LAB 9 - Green Fluorescent Protein (GFP) Purification correctly answered 2023

LAB 9 - Green Fluorescent Protein (GFP) PurificationWhat is a protein? Proteins are long chains of amino acids, some of which are hydrophobic Explain the relationship between genes and proteins: Genes contain the genetic code which determine the amino acid composition of a protein. There is a unique gene for each protein within all of the cells of the body Using your own words, describe cloning: The duplication and propagation of a cell or organism. Describe how the bacterial cloned cells on your LB/amp plate differ from the cells on your LB/amp/ara plate. How would you design an experiment to show that both plates of cloned cells behave similarly and do contain the same DNA? There was no difference as far as the growth, but rather the appearance of the bacterial colonies which signifies both retain different colors in the presence of UV light. The LB/amp/ara plate glowed green primarily because of arabinose, which explains why the other plate did not glow. Describe how you might recover a cancer-curing protein from the bacterial cells: One can isolate a single fluorescent green colony of bacteria and grow large amounts of the bacteria in a liquid growth media. Bacteria in liquid media can be concentrated by centrifugation. After the bacterial cells are lysed to release the cancer curing protein, the protein can be isolated by passage through a chromatography column which has an affinity for the cancer-curing protein. What is a bacterial colony? A bacterial colony is a large group or cluster of bacterial cells that originated from a single, clonal cell. Why was one green colony and one white colony picked from your agar plates? What could this prove? If a green colony was streaked onto an LB/amp plate, the resulting colonies would be white. This plate does not contain arabinose which is needed to induce expression of the GFP gene and generate green fluorescent colonies. If a white colony was streaked onto an LB/amp/ara plate, the resulting colonies would be green. Explain how placing cloned cells in nutrient broth to multiply relates to your overall goal of purifying the fluorescent protein: This makes it favor for the bacteria granting it an environment where it can be grown to encourage the production of more proteins for observation. What is a good way to concentrate a large number of cells? To place it in a tube containing the liquid cell culture into a centrifuge and spin it As you spin the cell culture, where would you expect the cells to concentrate, in the liquid portion or at the bottom of the tube in a pellet? In the pellet Exercise 1: Bacterial Concentration and Lysis: Step 1 = Using a marker, label one new ? ? tube with your name and period Micro centrifuge tube Exercise 1: Bacterial Concentration and Lysis: Step 2 = Obtain ? ? cultures previously grown from the common prep-bench (two ? and one ? culture) and observe them in normal lighting and then with the UV light. Note any color differences that you observe. -> three liquid -> two + -> one - Exercise 1: Bacterial Concentration and Lysis: Step 3 = Using a clean pipette, transfer ?mL of a (?) liquid culture into the ?mL micro centrifuge also labeled (?), then cap it. You may now set aside your (?) for disposal. -> 2mL -> (+) -> 2mL -> (+) -> (-) Exercise 1: Bacterial Concentration and Lysis: Step 4 = Spin the (?) micro centrifuge tube for ? minutes in the centrifuge at ? speed. Be sure to balance the tubes in the machine. -> (+) -> 5 minutes -> maximum speed Exercise 1: Bacterial Concentration and Lysis: Step 5 = After the bacterial liquid culture has been centrifuged, open the tube and slowly pour off the liquid ? above the ?. -> supernatant -> pellet Exercise 1: Bacterial Concentration and Lysis: Step 6 = Observe the pellet under ? light. Note your observations UV Exercise 1: Bacterial Concentration and Lysis: Step 7 = Using a new pipette, add ?uL of ? ? to each tube. ? the bacterial pellet thoroughly by rapidly pipetting ? and ? several times. -> 250uL -> TE solution -> Resuspend -> up and down Exercise 1: Bacterial Concentration and Lysis: Step 8 = Using a new pipette, add ?uL (~1 drop) of ? to the resuspended bacterial pellet. Cap and mix the contents by ? and ? vigorously. The ? will start ? bacterial ? ?. Observe the tube under the UV light. Place the micro centrifuge tube in the ? for ?-? minutes. Freezing will cause the bacteria to ? and ? completely. -> 50uL -> lysozyme -> flicking -> inverting -> lysozyme -> digesting -> cell wall -> freezer -> 10-20 minutes -> explode -> rupture