Construction of a new shuttle vector for Lactobacillus.

Area of Science:

• Molecular Biology
• Microbiology
• Genetic Engineering

Background:

• The malolactic enzyme is crucial for malolactic fermentation in lactic acid bacteria.
• Efficient gene cloning and expression systems are needed for studying Lactobacillus sp. 89.
• Shuttle vectors are essential tools for genetic manipulation across different bacterial species.

Purpose of the Study:

• To clone the malolactic enzyme gene from Lactobacillus sp. 89.
• To construct a functional shuttle vector for expression in Lactobacillus sp. 89 and Escherichia coli.
• To characterize the stability and properties of the constructed shuttle vector.

Main Methods:

• Construction of the shuttle plasmid pLE16 by combining pBR328 and a plasmid from Lactobacillus bulgaricus 10.
• Transformation of Lactobacillus sp. 89 using electroporation and selection on chloramphenicol-containing MRS medium.
• Restriction mapping of the pLE16 plasmid using various restriction enzymes.

Main Results:

• Successful construction of the pLE16 shuttle vector (7600 bp) capable of replication and expression in both target organisms.
• Identification of transformants in Lactobacillus sp. 89, demonstrating successful gene transfer.
• Observation of instability in chloramphenicol resistance in 50% of clones after 28 generations without selection pressure.

Conclusions:

• The pLE16 shuttle vector is a viable tool for cloning and expressing genes in Lactobacillus sp. 89.
• The study provides a foundation for further genetic analysis of the malolactic enzyme in Lactobacillus.
• Further investigation is needed to enhance the stability of the shuttle vector in the absence of selection.