Perehinec Tania M, Qazi Saara N A, Gaddipati Sanyasi R, Salisbury Vyvyan, Rees Catherine E D, Hill Philip J
School of Biosciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics LE12 5RD, UK.
BMC Mol Biol. 2007 Sep 19;8:80. doi: 10.1186/1471-2199-8-80.
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.
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.
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.
Gateway重组克隆系统可实现DNA片段的轻松快速连接。在此，我们报告了三种不同革兰氏阳性载体的构建与评估，这些载体可与多位点Gateway克隆系统一起使用，以便在质粒构建体中快速产生新的基因排列，用于多种革兰氏阳性细菌。
将报告基因表达模式与传统构建的克隆进行比较表明，尽管基因表达的总体水平可能有所不同，但残留重组（att）位点的存在对基因表达模式没有影响。这些新载体的快速构建使得在不同细菌细胞中评估质粒构建体后能够优化载体/基因组合，并证明了使用Gateway克隆进行质粒构建的优势。
Gateway克隆后存在的残留att位点不影响革兰氏阳性细菌中启动子诱导模式，并且没有证据表明转录本的mRNA稳定性存在差异。然而，基因表达的总体水平可能会降低，这可能是由于某些转录后事件所致。本文所述的新载体能够在多种革兰氏阳性细菌中实现更快、更高效的克隆。
