Site‐specific recombination for cloning of large DNA fragments in vitro. Issue 6 (26th May 2015)

• Record Type: Journal Article
• Authors: Dai, Ruixue; Zhang, Bo; Zhao, Guoping; Ding, Xiaoming
• Is Part Of: Engineering in life sciences. Volume 15:Issue 6(2015)
• Page Start: 655
• Page End: 659
• Publication Date: 2015-05-26
• Subjects: Bioengineering -- Periodicals; 660.605
• DOI: 10.1002/elsc.201400267
• Languages: English
• ISSNs: 1618-0240

Abstract

<abstract abstract-type="main"> <title> <x xml:space="preserve">Abstract</x> </title> <p>Natural‐product biosynthetic gene clusters are composed of large DNA fragments, which are difficult to synthesize in vitro. In this study, a <italic>Streptomyces</italic> phage site‐specific ΦBT1 integrase‐mediated recombination reaction was employed to obtain the 55 kb erythromycin biosynthesis gene cluster of <italic>Saccharopolyspora erythraea</italic>. Genome fragments inserted by ΦBT1 integrase recognition <italic>att</italic>‐sites (attachment site of bacteria and attachment site of phage) and two selectable markers (<italic>aphII</italic> gene and <italic>aac(3)IV</italic> gene) on both sides of the target gene cluster were extracted and subjected to an in vitro site‐specific recombination reaction to yield a plasmid pZB201 carrying the erythromycin biosynthesis gene cluster. This technique avoids mutations caused by PCR and limitations caused by restriction enzyme sites, making heterologous expression much more effective and convenient.</p> </abstract>