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Biotech week 7,8

  1. What is a fusion protein?
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  2. How is a regulated promoter different than a constitutive promoter?
    Constitutive promoters are ALWAYS ON
  3. How is gene expression/translation different between eukaryotes and prokaryotes?
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  4. In terms of proteins, do eukaryotes or prokaryotes have more post-transnational processing?
    Eukaryotes
  5. T/F
    Prokaryotes give more proteins per amount of effort than Eukaryotes
    True
  6. Plasmids are a normal part of which organisms life? Prokaryotic/Eukaryotic
    Prokaryotic
  7. Which of the following often make it easier to express proteins in Prokaryotic systems compared to Eukaryotic systems?
    * Prokaryotic cells tend to grow and divide more rapidly than Eukaryotic cell cultures with less effort

    * Prokaryotic expression vectors don't require a lot of features to ensure proper RNA packaging and processing

    * Prokaryotes more readily replicate extra-chromosomal DNA and propagate it as compared to Eukaryotic cells
  8. Why do we want a regulated promoter? (Two conflicting desires make tight regulation necessary)
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  9. Why do we often use regulated expression when expressing proteins for biotechnology applications?
    To balance the goal of expressing lots of protein in each cell with the need to produce many healthy cells from which to produce enough healthy cells from which to extract enough protein for downstream applications
  10. The lac operon is (on/off) when left to itself?
    off, no lactase is produced.
  11. Define: Operon
    All genes involved in a particular process. In the case of the lac operon, the genes involved in producing lactase
  12. Define: polycistronic regulation
    when multiple genes are linked to one promoter
  13. Which of the following is a true statement about the Pi and Plac promoters?
    Pi is constitutive promoter, and PLac is regulative (does not turn on without presence of lactose)
  14. Which of the following is key for keeping the Plac promoter “Off” in the absence of Lactose?
    Lac-I must be bound to the Operator sequence

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  15. Why would we use IPTG to drive expression from the Plac promoter instead of Lactose?
    Unlike IPTG, Lactose would be metabolized. Eventually there would not be enough to bind Lac-I protein, which would be free to bind the operator and repress expression Plac.
  16. Which factor is shared between DNA and protein that affects migration of the molecule during electrophoresis?



    A) Size
  17. If the coding sequence of your gene is 441 base pairs then what is the approximate molecular weight of the protein?




    C) 16.8 kDa

    Remember to divide amino acid count by three before multiplying the ratio by 114 kDa/amino acid
  18. What is the approximate weight of the GST::CaMPARI fusion protein if we do not include the linker region?
    • 74.7 kDA
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  19. What is SDS-PAGE?
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  20. How does SDS-Page work?
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  21. In SDS-PAGE, which reagent coats proteins to give them a uniform negative charge?
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  22. Why is the reducing agent β-Mercaptoethanol added in Sample Loading Buffer?




    B) Break disulfide bonds
  23. Which element is not shared for both DNA and protein gel electrophoresis?
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  24. How do you visualize proteins in a gel?
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  25. What does the lacI gene encode for?
    the re-pressor protein that will repress expression of LacZ gene
  26. How is the default regulation of lac operon OFF? Hint: in the absence of lactose




    C) Lac1 protein binds the lac operon OPERATOR(lacOP), preventing RNA pol from transcribing lacZ, lacY, and lacA
  27. What is the goal of SDS-Page? (all that apply)



    A) Separate proteins based on size AND Visualize all proteins in a sample
  28. Which of the following is a true statement about the difference between SDS Page and western blots?




    C) Western blots use an antibody to a specific protein. SDS-Page allows visualization of all protins in a sample
  29. When preforming a western blot, which of the following best describes the role of the Non-variable portion of the primary antibody?




    B) It is recognized and bound by the secondary antibody

    It can be thought of as being the antigen for the secondary antibody
  30. Antibody overview: what is "Antigen"?
    Molecule you want to detect (like GST tag)
  31. Antibody overview: what is "variable region"?
    The region that binds to the antigen; each antibody has specificity for a different antigen
  32. Antibody overview: what is "constant regions"?
    the same between antibodies (of each class from the same species)
  33. Antibody overview: what is "epitope"?
    specific site on the antigen that the antibody recognizes
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  35. monoclonal antibodies are made with ________ ?
    hybridoma
  36. Polyclonal Antibodies (pros and cons)
    • Pros:
    • - less expensive to produce
    • - reagent often produces greater sensitivity/signal strength [binds target at multiple sites]

    • Cons:
    • - limited amount produced
    • - Variable animal to animal
    • - High background from non-specific binding
  37. Monoclonal Antibodies (pros and cons)
    • Pros:
    • - The antibody producing cells (hybridoma) can be cultured
    • - Allows large-scale production of a specific AB
    • - Reagent is highly specific [low background]

    • Cons:
    • - signal strength is limited by one antibody/protein
    • - not cheap to produce
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  39. How are western blots different than SDS-PAGE?
    Western blots use an antibody to a specific protein. SDS-Pages allows visualization of all proteins in a sample
Author
saucyocelot
ID
360679
Card Set
Biotech week 7,8
Description
Concepts and vocab from lectures 7-8
Updated

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