A plasmid vector with positive selection and directional cloning based on a conditionally lethal gene

Area of Science:

• Molecular Biology
• Bacterial Genetics
• Recombinant DNA Technology

Background:

• The bacterial ribonuclease barnase (Barn) is lethal to Escherichia coli (Ec) hosts upon full expression.
• The inhibitor barstar (Bars) can protect the host from Barn's lethal effects.
• Existing cloning vectors often require specific bacterial strains or complex selection methods.

Purpose of the Study:

• To develop a novel plasmid vector for positive selection of cloned DNA inserts in E. coli.
• To create a versatile cloning vector that simplifies the process of identifying successful transformations.
• To engineer a vector that does not require specialized E. coli strains for selection.

Main Methods:

• A modified expression plasmid (pMT416) was used as the base for the new vector.
• The multiple cloning site (MCS) from pUC19 was inserted into the barn gene.
• The resulting plasmid, pMT440, was designed for inducible expression of barnase, with barstar expressed constitutively for host protection.

Main Results:

• Plasmid pMT440 is lethal to E. coli under inducing conditions unless foreign DNA is inserted into the MCS.
• Insertion of DNA fragments into the MCS inactivates the modified barn gene product, allowing cell survival.
• This system provides positive selection for cloned inserts, enabling straightforward identification of transformed colonies.

Conclusions:

• Plasmid pMT440 functions as a highly effective positive selection cloning vector.
• The vector simplifies cloning procedures by eliminating the need for screening methods or special E. coli strains.
• This tool enhances the efficiency and ease of molecular cloning in E. coli.