Fig. 1: Excision and replication dynamics of PICMI115.
a Schematic representation of PICMI115 integrated between the zntR and fis genes encoding a HTH-type transcriptional regulator and the factor for inversion stimulation respectively. Arrows depict inward- and outward-directed primers used to detect the integration site after excision (gray) or the single circular and concatemeric forms of PICMI115 (black). For transduction experiments PICMI115 was marked by CmR (orange triangle). Orange forward and gray reverse primers were used to control of the integration of PICMI115 at the end of fis gene. b Estimation of phage and PICMI DNA size. DNA extracted from phage Φ115 or as control Φ115pure, were separated on a SYBR Green stained agarose gel and Southern blotted with PICMI115 or Φ115 probes. M: molecular marker (Smart ladder Eugentec). Images are representative of two independent experiments. Uncropped gels and blots are provided as a Source Data file. The dynamics of excision and replication following phage Φ115 infection were explored by qPCR (c) and Southern blot (d). c Standard curves allowed for the determination of the number of DNA copies per 20 ng of DNA. Source data are provided as a Source Data file. Bar charts show the mean ± Standard Error of Mean (SEM) with n = 3 independent replicates depicted by individual dots. The dashed line signifies the limit of detection for this assay. Spearman’s test for x versus time (p-value) indicates a significant increase in copy at 15 min for phage (0.9602, p < 0.001), the empty site (0.8183, p = 0.0002), and circular/concatemer PICMI (0.9274, p < 0.001). In contrast, a reversed trend is observed for vibrio (0.7419, p = 0.0015). At the 30-minute mark, a one-tailed t-test (p-value), transformed to Log10, demonstrated significance for phage (0.0060) and circular/concatemer PICMI (0.0059) only. d In the Southern blot C, S, and P indicate the concatemeric and single form of PICMI115 and phage genome respectively. Images are representative of two independent experiments.
