pKSS--a second-generation general purpose cloning vector for efficient positive selection of recombinant clones

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Plasmid vectors are essential tools in molecular biology for gene cloning and manipulation.
• Direct selection methods simplify the identification of recombinant clones, reducing screening time and effort.
• Existing positive selection vectors have limitations that necessitate further improvements.

Purpose of the Study:

• To introduce a new small plasmid vector, pKSS, for the direct selection of insert-containing plasmid clones.
• To present a novel selection strategy based on acquired sensitivity to p-chloro-phenylalanine (p-Cl-Phe).
• To offer a valuable alternative to existing positive-selection vectors for various cloning applications.

Main Methods:

• Development of the pKSS plasmid vector containing a pheS allele with relaxed substrate specificity.
• Utilizing the pKSS vector to transform wild-type Escherichia coli strains.
• Employing agar plates containing p-chloro-phenylalanine (p-Cl-Phe) and ampicillin for positive selection of recombinant clones.
• Assessing the efficiency of the positive selection strategy.

Main Results:

• The pKSS vector enables direct selection of insert-containing clones by conferring p-Cl-Phe sensitivity to transformed cells.
• Insertion or replacement of the plasmidial pheS gene by a cloned fragment allows transformed cells to survive on selective media.
• The positive selection of recombinant clones is highly efficient, exceeding 99% efficiency.
• The method does not require purification of the vector fragment prior to cloning and is host strain-independent.
• The pKSS vector possesses multiple restriction sites and features for fragment analysis, similar to pBluescript.

Conclusions:

• The pKSS plasmid vector provides a highly efficient and valuable tool for direct positive selection of recombinant clones.
• This method simplifies cloning procedures, particularly when a high proportion of non-recombinant vectors is expected.
• pKSS is particularly useful for constructing DNA libraries and offers a robust alternative to previous selection vectors.