Biochemical foundation for each of the four enzymatic assays performed on each cellular fraction. We performed various assays on each of the four log phase culture fractions, as well as the extracellular fraction from a stationary phase culture, to test the localization of the enzymes. For the protease assay, a specific soluble chromogenic substrate called azocasein was employed to determine presence of enzyme activity. The degradation of this substrate in trichloroacetic acid produces a colour change that can be registered using a spectrophotometer, and this change in absorbance over time is proportional to enzyme activity (Charney and Tomarelli, 1947). We measured the absorbance of the sample at 370nm as the maximum wavelength at which this
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The purpose of the cell fractionation protocol was to separate P. aeruginosa into four cellular components while maintaining the enzyme activity within each of the four fractions. The first step in fractionation is commonly centrifugation as it separates the components based on size, meaning that after the initial centrifugation, the pellet will contain whole cells, and the supernatant will contain the extracellular component (Alberts et al., 2002). The sample we used was spun down and the supernatant was removed, which presumably contained any secreted molecules within the residual volume of liquid. A step that we did not perform, however, which may have led to improved separation of the components is to layer the surface of the homogenate with a dilute salt solution, which causes the components to have greater separation (Alberts et al., 2002). The periplasm can be isolated by spheroblasting, then centrifugation (Thein et al., 2010). To do this, the pellet was re-suspended in a solution containing Ethylenediaminetetraacetic acid (EDTA), tris buffer, lysozyme, and sucrose. Inner membranes of Gram-negative bacteria can be uncovered by using a mixture of lysozyme and EDTA, which causes the outer membrane to break down (Thein et al., 2010), liberating the periplasmic contents. The sucrose was used to maintain an isotonic solution to prevent lysis of the sphaeroplasts, which are simply cytoplasmic membrane bound cells. The sphaeroplasts were then spun down using centrifugation, and the supernatant was removed, which presumably contained the contents of the periplasm. Once the periplasmic components have been collected, one method to obtain the cytoplasmic components is to lyse the cells, and then spin down any of the membrane fragments that are present (Thein et al., 2010); to do this we osmolytically lysed the cells by subjecting them to a hypotonic solution. We also added DNase at this point, which degraded the DNA that
The purpose of the cell fractionation protocol was to separate P. aeruginosa into four cellular components while maintaining the enzyme activity within each of the four fractions. The first step in fractionation is commonly centrifugation as it separates the components based on size, meaning that after the initial centrifugation, the pellet will contain whole cells, and the supernatant will contain the extracellular component (Alberts et al., 2002). The sample we used was spun down and the supernatant was removed, which presumably contained any secreted molecules within the residual volume of liquid. A step that we did not perform, however, which may have led to improved separation of the components is to layer the surface of the homogenate with a dilute salt solution, which causes the components to have greater separation (Alberts et al., 2002). The periplasm can be isolated by spheroblasting, then centrifugation (Thein et al., 2010). To do this, the pellet was re-suspended in a solution containing Ethylenediaminetetraacetic acid (EDTA), tris buffer, lysozyme, and sucrose. Inner membranes of Gram-negative bacteria can be uncovered by using a mixture of lysozyme and EDTA, which causes the outer membrane to break down (Thein et al., 2010), liberating the periplasmic contents. The sucrose was used to maintain an isotonic solution to prevent lysis of the sphaeroplasts, which are simply cytoplasmic membrane bound cells. The sphaeroplasts were then spun down using centrifugation, and the supernatant was removed, which presumably contained the contents of the periplasm. Once the periplasmic components have been collected, one method to obtain the cytoplasmic components is to lyse the cells, and then spin down any of the membrane fragments that are present (Thein et al., 2010); to do this we osmolytically lysed the cells by subjecting them to a hypotonic solution. We also added DNase at this point, which degraded the DNA that