IMPROVEMENT OF TRANSFORMATION EFFICIENCY FOR Lactobacillus curvatus CRL705

TERAN, L. C.; FADDA, S.; CHAMPOMIER-VERGÈS, M.C.; ZAGOREC, M.; CHAILLOU, S.; RAYA, R.

Simposio; V International Symposium of Lactic Acid Bacteria; 2016

CERELA-CONICET

IMPROVEMENT OF TRANSFORMATION EFFICIENCY FOR Lactobacillus curvatus CRL705L.C. Terán1, S. Chaillou2, M-C. Champomier-Vergès2, S. Fadda1, R. Raya1, M. Zagorec31CERELA-CONICET. Chacabuco 145. 4000 San Miguel de Tucumán, Argentina. E-mail: lteran@cerela.org.ar2MICALIS. INRA AgroParisTech UMR1319. Domaine de Vilvert. Bat526. F78352 Jouy-en-Josas, France. 3 UMR1014 SECALIM, INRA, Oniris, 44307, Nantes, FranceApplying 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 be obtained but no strain totally cured of 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 106 transformant 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. curvatus CRL705 properties.