Study Questions Amgen Lab 6 (part I and part II): Overnight Culture of E. coli / Purification of mFP—CLASS SET
Part I: Overnight Culture of E. coli
Directions: Answer the following questions ON YOUR OWN PAPER and in COMPLETE SENTENCES.
1. What did your teacher do for this lab?
2. The original mFP was mutated to ______.
3. What are the three important regions required in order to “express” the rfp gene?
4. What is the purpose of the AraC protein?
5. What is the promotor site? What is the function of a promotor site (look in notes)?
6. Define transcription.
7. Define translation.
8. Describe what happens to protein production when arabinose is not available in the bacterium’s environment. (explain in detail)
9. Describe what happens to protein production when arabinose IS PRESENT in the bacterium’s environment. (explain in detail)
10. What is the dual function of AraC protein?
11. What are “inclusion bodies”?
Part II: Purification of mFP
Directions: Answer the following questions ON YOUR OWN PAPER and in COMPLETE SENTENCES.
1. What is a “cloning vector”?
2. What is an “expression vector”?
3. Explain the two objectives that most biotech companies like Amgen would do when they have identified a promising therapeutic protein. (think about what we have done in this series)
4. Explain how we LYSED the bacterial cells.
5. What is in the tube when we broke open the bacterial cells? Why did we centrifuge the sample?
6. How big is the mFP protein?
7. DRAW and DESCRIBE what the mFP protein looks like.
8. What is a fluorophore?
9. Define hydrophobicity. What is it important in this lab?
10. Describe how proteins react in water.
11. Define conformation.
12. What groups interact in the protein to provide this conformation? (in protein notes)
13. What has made the isolation of the mFP easier when there are lots of other proteins in our sample?
14. Describe column chromatography.
15. Draw a picture of the column that we used in the lab. LABEL the “resin bed”.
16. What types of beads were used in the resin bed of our columns?
17. How did we get the mFP to “stick” to the column? (explain in detail)
18. What was the purpose of the wash buffer (low salt concentration)?
19. What was the purpose of the elution buffer (very low salt concentration)?
20. Explain HOW the mFP is released from the resin bed.
21. Does our tube of mFP contain ONLY mFP protein? Explain.
22. What does it mean when our tube of mFP fluoresces very brightly under UV light?
23. What difficulties did you encounter in this lab?
24. What did you enjoy about this lab series? What improvements could be made to this lab series?