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<b>Abstract</b>— <p>Efficient transformation of mycobacteria, in particular, <i>M. abscessus</i>, is significantly complicated by the specific structure of their cell wall. The most widely used and effective method of introducing plasmid and phage DNA into mycobacterial cells is electroporation. The efficiency of electroporation is significantly affected by many factors, such as the nature of the DNA, the selective marker, growth supplements, the parameters of the electrical impulse, the species and the strain of the recipient mycobacterium. Although conditions for efficient electroporation for the slow-growing pathogen <i>M. tuberculosis</i> and the fast-growing saprophyte <i>M. smegmatis</i> have been described in details, recommendations for <i>M. abscessus</i> are scattered and even contradictory. Here it was established that efficient transformation of <i>M. abscessus</i> ATCC 19977 with the replicative vector pMV261 by electroporation is possible when using a logarithmic growth phase culture in a fairly wide range of optical density values OD<sub>600</sub> = 0.8–4.2, while cooling has little effect on the transformation frequency. A critical parameter is the mass of the introduced DNA. It has been established that the number of transformants obtained per 1 µg of DNA increases proportionally to the square of its mass. In case of introducing less than 0.5 μg of plasmid DNA the efficiency of electroporation is insufficient.</p>

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Optimization of Transformation Conditions by Electroporation for Mycobacterium abscessus

  • 期刊论文

  • 发表日期：

    2025年9月24日

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  • E. V. Zakhareva,
  • B. A. Martini,
  • E. G. Salina

摘要

Abstract—

Efficient transformation of mycobacteria, in particular, M. abscessus, is significantly complicated by the specific structure of their cell wall. The most widely used and effective method of introducing plasmid and phage DNA into mycobacterial cells is electroporation. The efficiency of electroporation is significantly affected by many factors, such as the nature of the DNA, the selective marker, growth supplements, the parameters of the electrical impulse, the species and the strain of the recipient mycobacterium. Although conditions for efficient electroporation for the slow-growing pathogen M. tuberculosis and the fast-growing saprophyte M. smegmatis have been described in details, recommendations for M. abscessus are scattered and even contradictory. Here it was established that efficient transformation of M. abscessus ATCC 19977 with the replicative vector pMV261 by electroporation is possible when using a logarithmic growth phase culture in a fairly wide range of optical density values OD600 = 0.8–4.2, while cooling has little effect on the transformation frequency. A critical parameter is the mass of the introduced DNA. It has been established that the number of transformants obtained per 1 µg of DNA increases proportionally to the square of its mass. In case of introducing less than 0.5 μg of plasmid DNA the efficiency of electroporation is insufficient.