Lab 8 Flashcards
Q: What is gene expression?
The process by which cells use the instructions in their DNA to synthesize proteins. It involves transcription (DNA to RNA) and translation (RNA to protein)
What is lactase and what does it do?
an enzyme produced by E. coli that breaks down lactose into glucose and galactose for energy.
Why do we use a spectrophotometer in this lab?
To measure the absorbance of the yellow product formed when lactase breaks down ONPG. The absorbance indicates how much enzyme activity is present.
What is the purpose of centrifugation in this experiment?
to separate the E. coli cells from the growth media and later to separate the cell debris after lysis.
Q: What is the function of the lysis buffer?
A: It breaks open the E. coli cells without denaturing the lactase enzyme.
Q: What is ONPG, and why is it used?
ONPG (2-nitrophenyl β-D-galactopyranoside) is a substrate for lactase. When lactase breaks it down, it produces a yellow product, which can be measured with a spectrophotometer.
Q: Why do we measure absorbance at 420 nm?
420 nm is the wavelength that corresponds to the yellow color produced by the breakdown of ONPG, allowing us to measure lactase activity.
Q: What does the standard deviation tell you in this lab?
A: It tells you how variable your data is, providing an indication of how consistent your measurements are across different trials.
Q: What is centrifugation?
Centrifugation is a process where a sample is spun rapidly in a machine (centrifuge) to separate its components based on their size and density.
What happens during centrifugation?
During centrifugation, heavier components (like cells or large particles) form a pellet at the bottom of the tube, while lighter components (like liquids or small particles) remain in the supernatant (the liquid above the pellet).
Why do we centrifuge before lysing the E. coli cells?
to separate the E. coli cells from the growth media, which does not contain lactase enzyme. The cells form a pellet at the bottom, and the supernatant (growth media) is discarded.
: What is the purpose of removing the growth media before lysing the E. coli cells?
Removing the growth media ensures that only the cells (which contain the lactase gene) are used for lysis, so we can focus on breaking open the cells to extract the lactase enzyme.
Q: Why do we centrifuge after lysing the E. coli cells?
After lysis, centrifugation is used to separate the cell debris (broken cell parts like membranes) from the liquid lysate that contains the enzyme lactase.
What is the purpose of separating the cell debris after lysis?
The purpose is to isolate the enzyme-containing lysate from the cell debris so we can accurately measure enzyme activity (lactase) in the liquid portion of the sample.
What does the supernatant contain after centrifugation post-lysis?
soluble components, including the lactase enzyme, while the pellet contains the cell debris.
