A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes

Area of Science:

• Microbiology
• Molecular Biology
• Genetic Engineering

Background:

• Bacterial strain construction is crucial for various biotechnological applications.
• Efficient methods for introducing multiple exogenous DNA fragments into bacterial chromosomes are needed.

Purpose of the Study:

• To develop a novel, efficient, and time-saving method for constructing bacterial strains with multiple DNA insertions.
• To demonstrate the applicability of the method for sequential gene integration.

Main Methods:

• Utilized mini-Mu transposon for initial DNA integration via an FRT cassette.
• Employed FLP/FRT recombination for excision of selection markers.
• Repeated transposition and excision cycles for sequential insertion of foreign genes.

Main Results:

• Successfully integrated chloramphenicol, tetracycline, and gentamicin resistance genes into Escherichia coli BW25113 chromosome.
• Demonstrated a simple and time-saving process for multiple DNA insertions.
• Validated the method's applicability across different microbial species.

Conclusions:

• The developed method offers a robust platform for engineering bacterial genomes.
• This technique facilitates the construction of custom bacterial strains with desired genetic modifications.
• The approach is versatile and applicable to a broad range of microorganisms.