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Related Experiment Videos

Spheroplast formation and plasmid isolation from Rhodococcus spp

N A Assaf1, W A Dick

  • 1Purdue University, West Lafayette, IN.

Biotechniques
|December 1, 1993
PubMed
Summary

Researchers developed a new method for lysing Rhodococcus cells and isolating large plasmid DNA. This technique uses mutanolysin and lysozyme enzymes for efficient cell wall degradation and DNA extraction.

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • Rhodococcus are diverse aerobic gram-positive bacteria.
  • They play roles in plant diseases and pollutant degradation.
  • Efficient plasmid DNA isolation from Rhodococcus is challenging.

Purpose of the Study:

  • To develop an effective method for cell lysis in Rhodococcus.
  • To enable the isolation of large plasmid DNA from Rhodococcus strains.

Main Methods:

  • Cells were pre-digested with mutanolysin to create lysozyme-susceptible cells.
  • Mutanolysin degrades peptidoglycan, releasing sugars and amino acids.
  • Subsequent lysozyme treatment formed spheroplasts, leading to cell lysis.

Main Results:

  • A 1-hour mutanolysin pre-digestion followed by 30 minutes of lysozyme incubation efficiently lysed Rhodococcus cells.
  • Intact large plasmid DNA (108 kb) was successfully isolated from wild-type strains.
  • The method confirmed complete cell lysis and high-quality DNA recovery.

Conclusions:

  • The described enzymatic method provides efficient cell lysis for Rhodococcus.
  • This technique facilitates the isolation of intact large plasmids from Rhodococcus.
  • This advancement supports further research into Rhodococcus genetics and applications.

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