Abstract
BackgroundThe development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. The construction of plasmid-less marker-less strains has many advantages for the further practical exploitation of these bacteria in industry. Such producer strains are usually constructed by sequential chromosome modifications including deletions and integration of genetic material. For these purposes complex methods based on in vitro and in vivo recombination processes have been developed.ResultsHere, we describe the new scheme of insertion of the foreign DNA for step-by-step construction of plasmid-less marker-less recombinant E. coli strains with chromosome structure designed in advance. This strategy, entitled as Dual-In/Out, based on the initial Red-driven insertion of artificial φ80-attB sites into desired points of the chromosome followed by two site-specific recombination processes: first, the φ80 system is used for integration of the recombinant DNA based on selective marker-carrier conditionally-replicated plasmid with φ80-attP-site, and second, the λ system is used for excision of inserted vector part, including the plasmid ori-replication and the marker, flanked by λ-attL/R-sites.ConclusionThe developed Dual-In/Out strategy is a rather straightforward, but convenient combination of previously developed recombination methods: phages site-specific and general Red/ET-mediated. This new approach allows us to detail the design of future recombinant marker-less strains, carrying, in particular, rather large artificial insertions that could be difficult to introduce by usually used PCR-based Recombineering procedure. The developed strategy is simple and could be particularly useful for construction of strains for the biotechnological industry.
Highlights
The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls
Construction of plasmid-less marker-less strains has advantages for extending the practical exploitation of these bacteria in industry [1,2,3,4,5]. Such producer strains are usually constructed by sequential chromosome modifications, mainly including deletions and integration of genetic material
The desired locations of φ80-attB sites can be spread in non-essential parts of bacterial genome at different distances from oriC
Summary
The development of modern producer strains with metabolically engineered pathways poses special problems that often require manipulating many genes and expressing them individually at different levels or under separate regulatory controls. Construction of plasmid-less marker-less strains has advantages for extending the practical exploitation of these bacteria in industry [1,2,3,4,5] Such producer strains are usually constructed by sequential chromosome modifications, mainly including deletions and integration of genetic material. This method has been named Recombinogenic Engineering or Recombineering and reviewed in several papers [12,13,14,15] It usually (but not always) based on λRed- or RecET-mediated recombination between bacterial chromosome and amplified DNA fragment carrying the removable selective marker, in which PCR primers provide the rather short homology to the targeted sequence. Site-specific insertion of cassettes can be carried out in preliminary randomly integrated recombinogenic sites [3], or the "marked" cassettes can be randomly integrated, followed by excision of the marker by a site-specific recombination system [1]
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