In-vivo cloning

• What is the purpose of in vivo cloning?

  To amplify isolated DNA fragments
• What are sticky ends in DNA cloning?

  Overhangs created by staggered cuts
• What are plasmids used for in cloning?

  They act as vectors
• Why are plasmids considered vectors?

  They carry DNA fragments into host cells
• What is the role of restriction endonuclease enzymes?

  To cut double-stranded DNA
• What is the promoter region in DNA?

  A sequence where RNA polymerase binds
• What is the function of the terminator region in DNA?

  To signal RNA polymerase to detach
• Why is it important to add a promoter region to a gene?

  It allows RNA polymerase to initiate transcription
• What happens after the DNA fragment is modified?

  It is inserted into a vector
• What is the most common vector used in cloning?

  Bacterial plasmids
• How are plasmids characterized?

  They are circular loops of DNA
• Why do we use the same restriction endonuclease for the plasmid and DNA fragment?

  To create compatible sticky ends
• What is the role of DNA ligase in cloning?

  To glue DNA fragments together
• What does the term "annealing" refer to in DNA cloning?

  Sticking together two pieces of DNA
• What is transformation in the context of cloning?

  Getting plasmids into host cells
• How do we make bacterial cell membranes permeable for plasmids?

  By adding calcium ions and heat shock
• What is the effect of heat shock on bacterial cells?

  It increases membrane fluidity
• What happens after plasmids enter bacterial cells?

  The inserted gene is expressed
• Why is it necessary to identify which bacteria have taken up the recombinant plasmid?

  Not all bacteria incorporate the plasmid
• What are the three key reasons why bacteria may not take up the recombinant plasmid?

  Plasmid doesn't enter, rejoining, self-sticking
• What are the three key methods used to identify transformed cells?

  • Antibiotic resistance genes
  • Fluorescent markers (GFP)
  • Enzyme markers (lactase)
• What antibiotic resistance genes are mentioned for the identification method?

  Tetracycline and ampicillin
• What happens to the tetracycline gene when the DNA fragment is inserted?

  The tetracycline gene is disrupted
• What does it indicate if a colony grows on ampicillin but not on tetracycline?

  It contains the recombinant plasmid
• How do fluorescent markers help in identifying transformed cells?

  Non-glowing colonies contain the recombinant plasmid
• What protein do jellyfish produce that is used as a fluorescent marker?

  Green fluorescent protein (GFP)
• What is the role of the lactase enzyme in the enzyme marker method?

  It turns a colorless substance blue
• What indicates that a colony contains the plasmid of interest in the enzyme marker method?

  It cannot turn the colorless substance blue
• What is the purpose of growing bacteria in fermenters?

  • Control temperature
  • Produce large quantities of clones
  • Ensure all clones contain the gene of interest
• What does "in vivo" mean in the context of cloning?

  Cloning in a living organism
• Why is it important that all bacteria grown in fermenters are clones?

  They all contain the gene of interest