A strategy for seamless cloning of large DNA fragments from Streptomyces.

We report a novel method for the seamless cloning of large DNA fragments (SCLF) of up to 44 kb or larger from Streptomyces chromosomal DNA. SCLF is based on homologous recombination in Streptomyces and is easy to perform. The strategy of SCLF is to flank the target sequence in the chromosomal DNA with two identical restriction sites by the insertion of plasmids containing that site at either end of the fragment, which is then isolated by plasmid rescue through the self-ligation of restriction digested genomic DNA. The method involves three steps: (i) placing a certain restriction site (CRS) at the 3'-end of the target sequence by insertion through homologous recombination of a plasmid containing the CRS; (ii) inserting through homologous recombination at the 5'-end of the target sequence a linearized self-suicide vector with the identical CRS; (iii) digesting the genomic DNA with the certain restriction enzyme followed by self-ligation in order to plasmid rescue the target fragment. SCLF can be applied to other Actinomycetales, and further optimizations may reduce the amount of time required to perform this technique.