Rapid customised operon assembly by yeast recombinational cloning.
We have developed a system called the Operon Assembly Protocol (OAP), which takes advantage of the homologous recombination DNA repair pathway in Saccharomyces cerevisiae to assemble full-length operons from a series of overlapping PCR products into a specially engineered yeast-Escherichia coli shuttle vector. This flexible, streamlined system can be used to assemble several operon clones simultaneously, and each clone can be expressed in the same E. coli tester strain to facilitate direct functional comparisons. We demonstrated the utility of the OAP by assembling and expressing a series of E. coli O1A O-antigen gene cluster clones containing various gene deletions or replacements. We then used these constructs to assess the substrate preferences of several Wzx flippases, which are responsible for translocation of oligosaccharide repeat units (O units) across the inner membrane during O-antigen biosynthesis. We were able to identify several O unit structural features that appear to be important determinants of Wzx substrate preference. The OAP system should be broadly applicable for the genetic manipulation of any bacterial operon and can be modified for use in other host species. It could also have potential uses in fields such as glycoengineering.
School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia.
Michael A
MA
Liu
School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia.|School of Biomedical Sciences and Infectious Diseases Program, Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology, Brisbane, 4001, QLD, Australia.
Johanna J
JJ
Kenyon
School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia.
Jason
J
Lee
School of Life and Environmental Sciences, The University of Sydney, Sydney, NSW, 2006, Australia. peter.reeves@sydney.edu.au.
Peter R
PR
Reeves
D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics; D017353:Gene Deletion; D005822:Genetic Vectors; D059765:Homologous Recombination; D026901:Membrane Transport Proteins / Q000235:genetics; D005810:Multigene Family; D019081:O Antigens / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D009876:Operon; D016133:Polymerase Chain Reaction / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D019081:O Antigens; C543926:Wzx protein, E coli
Cloning; Flippase; O-antigen; Operon; Recombination; Translocase