Journal: Applied Microbiology and Biotechnology

Article Title: A new-engineered integrative tool to target the terminal compartment of the Streptomyces chromosome

doi: 10.1007/s00253-026-13707-2

Figure Lengend Snippet: Characterization of Samy phage attachment and integration sites in a panel of six Streptomyces species. A Schematic representation of pOJ260_ SAMYPH94 vector. The two black dashed lines mark the boundaries of the Samy phage region, which was cloned into the pOJ260 vector (Bierman et al. ) using a Golden Gate Assembly approach. The predicted promoter and att P site sequences are presented in Figure . ApraR, apramycin resistance; ColE1 Ori, ColE1 origin of replication in E. coli ; lacZα , gene encoding the LacZ alpha peptide; OriT, origin of transfer; SAMYPH93’ , fragment of SAMYPH93 gene). B Integration of pOJ260_ SAMYPH94 into the SAM40697_5543 gene or its orthologs in a panel of six Streptomyces species. PCR amplification of the att L and att R regions defined based on Samy prophage orientation in its native S. ambofaciens ATCC 23877 host is shown, with expected sizes indicated in brackets. The integration of pOJ260_ SAMPHY_94 in S. ambofaciens DSM 40697 is represented at scale. No PCR amplification is expected when gDNA from wild-type Streptomyces strains is used as the template (“T-”) or in absence of template (“∅”). The number “1” represents clone #1 analyzed for each strain. “L” denotes the molecular weight ladder (Thermo Scientific GeneRuler DNA Ladder 1 Kb Plus). The results for three independent clones per strain are presented in Figure . C Alignment of Samy- att P and att B sites from six Streptomyces strains against the consensus att B sequence. Sequences of 50 bp centered on the repeat (black hatched rectangle) identified in the integrated form of the Samy prophage (Jaffal et al. ) were analyzed. Strain names are presented in abbreviated form before each sequence, with the full names provided in panel B. The logo was generated using the online tool WebLogo ( https://weblogo.berkeley.edu/logo.cgi ) (Crooks et al. ). Gray lines indicate positions that are perfectly conserved across all analyzed sequences. The att P site is surrounded by imperfect inverted repeat sequences shown as arrows of the same color, indicating complementary sequences. During integration, a minimal sequence of 2-bp identical between att P and att B sites is referred to as the crossover sequence (Smith ). The repeat region contains several identical 2 bp positions, leaving the exact cleavage site yet to be determined. Please note that S. coelicolor and S. lividans att B sites are identical

Article Snippet: The Thermo Scientific GeneRuler 1 kb Plus DNA Ladder was used as the molecular size marker.

Techniques: Plasmid Preparation, Clone Assay, Amplification, Molecular Weight, Sequencing, Generated