Team:Austin UTexas/LPlantarum
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 the metabolic pathway would not benefit our ultimate goal.
Once we could successfully grow our chosen bacteria, In order to transform the gram positive bacteria, Lactobacillus plantarum, with pMSP3535 we needed to identify and work with a different protocol than we were familiar with from other model organisms we have worked with. We attempted several protocols including Landete [b] and Speer [c]. However, we found success using a variation of the Welker protocol [a] in which they transformed Lactobacillus casei (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.
