The resulting cDNA was diluted 1:25 or 1:1250 for probing target gene and 16s rRNA templates respectively. Primers were designed to amplify a region of 150 bp within each transcript, using the Power SYBR Green PCR 2× Master Mix kit (Applied Biosystems). qRT-PCR was performed using the Applied Biosystems 7900HT Real-Time system. The run was computer controlled by SDS 2.3 (Applied Biosystems). A no template control (NTC) was performed to provide a value for the background fluorescence present in a negative reaction. Three replicates for both the target and endogenous control www.selleckchem.com/products/ulixertinib-bvd-523-vrt752271.html were analyzed, and the target quantitation was normalized to the endogenous control for each replicate. The NTC was automatically
subtracted from each RT-PCR reaction prior to averaging the replicates. The resulting data for each sample were calibrated to the WT expression Crenigacestat clinical trial levels and are shown as a relative quantity to the WT. A gene expression plot based on relative quantitation was generated using RQ Manager 1.2 (Applied Biosystems). Motility and developmental assays Motility phenotypes of mutants were compared with that GSK2879552 nmr of the WT strain using swarm assays [58], by microscopic examinations of colony edges, and by time-lapse microscopy [59]. Swarm assays were performed in triplicate as described by Shi and Zusman [58]. Photomicrographs of the edges of isolated colonies were obtained using a Nikon FXA microscope with
the 10× objective and captured by a Coolsnap Cf camera. Time-lapse microscopy was performed on CTPM medium with Beta adrenergic receptor kinase 1.5% Ultra-Pure agarose (Invitrogen) slabs.
Cells were taken from mid-log phase liquid cultures and 50 μl of cell culture was pipetted onto the surface. Slabs were covered with a coverslip and incubated at 32° for 30 min prior to microscopic examination. For MC assays, 50 μl of mid-log phase cells were pipetted directly onto a slide inside a silicone gasket. After 20 min adherence at room temperature, the excess media were removed and the cells were overlaid with CTPM broth and 1% MC, (final concentration 0.5× and 0.5%, respectively). After a coverslip was placed, the slide was incubated at 32° for 30 min. Cells were photographed at 200× magnification, every 30 seconds for 30 min, yielding 61 time points for measurement. Time-lapse data are based on 25 randomly chosen cells tracked for each strain and each condition. Strains that had fewer than 10% motile cells are classed as non-motile and their reversal rates were not determined. Motile cells were tracked in Metamorph, and their position data was used to generate velocity rates, but only reversing cells factored into cell reversal frequency by the Motility Macro v2.2 [60]. Cells were considered to reverse if they progressed one cell length then paused and moved in a new direction at least 110 degrees from the original direction of motion. Speeds are related in the text as the average of 25 cells ± the standard deviation.