Abstract
BackgroundThe Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. Here we report the construction and evaluation of three different Gram-positive vectors that can be used with the Multisite Gateway cloning system to rapidly produce new gene arrangements in plasmid constructs for use in a variety of Gram-positive bacteria.ResultsComparison of patterns of reporter gene expression with conventionally constructed clones show that the presence of residual recombination (att) sites does not have an effect on patterns of gene expression, although overall levels of gene expression may vary. Rapid construction of these new vectors allowed vector/gene combinations to be optimized following evaluation of plasmid constructs in different bacterial cells and demonstrates the benefits of plasmid construction using Gateway cloning.ConclusionThe residual att sites present after Gateway cloning did not affect patterns of promoter induction in Gram-positive bacteria and there was no evidence of differences in mRNA stability of transcripts. However overall levels of gene expression may be reduced, possibly due to some post-transcriptional event. The new vectors described here allow faster, more efficient cloning in range of Gram-positive bacteria.
Highlights
The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments
While many cloning and expression vectors have been developed for use in Gram-negative bacteria, there is a paucity of equivalent materials for use when working in Gram-positive bacteria
The Gateway system uses directed recombination between modified attachment sites derived from E. coli bacteriophage λ
Summary
The Gateway recombinatorial cloning system allows easy and rapid joining of DNA fragments. While many cloning and expression vectors have been developed for use in Gram-negative bacteria, there is a paucity of equivalent materials for use when working in Gram-positive bacteria Recombinatorial cloning systems, such as Invitrogen's 'Gateway' provide an alternative to conventional cloning that uses restriction enzymes and ligation. During integration the phage attP site recombines with the related, but non-identical, bacterial attB site producing hybrid attL and attR sequences located to the left and right of the phage genome, respectively. This recombination is mediated by integrase (Int) and integration host factor (IHF). To facilitate the recombinatorial cloning, the appropriate enzymes are supplied by the manufacturer as BP clonase, that mediates attB/attP recombination events, and LR clonase to mediate recombination between attL/attR sequences
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