Fig. 3
PCR from genomic DNA (gDNA) to prove vector integration of pKC1139-based constructs by homologous recombination. a Scheme of tested primer combinations and expected PCR products in case of no vector integration: Primer A and B form PCR-product AB indicating presence of the insert. Primer C and D form PCR-product CD flanking the genomic location of the gene of interest. Both PCR-products occur in the mutant Actinoplanes sp. SE50/110 [pKC1139:: PermE*::::acbL] (L) (b). c Scheme of tested primer combinations and expected PCR products in case of vector integration by homologous recombination (HR) leading to the PCR-products CB (from Primer C and B) and AD (from Primer A and D). Both PCR-products occur in the tested mutant Actinoplanes sp. SE50/110 [pKC1139::PermE*::acbL] (L) indicating, that vector integration by homologous recombination has occurred (d). The primer combination AB does not form a PCR product in this constellation. Primer combination CD might theoretically produce a larger product (grey dashed lines), but due to the size, GC-content and complex structures within the vector sequence leading to premature termination of the polymerase reaction, this product does not emerge during PCR. As control for all PCR experiments the empty vector control Actinoplanes sp. SE50/110 [pKC1139] (C1) and the wild-type (C2) were tested. Here, only primer combination C and D led to the expected fragment CD (b)