Mitchell M A, Skowronek K, Kauc L, Goodgal S H
Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia 19104-6076.
Nucleic Acids Res. 1991 Jul 11;19(13):3625-8. doi: 10.1093/nar/19.13.3625.
Electroporation of plasmid and chromosomal DNAs were tested in Haemophilus influenzae because of an interest in introducing DNA into mutants that are deficient in competence for transformation. The initial experiments were designed to investigate and optimize conditions for electroporation of H. influenzae. Plasmid DNA was introduced into the competence proficient strain Rd and its competence-deficient uptake mutants com-52, com-59, and com-88, and the recombination deficient mutant rec1. Plasmid DNA could also be electroporated into the non-transforming strains Ra, Rc, Re and Rf. Plasmid DNA without sequences that are involved in tight binding (uptake) of DNA by competent cells of H. influenzae Rd was electroporated into both competent and non-competent cells. Competent cells were several orders of magnitude less efficient than non-competent cells for electroporation of plasmid DNAs. Electroporation of H. influenzae chromosomal DNA was not successful. Low levels of integration of chromosomal markers were observed following electroporation and these could be ascribed to transformation. The treatment of cells with DNasel following electroporation separated the effects due to electroporation from those due to transformation. The DNasel treatment did not affect the efficiency of plasmid incorporation, but severely restricted effects due to natural DNA transformation.
由于对将DNA导入转化能力缺陷的突变体感兴趣，因此在流感嗜血杆菌中测试了质粒DNA和染色体DNA的电穿孔。最初的实验旨在研究和优化流感嗜血杆菌电穿孔的条件。将质粒DNA导入具有转化能力的Rd菌株及其缺乏摄取能力的突变体com-52、com-59和com-88，以及缺乏重组能力的突变体rec1。质粒DNA也可以通过电穿孔导入非转化菌株Ra、Rc、Re和Rf。将不含流感嗜血杆菌Rd感受态细胞紧密结合（摄取）DNA所需序列的质粒DNA导入感受态细胞和非感受态细胞。对于质粒DNA的电穿孔，感受态细胞的效率比非感受态细胞低几个数量级。流感嗜血杆菌染色体DNA的电穿孔未成功。电穿孔后观察到染色体标记的低水平整合，这些可能归因于转化。电穿孔后用DNA酶处理细胞，将电穿孔的影响与转化的影响分开。DNA酶处理不影响质粒掺入的效率，但严重限制了天然DNA转化的影响。
