Question

These questions are based off the General Biology 2 Lab: PCR and DNA Fingerprinting. Here is some background:

"Previously in this course you performed a genetic transformation of E. coli bacterial cells. The results of this procedure were colonies of cells that fluoresced when exposed to ultraviolet light. This is not a normal phenotype (characteristic) for E. coli. You were then asked to figure out a way to determine which molecule was becoming fluorescent under UV light. After determining that the pGLO plasmid DNA was not responsible for the fluorescence under the UV light, you concluded that it was not the plasmid DNA that was fluorescing in response to the ultraviolet light within the cells. This led to the next hypothesis that if it is not the DNA fluorescing when exposed to UV light, then it must be a protein that the new DNA produces within the cells." Some of the steps of this experiment:

Exercise 1: Bacterial Concentration and Lysis

Exercise 2: Protein Chromatography

a. 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?

b. Describe how you might recover a cancer-curing protein from the bacterial cells.

c. Why was one green colony and one white colony picked from your agar plates?

d. Explain how placing cloned cells in nutrient broth to multiply relates to your overall goal of purifying the fluorescent protein.

e. Why did you discard the supernatant in this part of the protein purification procedure?

f. What was the purpose of rupturing or lysing the bacteria?

g. Why did you discard the pellet in the chromatography of the protein purification procedure?

h. Briefly describe hydrophobic interaction chromatography and identify its purpose in this lab.

i. As a summary, provide a definition or purpose for chromatography in general

PLEASE ANSWER EVERYTHING or at least 80% of the questions!!!

Answer 1

A) 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.

B) This is done by breaking the cell wall of the bacterial cell using lysozyme. Then, freeze the substance which allows the cytoplasm to expand making the cell burst, releasing all of the proteins and genetic material into the supernatant.

C) To show how the genes were expressed.

D) 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.

E) It did not contain the required the protein.

F) This is to extract the protein from the cell. However, this requires another procedure the obtain the GFP by separating it from other endogenous bacterial proteins.

G) The proteins are now stored in the supernatant because of how the cell bursted with everything inside going outside into the supernatant.

H) The hydrophobic property of the GFP matches with the high concentration of salt in the column which captures the GFP instead of eluting. This separation of the proteins is linked with gel infiltration or the difference of polarity.

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