Stemi 2000 c dissecting microscope

Gene Tools, Inc. designed all the morpholinos. Sequence is provided (Figure 2C). Control MO sequence used in this study is 5′-CCTCTTACCTCAGTTACAATTTATA-3′. All MOs were each injected into Transgenic (Tg: flk:EGFP) (Choi et al., 2007 (link)) one cell stage embryos. The 1 mM MO stock solutions were diluted to a final concentration of 2 ng/nL (250 μM), and appropriate concentrations as shown in the figure panels were injected into each embryo. For phenotypic imaging, 30 and 52 hpf embryos were anesthetized in 0.02% tricaine, mounted on a depression slide, and imaged with a Zeiss Stemi 2000-C dissecting microscope.

To test the ability of SypA variants to induce biofilm formation despite the presence of active SypE, the sypG overexpression plasmid pEAH73 [25 (link)] was introduced into the strains by conjugation and selection for Cm^R. These strains were grown overnight in LBS containing Cm, then subcultured into the same medium at 28°C. After growth to mid-log phase, cultures were normalized to an OD₆₀₀ of 0.2 and an aliquot of 10 μL was spotted onto LBS Cm plates. After the spots dried, the plates were inverted and incubated for 48 h at 28°C. After incubation, images of the spots were captured using a Zeiss Stemi 2000-C dissecting microscope.
To test the activity of SypA variants, we evaluated their ability to complement a ΔsypA mutant under conditions in which SypE is non-inhibitory, i.e., during overexpression of rscS from plasmid pKG11 [23 (link)]. When overproduced under these LBS conditions, RscS inhibits the kinase activity of SypE, permitting SypA to be unphosphorylated and thus active [26 (link)]. The pKG11-containing strains were grown and analyzed as described above for SypG overproduction, with the exception that the plates were incubated at 24°C.

V. fischeri strains were grown overnight at 28°C in LBS broth with appropriate antibiotics. The following day, cultures were diluted 1:100 in LBS and grown at 28°C with shaking. After reaching mid-log phase, cultures were diluted to an OD₆₀₀ of 0.2. Cultures were spotted in 10 μl aliquots onto the appropriate solid media (as indicated in the figure legends) and grown at room temperature (approximately 24°C). Spotted cultures were imaged at the indicated times using a consistent magnification with a Zeiss Stemi 2000-C dissecting microscope. At the final time point colonies were disrupted by dragging a toothpick across the center of the colony to assess cohesiveness; this is a previously described method for detecting Syp polysaccharide production [16 (link), 17 (link)]. The images presented here are representative of at least two independent experiments. Due to the variability between experiments, a difference of 2 hours or less was not considered significant when determining differences in time to initiation of wrinkling. Between replicates, the time to initiation of wrinkling by V. fischeri under any given condition was consistent relative to the different conditions being tested in each experiment.