Using CRISPRi to knock down acm2 expression in L. plantarum WCFS1.

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Using CRISPRi to knock down acm2 expression in L. plantarum WCFS1. Using CRISPRi to knock down acm2 expression in L. plantarum WCFS1. (A) Quantifying the expression of acm2 using droplet digital PCR. The transcriptional levels of acm2 in wild-type L. plantarum WCFS1, in control cells with nontargeting sgRNA (IM133), and in CRISPRi strain cells with sgRNA targeting acm2 (IM134) are indicated. For the latter, cultures with SppIP inducer concentrations of 10 and 25 ng/ml were compared to uninduced culture, and those results are also shown (inset). The error bars represent standard deviations of results from at least two biological replicates, each of which was analyzed with three technical replicates. (B) Growth of L. plantarum with various expression levels of acm2. (Top panel) Culture tubes of L. plantarum grown for 16 h. Sedimentation of cells was clearly observed when acm2 was deleted (Δacm2 [34]) or knocked down (IM134 with or without added SppIP) but not in the wild-type or control cells (IM133). (Bottom panel) Quantification of cell sedimentation by measuring OD600 in the upper layer of the medium (see arrow in top panel) before and after vortex mixing. The ratios are plotted in the bar plot. The error bars represent standard deviations of data from three parallel measurements. (C) Phase-contrast images of the corresponding strains. The CRISPRi strains were imaged at the exponential-growth phase. The scale bar is 5 µm. Ine Storaker Myrbråten et al. mSphere 2019; doi:10.1128/mSphere.00007-19