Manipulation of operons in Gram-negative bacteria using classic methods such as the lambda recombinase system can be a slow and cumbersome process, particularly in situations where several genes need to be knocked out or replaced sequentially in the same strain. To overcome this issue, we have developed a system (called the Operon Assembly Protocol or OAP) that takes advantage of the homologous recombination DNA repair pathways that exist naturally in Saccharomyces cerevisiae to assemble whole operons into a specially-engineered yeast-E. coli shuttle vector (the Operon Assembly Vector or OAV). By introducing the linearised OAV into yeast along with a series of PCR products engineered to contain short sequence overlaps, multiple customised operons with any combination of genes in any given order can be assembled in parallel in a single step. Each of these operon clones can be expressed in the same E. coli tester strain, thus allowing for direct functional comparisons between different operons.
Over the past 12 months, we have been using the OAP system to assemble both wild-type and mutant O-antigen gene cluster clones from various Gram-negative bacteria. We have used these clones to examine several aspects of the nature of the substrate preference exhibited by the Wzx flippase, which is responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. This has allowed us to identify several O-unit structural features that appear to be important determinants of Wzx substrate preference. We believe that the ability of the OAP system to quickly and conveniently assemble whole operons can be utilised broadly for a wide variety of applications, including biotechnology, glycoengineering and vaccine development.