Journal: Frontiers in Microbiology
Article Title: The stability region of the Streptomyces lividans plasmid pIJ101 encodes a DNA-binding protein recognizing a highly conserved short palindromic sequence motif
Figure Lengend Snippet: Electrophoretic mobility shift assays demonstrating binding of SpdA2-His to the palindromic sequence motif sps . Different PCR fragments, as schematically illustrated, were incubated with ~2 pmol purified SpdA2-His protein (+) or without SpdA2-His (−), run on a 6% Tris-acetate polyacrylamide gel and stained with EtBr. The gray arrow indicates the band corresponding to a PCR fragment (FTraR pSVH1 ) added as a negative control for binding specificity. Black arrows mark retarded DNA fragments. Interaction of SpdA2-His with the FA2 fragment resulted in several retarded bands (A) . Whereas SpdA2-His did not interact with the FA2-sps fragment (B) , SpdA2-His shifted fragment F1sps, containing a single palindromic sps sequence (C) . SpdA2-His also bound to the PCR fragment FSpdA up , containing a T insertion within the palindromic sequence sps (D) , but did not recognize FSpdA, lacking sps (E) .
Article Snippet: Construction of plasmids pIJ303ΔspdA2, pIJ303-sps, and pIJ303spdA2c To construct pIJ303Δ spdA2 we performed an XL-PCR (KAPA Long Range DNA Polymerase, Kapa Biosystems) with primers 303DelAl/303DelBu (Table ).
Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Sequencing, Polymerase Chain Reaction, Incubation, Purification, Staining, Negative Control