Sample preparation and lysis time determination Lysogens

Sample preparation and lysis time determination Lysogens eFT508 supplier were cultured overnight in LB or minimal salts media (see below) at 30°C on a rolling drum. Stationary phase cultures were diluted 100-fold in LB or minimal salts media, then grown to A550 ~ 0.2. 200 μL of exponentially growing cells were immobilized on a 22 mm square glass coverslip that has been pretreated with 0.01% tissue-culture tested poly-L-lysine (mol. wt. 150 K – 300 K, Sigma, St. Louis, MO) at room temperature for 30 min. After assembling the perfusion chamber, the device was immediately placed on the heating platform and CH5424802 molecular weight infused

with heated medium to maintain the chamber temperature at 30°C for 30 min to stabilize the cells. To induce lysis, the chamber temperature was raised to 42°C for 15 min, and then dropped to 37°C for the duration of the observation period (i.e., until ~95% of cells are lysed). Video recording was initiated at the time when the temperature was raised to 42°C. Under these conditions, it usually takes less than 5 min for the temperature to rise from 30°C to 42°C, a transition comparable to shifting culture flasks from a 30°C to 42°C

waterbath shaker. Some experiments were performed by adding KCN to the growth medium in the sidearm feeder bottle to a final concentration of 20 mM. Videos were subsequently analyzed using Windows Media Player™ BIRB 796 molecular weight playback. The times of individual lysis events were then noted visually and recorded manually. The lysis time was defined as the time Ureohydrolase from the initiation of the first temperature shift to when the image of the cell disappeared from view. In general, it takes about a few seconds (frames) for lysing cells to fully disappear from view (Figure 1A). Determination

of lysogen growth rate Lysogen growth rate was manipulated by using different growth medium formulations: (i) full-strength LB (10 g tryptone, 5 g yeast extract, 10 g NaCl per L dH2O), (ii) one-fifth-strength LB (2 g tryptone, 1 g yeast extract, 10 g NaCl per L dH2O), (iii) 20 mM glucose in Davis minimal salts (7 g K2HPO4, 2 g KH2PO4, 1 g (NH4)2SO4, 0.5 g sodium citrate•2H2O, and 0.2 g MgSO4•7H2O), and (iv) 40 mM glycerol in Davis minimal salts. We assessed the growth of the lysogen strain IN56 by culturing it overnight at 30°C in each growth media. The next day, 90 μL of the overnight culture was used to inoculate 25 mL growth medium and the culture was placed in a 30°C waterbath shaker at 220 rpm. Culture growth was followed with a sipper-equipped spectrophotometer at A550. The growth rate was calculated as the slope of the linear regression of natural-logarithm transformed A550 values over time. Statistical analysis In most cases, data collection for a given strain or treatment spanned several days. Therefore, even for the same lysogen strain or experimental treatment the means and/or variances may be significantly different among data collected from different dates.

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