Investigating Nuclease Processing with Variably Phosphorothioated Cassettes. A)

<p>Diagram of the variably phosphorothioated (VPT) cassette series. Homology arms to <i>lacZ</i> are shown in blue, and the inserted heterologous <i>kanR</i> gene is shown in gold; the lagging-targeting strand is represented as the top strand, and the leading-targeting strand is represented as the bottom strand. The red asterisk indicates the presence of 4 consecutive phosphorotiohate bonds. <b>B)</b><i>In vitro</i> Lambda Exo digestion of non-phosphorothioated (VPT1, left), single end phosphorothioated (VPT2, center), and dually phosphorothioated (VPT4, right) dsDNA cassettes. Samples were digested with no enzyme (lanes 1, 5, & 9) and tenfold-increasing amounts of enzyme, from left to right. The bottom band is dsDNA, and the top band is the ssDNA product after Lambda Exo degrades one of the two strands. PAGE analysis confirms that dually phosphorothioated cassettes are protected from degradation. <b>C–E)</b> Recombination frequencies for the VPT cassette series in EcNR2 (C), nuc4⁻ (D), and EcNR2.<i>xseA</i>⁻ (E). Insertion frequencies were measured as the number of kanamycin resistant recombinants over the total number of cfu (as plated on non-selective media). These data are presented as the mean with the error bars as standard deviation; n = 3 for EcNR2 and EcNR2.<i>xseA</i>⁻, and n = 2 for nuc4⁻. Numerical data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044638#pone.0044638.s002" target="_blank">Table S2</a>. <b>F& G)</b> Cassette insertion frequencies were measured in technical replicates for EcNR2, nuc4⁻, and EcNR2.<i>xseA</i>⁻. Data in F) & G) are presented as the mean with the standard error of the mean. The ratio between VPT4 and VPT1 (F) indicates a strain’s ability to recombine cassettes with terminal PT bonds preventing the direct action of Lambda Exo. The ratio between VPT4 and VPT7 (G) indicates whether a strain is more able to process terminal PT bonds (VPT4) in comparison with internal PT bonds (VPT7).