A high efficient electroporation of Pseudomonas sp. QDA pretreated with alginate lyase
Most of Pseudomonas strains have copious alginates enclosed the bacteria cells, which makes it difficult to transfer exogenous DNA into the cells during transformation. We report here that pretreatment of Pseudomonas sp. QDA with alginate lyase before electroporation obviously increased transformation efficiency approximately 104-fold than that without pretreatment of alginate lyase, and a high transformation efficiency nearly the same as that of alginate production deficient mutant was obtained. Among the alginate lyases tested, AL2 was the most effective enzyme for pretreatment. The optimized transformation conditions were that the cells of Pseudomonas sp. QDA in the middle of logarithmic stage (OD600 = 0.8) were incubated at 40 °C for 20 min with 1.28 U ml−1 of alginate lyase AL2, then harvested, washed twice and electroporated at 10 kV cm−1 in electroporation buffer III (300 mM glucose, 5 mM CaCl2, 25 mM HEPES in ultrapure water, pH 7.0). At this condition, the maximum transformation efficiency obtained was 8.5 × 107 transformants per μg of plasmid DNA for Pseudomonas sp. QDA with broad-host-range vector pMF36-Gm (8.55 kb). This electroporation procedure was also efficient for Pseudomonas aeruginosa FRD1 (mucoid) and PAO1 (non-mucoid).
Journal: Enzyme and Microbial Technology - Volume 39, Issue 4, 2 August 2006, Pages 677–682