Rapid customised operon assembly by yeast recombinational cloning.

Area of Science:

• Molecular Biology
• Synthetic Biology
• Microbial Genetics

Background:

• Bacterial operons are crucial genetic elements controlling gene expression.
• Assembling and functionally analyzing operons can be challenging.
• Understanding O-antigen biosynthesis requires tools for genetic manipulation.

Purpose of the Study:

• To develop a novel system for efficient operon assembly.
• To enable direct functional comparisons of engineered operons.
• To investigate Wzx flippase substrate preferences in O-antigen biosynthesis.

Main Methods:

• Utilized Saccharomyces cerevisiae's homologous recombination DNA repair pathway.
• Developed the Operon Assembly Protocol (OAP) using overlapping PCR products.
• Constructed a yeast-Escherichia coli shuttle vector for cloning and expression.
• Assembled and expressed E. coli O1A O-antigen gene cluster variants.

Main Results:

• Successfully assembled and expressed multiple E. coli O-antigen operon clones.
• Identified key structural features influencing Wzx flippase substrate specificity.
• Demonstrated the utility of OAP for assessing enzyme function.

Conclusions:

• The Operon Assembly Protocol (OAP) is a flexible and efficient system for bacterial operon assembly.
• OAP facilitates direct functional comparisons of gene variants.
• The system has broad applicability for genetic manipulation and potential uses in glycoengineering.