Lecture Five - Cloning Vecotrs

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Lecture Five - Cloning Vecotrs
2013-09-24 12:27:45
Experimental Biology Cloning Vectors Lecture Five Plasmid pUC Blue White Screening

Experimental Biology
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  1. What is a Vector?
    A molecule used as a Vehicle to carry foreign DNA into a host cell
  2. What is the simples vector?
    A plasmid
  3. What are some features of Plasmids?
    • Extra Chromosomal
    • Not found in Eukarya
    • Mostly Circular
    • Encode or Environment-Specific characteristics
  4. Plasmids are not often seen in Eukaryotes. What is an organism that is an exception to this rule?
  5. Episomes are found in Eukaryotes. What is an Episome?
    A Viral Genome found outside of the chromosomes before transcription or replication.
  6. Are Episomes Plasmids?
    No, they are different
  7. What does the "R" in R-Plasmids Stand for?
  8. What are the two naming conventions of plasmids?
    • By funcion -> Colicin ->ColEI
    • By Discoverer -> pMBI
  9. What is the Replicon?
    The Origin of Replication of a Plasmid (OriC) and all accociated cis-acting elements
  10. What is the Host Range of a plasmid?
    The range of accpeted hosts
  11. What are some examples of plasmids with a Narrow host range?
    • ColE1
    • pMB1
  12. What are some Hosts of ColE1 and pMB1?
    E.coli, Salmonella and other closely related Enterpbacteriaceae
  13. What is an ecample of a Broad Host Range plasmid?
    R-Plasmids, specifically RK2
  14. Why can Broad Host Range Plasmids function in such diverse hosts?
    • THey encode all proteins required or initiation of replication themselves
    • Promoters Recognized in a Variety of Hosts
  15. What is the Copy Number of a plasmid?
    The number of plasmids that are maintained in one cell
  16. What are the two types of Plasmid Copy No.?
    • Stringent
    • Relaxed
  17. How can a plasmid control it's copy number?
    By having a degree of control of initiation of DNA synthesis
  18. What is the correlation between plasmid size and copy number?
    Larger Plasmid -> Smaller Copy Number
  19. What are some examples of Stringent plasmids?
    • ColE1
    • pMBI
  20. Generally how many copies of a stringent plasmid might you find in a cell?
  21. What is an example of a Relaxed Plasmid?
  22. What is the general relationship between plasmid size and copy number?
    Larger plasmids have lower copy numbers
  23. What happens to two plasmids (with the same replicon) in a cell?
    They will compete with eachother
  24. When do like-plasmids compete?
    • During DNA Replication
    • Partitioning into Daughter Cells (Telophase)
  25. How many classes of Incompatible plasmids does E.coli have?
  26. Why are plasmids used in cloning?
    • Their small size makes them easy to purify
    • Small Plasmids are easier to integrate
  27. What are the two types of Plasmid?
    A) Large, Small
    B) Fast, Slow
    C) Alpha, Gamma
    D) Artificial, Natural
    D) Artificial, Natural
    (this multiple choice question has been scrambled)
  28. Why are Natural Plasmids not used in cloning?
    • Low Copy Number
    • Potential to produce Toxins
    • Difficulty in Selection
    • Different Cut Sites
  29. Why might a Natural Plasmid be harder to select for?
    They may not have distinct features (such as Antibiotic Resistance) that allow confirmation of Transformation
  30. What are some advantages of Artificial Plasmids?
    • Controllable Copy Number
    • Small
    • Selectable Markers
  31. What is an example of a selectable marker?
    • Ampicillin Resistance
    • Tetracyclin Resistance
    • Lactose Metabolism
  32. Is a high or low copy number favoured for Artificial Plasmids?
    High as it gives more DNA
  33. What is the maximum size for a plasmid to be Transformed?
  34. Who discovered pBR322?
    Bollivar and Rodriguez
  35. What are the two common cloning vecors?
    • pBR322
    • pUC
  36. What are some features of pBR22?
    • Narrow Host Range
    • 16 Copies Per Cell (Copy Number)
    • Ampicillin and Tetracycline Resistance
  37. What is the size of a pBR322 plasmid?
  38. Plasmids are costly, therefore Bacteria only adopt them if there is an incentive. How is this overcome in cloning?
    Plasmids will encode resistence to an Antibiotic that the Bacteria will be grown on
  39. What is Insertional Activation/Inactivation?
    A way of screening a bacterial culture to determine if any have transformed.
  40. In what region of the pBR322 plasmid is the DNA inserted? What Restriction site is this?
    • Into the Tetracycline Resistance Gene
    • The Restriction Site is BamHI
  41. What is Replica Plating?
    • Plating whereby two plates are created simultaneously from a master plate
    • Both plates will have different conditions for bacterial growth, the presence/absence of bacterial colonies can give information,