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Updated: Jul 11, 2026
Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria
Tania M Perehinec1, Saara N A Qazi, Sanyasi R Gaddipati
1School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK. tania.perehinec@nottingham.ac.uk
Background:
The 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.
Results:
Comparison 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.
Conclusion:
The 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.
