An strategy to enhance electroporation competence of Streptococcus mutans

Objectives: To increase the efficiency of Streptococcus mutans electroporation transformation, this study aimed to optimal the transformation conditions and compared the effeiciency of different electroporation profiles. Methods: Streptococcus mutans UA159 were incubated in a 200 ml BHI culture media anaerobically till the optical density arrived OD660nm=0.5. Then different concentrations of glycine (0，2%，5%，10%，20%) were added into culture solutions to be incubated for one more hour. Bacteria cells were collected by centrifuge at 4℃ 3000rpm for 10 minuets. Cell pellets were washed twice with electroporation buffer (EP buffer: 4M sorbitol, 0.4 mM K3PO4 and 10% glycerol). Competence cells were condensed in 2ml EP buffer, and stored in -70℃ for later use. During the electroporation, 1µg linerized DNA from plasmid of pGL3 basic as foreign DNA was added into 50µl competence cell solution. Two electroporation profiles were compared (A. 1.6KV，200Ω，25µF；B. 2.2KV，200Ω，25µF). After transformation, cells were recovered in BHI (0.4M sorbitol contained) for 1 hour then plated on BHI agar media (with the concentration of ampicillin at 1µg/ml). After incubation at 37℃ for 12 hours, transformation clones were counted and differences of transform efficiency among different concentrations of glycine and different electroporation profiles were compared. Results: Among the five concentration groups, 10% of glycine can significantly improve the transformation effeiciency and there are no significance in their transform efficiency between electroporation profiles. Conclusion: To the electroporation transform, this study suggest to add 10% glycine into the bacteria culture when its optical denstity arrives OD660nm=0.5 to prepare the competence cell. Completely suspend 50µl competence cell and 1µg DNA, then electroporated for 4.5 ms in 200Ω, 25µF, 16A. This study was supported by Chinese NSFC (Grant No. 30400497)