Gene Cloning

• What are the 3 general steps to gene cloning???

  Form recombinant DNA

  Transformation

  Selection
• What is the first step to forming the recombinant?

  Cut the plasmid and gene of interest with a restriction enzyme
• What is the second step to forming the recombinante?

  Hybridization of the complementary sticky end of the gene of interest and plasmid
• What is the third step of forming the recombinant?

  Ligating the gene of interest and plasmid together
• What enzyme ligates the gene of interest and plasmid together?

  DNA ligase
• What is a recombinant?

  When 2 genes from different sources are combined into 1 molecule
• Where are you putting the recombinant in transformation?

  Into a bacteria cell
• What happens to the recombinant as the bacteria multiplies?

  The gene of interest replicates each time the bacteria multiplies
• How can we alter the bacteria's genotype?

  By the cell absorbing foreign DNA
• What is the foreign DNA often known as?

  Naked DNA
• What are the characteristics of naked DNA

  DNA not inside a cell

  Meaning that the donor cell can be dead or nonexistent
• What is something that Griffith's experiment said about 2 bacteria strains when mixed together?

  When 2 different strains of bacteria are mixed together, eventually both characteristics of the strains show
• What are the 2 methods of transformation?

  Natural and artificial
• How does a bacteria naturally transform?

  They have proteins on the cell surface that recognise and transport DNA from similarly related species
• What are the 2 methods of artificial transformation?

  Chemical

  Electroporation
• Explain how the chemical method of artificial transformation works

  Cold CaCl2 treatment and that it followed by heat shocking
• Explain how electroporation works in artificial transformation

  Cell is shocked with electric current which makes holes in the bacteria's membrane
• Define transformation

  Introducing the recombinant to the bacteria cell
• How does CaCl2 affect the permeability of the cell membrane?

  It makes the membrane more permeable
• Explain DNA amplification in vivo

  Once the bacteria cells are transformed, they are transferred to a liquid medium to grow

  • The total number of bacteria cells = increase total amount of DNA
• What is the final step of gene cloning?

  Selection
• Explain how selection works

  Selection identifies colonies of bacteria that have the recombinant DNA
• What is plating?

  Taking a sample of bacteria and allowing it to grow on a plate
• What does the plate contain?

  Agar
• What is agar made of?

  Antibiotics

  x-gal
• What are 2 components of the cloning vector that we haven't addressed yet?

  An ampR gene

  Lac Z gene
• Why is the ampR gene useful?


  Allows the cell to be resistant against ampicillin (an antibiotic)
• What is the first selection mechanism?

  Antibiotic Resistance
• Explain step by step how antibiotic resistance works

  • Bacteria is placed in a petri plate containing a specific antibiotic (ampicillin)
  • cloning vector gives antibiotic resistance to the bacteria because it contains the ampR gene
• How do we know that the selection for transformation was successful?

  Bacteria that successfully transformed the vector will live and grow on the plate

  Because living bacteria MUST contain a cloning vector
• What is the second method of selection mechanism?

  B-Gal screening
• Explain step by step how B-Gal screening works


  Bacteria is grown on petri plates containing X-Gal

  If the cloning vector has a LacZ gene then it will result in a blue precipitate

  If the cloning vector has the gene of interest then it will make a white precipitate
• What does Lac Z gene code for

  B-Gal enzyme
• True or false: vectors with a gene of interest will have a non functioning Lac z gene

  True
• Why do vectors with the gene of interest have a non functioning Lac Z gene?

  B/C the lac Z gene is removed to add the gene of interest into the vector
• How do you know there was a successful ligation

  Bacteria with a gene of interest and vector will have a non functioning Lac z gene therefore they won't code for the B-Gal enzyme

  Meaning it can't process X-Gal

  This forms a white precipitate and that's how you can tell there was a successful ligation
• How do you know there was an unsuccessful ligation?


  Bacteria with the Lac Z gene codes for the B-Gal enzyme

  B-Gal enzyme processes X-Gal on the petri plate

  Resulting in a blue precipitate

  Meaning there was an unsuccessful ligation
• What does the bacterial genome contain?

  Chromosomal DNA

  Many plasmids
• What are plasmids?

  Small, circular DNA molecules found in bacteria that are self replicating
• Are plasmids the same as chromosomes or different?

  They are separate from bacterial chromosomes