INVESTIGADORES
FADDA Silvina Graciela
congresos y reuniones científicas
Título:
IMPROVEMENT OF TRANSFORMATION EFFICIENCY FOR Lactobacillus curvatus CRL705
Autor/es:
TERÁN, L; CHAILLOU, S; CHAMPOMIER-VERGÈS, M.,; FADDA S,; RAYA, R; ZAGOREC, M
Lugar:
San Miguel de Tucuman
Reunión:
Simposio; V Simposio Internacional de Bacterias Lácticas-SIBAL CERELA- CONICET; 2016
Institución organizadora:
CERELA CONICET
Resumen:
Applying molecular biology tools to modify bacterial cell properties requires an efficient transformation system. For Lactobacillus the most frequent (and almost unique) way of transformation is electroporation. Protocols for preparing competent cells and for the incorporation of foreign DNA can vary among species and strains. Also, the transformation of this genus can be a hard task as cell wall sometimes represents a barrier for the entrance of DNA. Here, we present an optimized transformation protocol for Lactobacillus curvatus CRL705, a strain isolated from an Argentinian traditional sausage. Different parameters were evaluated such as the electrical parameters used for electroporation, the gap of the cuvettes, the incubation time applied after electroporation (between 2 h - 3 h), the antibiotic resistance used for selection (chloramphenicol or erythromycin) and the compatibility between the plasmids used for transformation (pRV566, pRV085, pGK12) and the native plasmids of the bacteria. In particular, L. curvatus CRL705 harbors two plasmids: pRC18 and pRC12. Trials for curing the strain and its derivatives were performed using novobiocin, a gyrase inhibitor. Derived clones, having lost one or the other plasmid could beobtained but no strain totally curedof both plasmids. Results showed that the most meaningful parameter during the transformation trials was the electrical one. The efficiency was improved up to 103 times more by changing the voltage and resistance. The higher efficiencies with 106transformant per µg of DNA were obtained at: 2.0 kV, 1000 Ω and 2.5 kV, 600 Ω keeping 25 µF constant. The optimized transformation protocol will enable the construction of mutants in various genes of interest and therefore contribute to a better knowledge and understanding of L. curvatusCRL705 properties.