4E: Recombination and Transformation

Question 1

Plasmid

Answer 1

a small circular loop of DNA separate from the chromosome, typically found in bacteria

• Plasmids are excellent cloning vectors as they can self replicate, are small and can be taken up by bacteria

Question 2

Recombinant Plasmids

Answer 2

a circular DNA vector that is ligated to incorporate a gene of interest

- A plasmid that has a foreign gene within it

Question 3

Bacterial Transformation

Answer 3

the process by which bacteria take up foreign DNA from their environment.

- Scientists use this process to introduce recombinant plasmids into bacteria

Question 4

Uses of Bacterial Transformations

Answer 4

• to create Insulin to manage diabetes

Question 5

Vector

Answer 5

a means of introducing foreign DNA into an organism

Question 6

Materials needed to created a recombinant plasmid

Answer 6

• Gene of interest
• Plasmid Vector
• restriction endonuclease
• DNA ligase

Question 7

Gene of Interest

Answer 7

a sequence of DNA, encoding the protein we wish to produce

- bacteria are able to use their DNA to synthesise an identical protein because the genetic code is universal

Question 8

Restriction Endonuclease

Answer 8

an enzyme that acts as a pair of molecular scissors and act on a specific sequence of DNA

• Cut both the plasmid and gene of interest to generate sticky ends which are now complementary to each other

Question 9

DNA ligase

Answer 9

an enzyme that joins molecules, specifically DNA and RNA by catalysing the formation of phosphodiester bonds

- Joins the gene of interest to the plasmid vector

- Not every plasmid takes up the gene of interest, most wont and will just ligate back with themselves = non-recombinant plasmids

Question 10

Plasmid Vector

Answer 10

a piece of circular DNA that is modified to be an ideal vector for bacterial transformation experiments.

Question 11

Reporter Genes

Answer 11

a gene with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the genes of interest

- If the reporter gene is identifiable, the gene of interest has not been taken up and the plasmid is therefore non-recombinant

    - however if the reporter gene does not work, then it has been cut to fit in the gene of interest and therefore the plasmid has become recombinant

Question 12

what is Heat Shock & the steps involved

Answer 12

a method that involves rapidly increasing and decreasing the temperature to increase the membrane permeability in order to enhance the likelihood of bacterial transformation.

- First requires the plasmid and bacteria to be placed in a calcium ion solution on ice as the positive calcium ions make the membrane more permeable. 

- Solution is then heated to 37-42 degrees for 25-45 seconds before being returned to ice

- The sudden change makes the plasma membrane even more permeable, allowing the plasmid vector to cross the phospholipid bilayer.

Question 13

Electroporation

Answer 13

a method that involves delivering an electric shock to bacterial membranes to increase their membrane permeability and increase the likelihood of bacterial transformation.

Question 14

Steps in Transforming Bacteria

Answer 14

1. Increase likelihood of uptake of recombinant plasmids through either heat shock or electroporation
2. Distinguish between transformed and untransformed bacteria by placing the cells onto a antibiotic-rich plate and all the cells that continue to grow have taken up a plasmid with the antibiotic resistance gene on it
3. transformed bacteria are cultured to produce the target protein, which is then extracted and purified for use in science and medicine

Question 15

Insulin

Answer 15

a hormone responsible for regulating our blood glucose levels

- Insulin protein has quaternary structure consisting of an alpha and beta polypeptide chain therefore we need two recombinant plasmids and thus two transformed bacteria samples to produce insulin

Question 16

Steps in creating the recombinant plasmid(Insulin)

Answer 16

1. .
   
   • 2 Plasmid Vectors are prepared which already contain the ampr gene for antibiotic resistance and lacZ the reporter gene and has the specific recognition site for the restriction endonuclease
   • The lacZ produces Beta-galactosidase, an enzyme which converts X-gal, a colourless compound to a blue
   2. .
      - Two plasmid vectors are used, one for insulin subunit A and B
   • An endonuclease such as BamHI cuts both the plasmids and the subunit A and B genes to form sticky ends

     - DNA ligase then joins each subunit gene to a separate plasmid at the sugar-phosphate backbone to create two recombinant plasmids.

Question 17

Steps in Creating Transformed Bacteria(Insulin

Answer 17

3. .
   
   • The plasmids are now added to a solution of E.coli bacteria and then either heat shock or electroporation can be used to increase the uptake of plasmids into the bacteria
   4. .
      - The bacteria cultures are spread and incubated onto agar plates containing X-gal and the antibiotic ampicillin
   • The colonies that for which are colourless can then be determined to be transformed bacteria with recombinant plasmids as the lacZ reporter gene is dysfunctional, meaning the gene of interest has been taken up
   5. .
      The recombinant plasmids will produce an insulin subunit with a beta-galactosidase tail formed from the half of the lacZ gene which is transcribed and translated

Question 18

Steps in Protein Production and Extraction (Insulin)

Answer 18

6. .
   
   • Transformed Bacteria that contain the recombinant plasmids are then placed into conditions to exponentially reproduce before their membranes are broken down to isolate the human insulin
   7. .
      The Alpha and Beta chains have their beta-galactosidase tails removed and are mixed together allowing the connecting disulphide bonds to form and create functional human insulin

Question 19

What is within a plasmid Vector

Answer 19

• Restriction endonuclease sites: sticky ends which are complementary to sticky ends on gene of interest
  • Antibiotic resistance genes: genes which confer antibiotic resistance
  • Origin of replication: a sequence in prokaryotes which signals the start site of DNA replication
    • Reporter Gene: a gene with an easily identifiable phenotype that can be used to identify whether a plasmid has taken up the genes of interest