A high-transformation-efficiency cloning vector for Thermus thermophilus

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Development of efficient cloning vectors is crucial for genetic manipulation of thermophilic bacteria.
• Existing vectors may have limitations in transformation efficiency or host range.
• Thermus species are important industrial microorganisms, necessitating robust genetic tools.

Purpose of the Study:

• To develop a novel cloning vector, pMK18, for use in Thermus species.
• To evaluate the transformation efficiency of pMK18 in Thermus thermophilus strains.
• To demonstrate the utility of pMK18 as a shuttle vector for gene cloning and transfer.

Main Methods:

• Construction of pMK18 by fusing elements from indigenous plasmids and pUC18.
• Transformation of Thermus thermophilus HB8 and HB27 using natural competence and electroporation.
• Testing pMK18 modified by the Escherichia coli methylation system.
• Cloning of a thermostable nitrate reductase beta-subunit gene in T. thermophilus.
• Transfer of the cloned gene to Escherichia coli.

Main Results:

• pMK18 demonstrated high transformation efficiencies (10^8 to 10^9 per microgram) in T. thermophilus.
• Efficient transformation was achieved via both natural competence and electroporation.
• T. thermophilus HB27 effectively accepted pMK18 modified by E. coli methylation.
• Successful direct cloning of a target gene in T. thermophilus.
• Demonstrated utility as a shuttle vector by transferring the gene to E. coli.

Conclusions:

• pMK18 is an effective and versatile cloning vector for Thermus species.
• The vector exhibits high transformation efficiencies in Thermus thermophilus.
• pMK18 functions as a shuttle vector, enabling gene manipulation across different bacterial genera.