Fig 1.
Potential applications to vaginal microbiota engineering and electroporation optimization.
A. Potential applications of engineering the vaginal microbiota.B. L. jensenii is a candidate to develop genetically engineered probiotics for the vaginal microflora. Electrotransformation is the most effective method to transform DNA into Lactobacillus spp. C. Parameters described as important to optimize electroporation in lactic acid bacteria that were tested in this study.
Table 1.
Designation, characteristics and transformation frequency of plasmids tested in Lactobacillus jensenii ATCC 25258.
Fig 2.
Optimization of competent cells preparation for L. jensenii ATCC25258 using pTRKH2.
A. Comparison of widely used electroporation protocols for Lactobacillus spp. Shown are transformation efficiency of Lactobacillus jensenii observed using original protocols described by Luchansky et al. (protocol 1) [22] and Berthier et al. (protocol 2) [21]. B. Increasing buffer concentration improves transformation efficiency. Shown are transformation efficiency of electrocompetent L. jensenii prepared at 1, 2 or 3X the concentration described in Luchansky et al. [16]. More concentrated buffers led to arcing. C. Addition of glycine improves transformability. Shown are transformation efficiency of electrocompetent L. jensenii prepared in 3X buffer with increasing titers of glycine (expressed as weight to volume percentage). Titers higher than 2.0% lead to markedly reduced growth rate. All electroporation were performed with 1 μg pTRKH2 in 0.1 cm electroporation cuvettes and electric parameters as follows: voltage: 6.5 kV/cm, resistance: 600 Ohm, capacitance: 25 μF. 3 biological replicates of 3 technical replicates were performed on different days (data are shown in S2 Table). Technical replicates are shown in the same color. (Wilcoxon signed-rank test p-value, ns p > 0.05, * p < = 0.05, ** p < = 0.01, *** p < = 0.001 and **** p < = 0.0001).
Fig 3.
Optimization of electroporation procedure for L. jensenii 25258 using pTRKH2 vector.
A. Increasing electroporation cuvette size yields a 2.5-fold increase in transformation efficiency. Shown are transformation efficiency of electrocompetent L. jensenii in 0.1 cm or 0.2 cm cuvette gap size. B. Increasing DNA quantity impacts transformation efficiency in a step-like manner. Shown are transformation efficiency of electrocompetent L. jensenii electroporated in 0.2 cm cuvettes with different DNA quantities, as indicated. Quantities above 4 μg (or volume above 5 μL) frequently result in electric arcs. C. Increasing electric pulses’ voltage improves transformation efficiency. Shown are transformation efficiencies for electrocompetent L. jensenii electroporated in 0.2 cm cuvettes with 1 μg DNA at different voltage intensities, as indicated. Increasing the voltage above 12.5 KV/cm causes arcing and kills the cells. All electroporations were carried with vector pTRKH2, 3X SMEB, 2% glycine, 6.5 kV/cm voltage (unless otherwise indicated), 400 Ω resistance, 25 μF capacitance. 3 biological replicates of 3 technical replicates were performed on different days (data are shown in S2 Table). Technical replicates are shown in the same color. (Wilcoxon signed-rank test p-value, ns p > 0.05, * p < = 0.05, ** p < = 0.01, *** p < = 0.001 and **** p < = 0.0001).
Fig 4.
Optimized transformations of various vectors and unrelated L. jensenii strains.
A. Transformation efficiency of different plasmids using the optimized protocol. Shown are transformation efficiencies obtained for plasmids pTRKH2, pTRK892 and pLEM415-ldhl-mRFP1. While pTRKH2 and pTRK892 yield similarly high efficiencies, the unrelated pLEM415 yields a measurable but moderate number of transformants. Numbers above bars represent the average number of transformants. B. Beta-glucuronidase assay with Lactobacillus jensenii pTRK892 transformants showed blue coloration after addition of X-GLU substrate. C. The optimized electroporation procedure is efficient across unrelated strains of L. jensenii. Shown is a comparison of transformation efficiencies obtained with the Luchansky [22] and optimized procedure with different clinical isolates. All electroporations were carried with 1.0 μg of DNA, 3X SMEB, 2% glycine, in 0.2 cm cuvettes and electric parameters as follows: voltage: 12.5 kV/cm, resistance: 400 Ω, capacitance: 25 μF. 3 biological replicates of 3 technical replicates were performed on different days (data are shown in S2 Table). Technical replicates are shown in the same color. (Wilcoxon signed-rank test p-value, ns p > 0.05, * p < = 0.05, ** p < = 0.01, *** p < = 0.001 and **** p < = 0.0001).