Specific-purpose broad-host-range vectors.

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The IncP4 plasmid RSF1010 is a broad-host-range vector widely used in molecular biology.
• Development of versatile cloning and promoter-probe vectors is crucial for genetic manipulation and gene expression studies.

Purpose of the Study:

• To construct and characterize novel plasmid derivatives of RSF1010.
• To create versatile cloning vectors with selectable markers.
• To develop promoter-probe vectors for assessing gene expression.

Main Methods:

• In vivo construction of plasmid derivatives.
• Insertion of transposons and DNA fragments.
• Selection of transformants based on gene inactivation.
• Modification of promoter regions for promoter-probe functionality.

Main Results:

• Construction of pAYC30 by inserting transposon Tn501 into RSF1010.
• Development of pAYC1, a 16.9 kb versatile cloning vector with dual replicons and five drug resistance markers.
• Creation of promoter-probe vectors pAYC36 and pAYC37 by modifying the aph gene promoter.
• Demonstration of selectable markers (HgCl2 sensitivity, gene inactivation) for cloned inserts.
• Establishment of a system for selecting clones with active promoters for gene aph expression.

Conclusions:

• The constructed plasmid derivatives expand the toolkit for broad-host-range cloning and gene expression analysis.
• These novel vectors offer enhanced features for genetic engineering, including selectable markers and promoter-driven expression systems.
• The promoter-probe vectors facilitate the identification and characterization of active promoters in various host organisms.