High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain.
High-efficiency transposon mutagenesis by electroporation of a Pseudomonas fluorescens strain.
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DOI：
10.1111/j.1574-6968.1997.tb12597.x
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发表时间：
1997-08
影响因子：
2.1
通讯作者：
F. Artiguenave;Roland Vilaginès;C. Danglot
中科院分区：
文献类型：
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作者：
F. Artiguenave;Roland Vilaginès;C. Danglot
本发明描述了一种通过电穿孔用转座子递送载体pUT/mini-Tn 5 Km诱变荧光假单胞菌菌株的方法。转座过程显示在12.5 kV cm-1下对于约4 ms的脉冲时间（Bowen和Koslak，1992）是最佳的。荧光假单胞菌L6.5靶菌株在指数生长期中期收获时表现出最大的电感受态。每皮摩尔递送载体（7.5kb）可获得多达7.7 × 10（5）个突变体，并且这些卡那霉素抗性突变体显示已丢失pUT质粒。通过使用大小不断增加的质粒（从11.5 kb到60.1 kb）进行外部校准，转化过程的效率评估为每皮摩尔递送载体约1.31 x 10（8）个转化子。转座过程的效率为0.58%。该方法可用于标记荧光假单胞菌L6.5株Alk+表型的3个独立的染色体位点。
A method is described for mutagenesis of Pseudomonas fluorescens strains by electroporation with the transposon delivery vector pUT/mini-Tn5 Km. The transposition process was shown to be optimal at 12.5 kV cm-1 for a pulse time (Bowen and Koslak, 1992) of about 4 ms. The Pseudomonas fluorescens L6.5 target strain exhibited maximal electrocompetence when harvested at the middle of the exponential growth phase. As many as 7.7 10(5) mutants per picomole of delivery vector (7.5 kb) could be obtained, and these kanamycin-resistant mutants were shown to have lost the pUT plasmid. By external calibration with plasmids of increasing size (from 11.5 to 60.1 kb), the efficiency of the transformation process was evaluated to be approximately 1.31 x 10(8) transformants per picomole of delivery vector. Efficiency of the transposition process was 0.58%. This rapid method was used to tag for the cloning three independent chromosomal loci responsible for the Alk+ phenotype of Pseudomonas fluorescens L6.5 strain.