Plasmids useable as gene-cloning vectors in an in vitro packaging by coliphage lambda: "cosmids"

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Cosmids are plasmids containing a cos site of lambda phage, enabling packaging into bacteriophage particles for gene cloning.
• Existing cosmid vectors have limitations in efficiency and require additional screening steps.

Purpose of the Study:

• To describe a new series of cosmids based on the ColE1 replicon.
• To optimize cosmid cloning properties for high-efficiency gene cloning.
• To develop vectors for specific restriction enzymes and assess their performance.

Main Methods:

• Development of small, temperature-sensitive cosmids lacking mobilisation functions and IS sequences.
• Construction of vectors for XmaI, BglII, BamHI, HindIII, PstI, KpnI, SalI, and EcoRI restriction enzymes.
• In vitro packaging and cloning into Escherichia coli to create a gene bank.

Main Results:

• Cosmids in specific size ranges provide high-efficiency cloning with predominantly hybrid clones, eliminating the need for secondary selection or screening.
• An Escherichia coli gene bank was generated with 5 x 10^5 clones per microgram of DNA, with unselected markers found in approximately 1 in 400 clones.
• Deletion of mobilisation functions did not affect copy number or segregation but led to loss of relaxation-complex formation.

Conclusions:

• The new cosmid vectors represent a significant advancement in high-efficiency gene cloning systems.
• These vectors facilitate the construction of comprehensive gene banks with improved accessibility to genetic markers.
• The described cosmids are amplifiable with chloramphenicol, constituting up to 50% of cellular DNA, enhancing their utility in molecular biology applications.