Journal: bioRxiv
Article Title: An Optimized Electrotransformation Protocol for Lactobacillus jensenii
doi: 10.1101/2022.10.31.514476
Figure Lengend Snippet: 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 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. Technical replicates are shown in the same color.
Article Snippet: We selected 3 shuttle vector plasmids described in the literature as being widely portable among lactobacilli. pLEM415-ldhl-mRFP11 (a gift from Sujin Bao, Addgene plasmid # 99842) is a pBLUESCRIPT derivative carrying an unnamed lactobacilli origin of replication, a high-copy version of the E. coli colE1 origin of replication, an erythromycin resistance gene and a mRFP1 expression cassette. pTRKH2 (kind gift from Michela Lizier, Addgene plasmid # 27168) is a theta replicative plasmid carrying a pAMβ1 origin, a p15A E. coli replication origin and an erythromycin resistance gene Erm(B) . pTRK892 (kind gift from Rodolphe Barrangou & Todd Klaenhammer, Addgene plasmid #71803) is a high copy shuttle vector bearing a pWVO1 replication origin that is functional in both gram negative and positive species. pTRK892 also carries a GusA reporter cassette under ppgm promoter and erythromycin Erm(C) resistance gene.
Techniques: Transformation Assay, Electroporation, Comparison
