Related Experiment Videos
Broad host range vectors derived from an RSF1010::Tn1 plasmid.
Plasmid
|November 1, 1986
Summary
New plasmid vectors derived from RSF1010 enable broad-range bacterial DNA cloning. These versatile vectors, including pAYC31, pAYC32, and cosmid derivatives, offer positive selection for inserted fragments and facilitate gene banking.
Area of Science:
- Molecular Biology
- Microbiology
- Biotechnology
Background:
- The IncQ/P4 plasmid RSF1010 offers a basis for developing cloning vectors.
- Broad-range bacterial cloning requires versatile and selectable plasmid systems.
Purpose of the Study:
- To construct novel plasmid vector derivatives of RSF1010 for efficient DNA cloning across diverse bacterial species.
- To engineer vectors with positive selection mechanisms and create cosmid derivatives for gene banking.
Main Methods:
- Construction of plasmid vectors pAYC31, pAYC32, and pAYC39 based on RSF1010.
- Insertion of DNA fragments into unique restriction sites (e.g., BamHI) to activate reporter genes (aph).
- Generation of cosmid vectors (pAYC51, pAYC52, pAYC53) by incorporating lambda cos sites and other genes (tet).
Main Results:
- pAYC31 utilizes a positive selection system based on aph gene expression upon fragment insertion, enabling selection on streptomycin media.
- pAYC32 and pAYC39 were developed as versatile cloning vectors.
- Cosmid vectors pAYC51, pAYC52, and pAYC53 were created by integrating lambda cos sites and tet genes into pAYC32.
- pAYC31 was successfully employed to construct a gene bank from Methylomicrobium flagellatum KT.
Conclusions:
- The developed RSF1010-derived plasmid and cosmid vectors provide robust tools for molecular cloning in a wide range of bacteria.
- These vectors facilitate positive selection and are suitable for constructing genomic libraries.