays on the overlapping regions of the pumAB genes generated a bicistronic messenger RNA, suggesting that they form an operon. When the pumAB genes were cloned into the pJET vector, recombinant plasmid pJET-pumAB was maintained under nonselective conditions in Escherichia coli cells after six daily subcultures, whereas pJET without pumAB genes was lost. These data indicate that pumAB genes confer post-segregational plasmid stability. In addition, overexpression of the PumA protein in the E. coli BL21 strain resulted in a significant growth inhibition, while BL21 co-expressing the PumA and PumB proteins did not show growth inhibition. These results indicate that pumAB genes encode a TA system where the PumB protein counters the toxic effects of the PumA toxin. Furthermore, P. aeruginosa PAO1 transformants with the pumA gene increased Caenorhabditis elegans and mouse mortality rate and improved mouse organ invasion, effects neutralized by the PumB protein. Moreover, purified recombinant His-PumA protein decreased the viability of C. elegans, indicating that the PumA protein could acts as a toxin. These results indicate that PumA has the potential to promoter the PAO1 virulence against C. elegans and mice when is expressed in absence of PumB. This is the first description, to our knowledge, of a plasmid-encoded TA system that confers plasmid stability and encoded a toxin with the possible ability to increase the P. aeruginosa virulence.
Asunto(s)
Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Genes Bacterianos/genética , Plásmidos/genética , Pseudomonas aeruginosa/genética , Sistemas Toxina-Antitoxina/genética , Factores de Virulencia/genética , Animales , Antitoxinas/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Caenorhabditis elegans/efectos de los fármacos , Modelos Animales de Enfermedad , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos , Masculino , Ratones , Ratones Endogámicos BALB C , Operón/genética , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/mortalidad , Pseudomonas aeruginosa/patogenicidad , ARN Bacteriano/análisis , Proteínas Recombinantes/genética , Análisis de Secuencia , Virulencia/genéticaRESUMEN
The fecal coliform can contaminate water of human consumption causing problems to public health. Many of these microorganisms may contain plasmid and transfer them to other bacteria. This genetic material may confer selective advantages, among them resistance to antibiotics. The objectives of this study were to analyze the presence of fecal coliforms in water and at drinker surface, to identify the existence of plasmid, conducting studies of resistance to antibiotics, plasmid stability and capacity of bacterial conjugation. Were collected microorganisms in water of drinker surface and were used specific culture media and biochemical tests for identification of organisms, tests were performed by checking the resistance to antibiotics (ampicillin 10 µg, tetracycline 30 µg, and ciprofloxacin 5 µg), was performed extraction of plasmid DNA, plasmid stability and bacterial conjugation. Was obtained results of 31% of Salmonella spp. and 51% for other coliforms. Among the samples positive for coliforms, 27 had plasmid stable and with the ability to perform conjugation. The plasmids had similar forms, suggesting that the resistance in some bacteria may be linked to those genes extra chromosomal.