[Phasmids. Properties and the use in genetic engineering. I. Construction of the phasmid vector]

Area of Science:

• Molecular Biology
• Genetic Engineering
• Microbiology

Background:

• Traditional plasmid and phage vectors have limitations in cloning efficiency and selection.
• There is a need for versatile vectors that combine the advantages of both plasmids and phages.

Purpose of the Study:

• To construct and characterize a novel phasmid vector molecule, lambda pMYF11.
• To evaluate the utility of lambda pMYF11 for gene cloning and library construction.

Main Methods:

• Construction of a hybrid phasmid vector (lambda pMYF11) by combining lambda 47.1 and pBR322 plasmid features.
• Modification of the CI- marker to cI+ via recombination with prophage 434.
• Utilizing positive selection based on Spi phenotype expression for recombinant molecules.
• Construction of an Escherichia coli gene library and detection of specific genes via complementation.

Main Results:

• The lambda pMYF11 phasmid vector was successfully constructed, integrating plasmid and phage functionalities.
• This vector supports replacement cloning for fragments up to 21 kilobase pairs using specific restriction enzymes (BamHI, HindIII, SalGI).
• Positive selection is achieved through the Spi phenotype, facilitating the identification of recombinant clones.
• A functional library of Escherichia coli genes was generated, and genes within the proline operon were identified.

Conclusions:

• The lambda pMYF11 phasmid vector offers a robust and efficient system for molecular cloning.
• Its design facilitates positive selection, enhancing the construction of gene libraries.
• This vector is valuable for genetic studies, particularly in the analysis of bacterial gene operons.