Team:Austin UTexas/LPlantarum

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Lactobacillus Plantarum

Lactobacillus plantarum is a gram-positive lactic acid producing bacteria, so it requires a different growth media than we typically use in our lab. In 1954, Briggs agar was developed (5). This media was designed for lactobacilli, but was not sufficient for many species, including Lactobacillus plantarum , so a different non-selective media for general lactobacilli was developed in 1960 by Man, Rogosa and Sharpe and named MRS(6). We have exclusively grown our Lactobacillus plantarum on MRS media. Further, we grew Lactobacillus plantarum in a CO2 incubator as referenced in most literature we studied (1)(2)(3). The metabolic pathways in the bacteria alters when grown aerobically to produce excess acetate (7) and less lactic acid. Because we intend to utilize this bacteria in a fermentable food, a change in this metabolic pathway would not benefit our ultimate goal.

Once we could successfully grow our chosen bacteria, we needed to transform the gram positive, Lactobacillus plantarum, with pMSP3535. In order to do this, we identified and worked with a different protocol than we had ever used in our lab. We attempted several protocols, including Landete [b] and Speer [c]. However, we found success using a variation of the Welker protocol[a]. Welker et al. transformed multiple strains of Lactobacillus casei using varying reagents and yielded different efficiencies across the species with each variation (3).

After preparing the necessary solutions, we followed the Welker protocol with some minor differences. We inoculated bacterial stocks with 10 mL of MRS broth [d] in a CO2 incubator, without shaking, overnight. After this, we subcultured the bacteria in 200 mL of prewarmed MRS broth with 0.9M NaCl from an OD600 of 0.1 until it reached an OD600 of 0.6 (~6 hours). After harvesting the cells by centrifugation and rinsing, we resuspended the cells in 4 mL of cold water and eight 0.5 mL aliquots were divided into 1.5 mL microcentrifuge tubes.

The cells were washed several more times with water and 30% PEG 8000, then stored in a -80°C freezer. According to the protocol, these cells will be viable for up to two years.To enhance the transformation efficiency, 600 μL of prepared cells were suspended 900 μL of cold, sterile, distilled water for 30 min as a pretreatment to electroporation.The cells were pelleted and washed several times with water and 30% PEG 8000. We added 100 ng of plasmid DNA to 100μL of cell suspension as opposed to 200 ng that the protocol recommends; our first plasmid of choice was pMSP3535. This plasmid expresses erythromycin resistance. The cells were electroporated in 2mm cuvettes using the Ec3 settings on the BioRad electroporator which corresponds to 600Ω and 3 kV, differing from the protocol recommendations. After cells were electroporated, they recovered overnight in the appropriate recovery media. They were plated on MRS agar plates [d] with 10μg/mL erythromycin and left to grow for 2 days.