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Phasmids as effective and simple tools for construction and analysis of gene libraries
N K Yankovsky1, Fonstein MYu, Lashina SYu
1Institute for Genetics and Selection of Industrial Microorganisms, Moscow, U.S.S.R.
Abstract:
Phasmid lambda pMYF131, a hybrid of phage lambda vectors and plasmid pUC19, was constructed. The phasmid and its derivatives were shown to be efficient vectors for construction and analysis of gene libraries in Escherichia coli cells. The lambda pMYF131 DNA molecule contains all the genes and regions essential for phage lytic development. The plasmid cannot be packaged either in the monomeric or the oligomeric form due to its specific length. Elongation of the DNA molecule by ligation with fragments of foreign DNA can make it packageable and this is easily detected by plaque formation. Hence, the procedures used to construct genomic libraries can be simplified by selection of only recombinant DNA molecules just at the time and on the basis of their packaging in vitro. The output of recombinant clones per vector molecule was several times higher for vector lambda pMYF131, compared to phage vector lambda L47.1AB, and attained 3 x 10(6) clones per micrograms DNA. Vector and recombinant phasmids can be obtained in large quantities in plasmid form. lambda pMYF131 contains nine unique restriction sites which allow the cloning of DNA fragments with blunt ends and of fragments with various types of cohesive ends, obtained by digestion with 14 prototype restriction enzymes. The maximal size of the cloned DNA fragments is approx. 20 kb for lambda pMYF131. Phasmid vectors were used to construct libraries of bovine, pig and quail genomes, and genomic libraries of 17 species of bacteria. Application of suitable methods allowed the identification 13 individual genes within these libraries.
Insights
A new hybrid vector, lambda pMYF131 (phasmid), efficiently constructs gene libraries in E. coli. This vector simplifies genomic library creation and gene identification through in vitro packaging and plaque selection.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- Gene library construction is crucial for genomic analysis.
- Existing phage and plasmid vectors have limitations in efficiency and selection.
Purpose of the Study:
- To develop a novel hybrid vector, lambda pMYF131 (phasmid), for enhanced gene library construction.
- To evaluate the efficiency and utility of this new vector system.
Main Methods:
- Construction of the lambda pMYF131 phasmid vector by combining phage lambda and plasmid pUC19.
- Utilizing in vitro packaging and plaque formation for selection of recombinant DNA molecules.
- Cloning DNA fragments using unique restriction sites and assessing insert size capacity.
Main Results:
- Lambda pMYF131 demonstrated high efficiency in constructing gene libraries, yielding 3 x 10^6 recombinant clones per microgram of DNA.
- The vector allows for simplified selection of recombinant DNA through plaque formation.
- It accommodates DNA fragments up to approximately 20 kb and possesses nine unique restriction sites for versatile cloning.
- Successfully used to create libraries for bovine, pig, quail, and bacterial genomes, leading to the identification of 13 genes.
Conclusions:
- The lambda pMYF131 phasmid vector is a highly efficient tool for constructing and analyzing gene libraries.
- Its unique properties simplify library construction procedures and enhance gene discovery.
- This vector system offers significant advantages over traditional phage vectors for genomic research.