[The study of optimal conditions of electroporation in Pseudomonas aeruginosa].
A P. aeruginosa strain PA68 isolated from the sputum of a patient suffering from bronchiectasis was used as the recipient strain. Optimum conditions including growth stage of the strain, electroshock voltage, concentration and preservation of competent cell were defined for the electroporation of PA68 with plasmid pSMC28. It was showed that the highest transformation efficiency was up to 1.68 x 10(3) CFU/microgram DNA under the optimum conditions in which the competent cells were collected at logarithmic growth phase (OD(540) = 0.7-0.8) and concentrated to about 10(11) cells/ml, the mixture of the competent cells and plasmid pSMC28 was eletroporated at 2.6 kV. With this optimal condition, Mu transponson complexes have been successfully transformed into P. aeruginosa strain PA68 and the obtained efficiency was 2.47 x 10(4) CFU/microgram DNA. This is the first time to electroporate Mu transposon complexes into Pseudomonas spp. The artificial Mu transposons could integrate into bacterial genomes at a single site randomly. Then the phenotype change was the result of the gene inactivation caused by Mu transposon insertion. That will be very helpful for the study of genomic function of Pseudomonas spp.
Shan
Zhi-Ying
ZY
College of Life Sciences, Nankai University, Tianjin 300071, China. zhiyingshan@163.com
Xu
Hai-Jin
HJ
Shi
Xing-Qi
XQ
Nie
Zhou
Z
Yu
Yan
Y
Zhang
Xiu-Ming
XM
Bai
Yan-Ling
YL
Qiao
Ming-Qiang
MQ
Gao
Cai-Chang
CC
D004251:DNA Transposable Elements; D018274:Electroporation; D006801:Humans; D011550:Pseudomonas aeruginosa / Q000235:genetics* / Q000254:growth & development; D014169:Transformation, Bacterial; D019895:Transposases / Q000502:physiology
D004251:DNA Transposable Elements; C114697:mu transposase; D019895:Transposases