U3A1: 4E recombinant plasmids + bacterial transformation

Question 1

plasmid

Answer 1

small, circular loop of DNA seperate from the chromosome, typically found in bacteria.

Question 2

recombinant plasmid

Answer 2

plasmid that has been edited to incorporate a target gene.

Question 3

4 things needed to create a recombinant plasmid

Answer 3

• gene of interest
• plasmid vector
• restriction endonuclease
• DNA ligase

Question 4

4 most important sites for a plasmid vector

Answer 4

• restriction endonuclease site
• antibiotic resistance genes
• origin of replication (ORI) (signals start site for DNA replication)
• reporter gene (identifiable phenotype)

Question 5

bacterial transformation

Answer 5

process in which bacteria take up foreign DNA from the environment.

Question 6

vector

Answer 6

the organism that is introduced to foreign DNA, typically plasmids.

Question 7

role of gene of interest in creating a recombinant plasmid

Answer 7

gene of interest is the sequence of DNA that encodes the protein we wish to produce.

Question 8

role of plasmid vector in creating a recombinant plasmid

Answer 8

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

Question 9

role of restriction endonucleases in creating a recombinant plasmid

Answer 9

cuts the plasmid and gene of interest to create identical sticky ends on either end of the DNA sequence. the overhanging ends of the gene of interest will be complementary to the plasmid vector.

Question 10

role of DNA ligase in creating a recombinant plasmid

Answer 10

joins the gene of interest to the plasmid vector by forming phosphodiester bonds in the sugar-phosphate backbone. creates the recombinant plasmid.

Question 11

2 methods that promote recombinant plasmid uptake

Answer 11

• heat shock
• electroporation

Question 12

process of heat shock

Answer 12

• bacteria and plasmids are placed in a (calcium ion) solution on ice.
• solution heated to 37-42 degrees for 25-45 seconds before being returned to ice.
• sudden change in temperature makes the plasma membrane more permeable and allows plasmid vectors to cross the phospholipid bilayer.

Question 13

process of electroporation

Answer 13

• bacteria and plasmids get electrically shocked.
• the electric shock stops.
• electrical current causes plasma membrane to become more permeable and allows plasmid vectors to cross the phospholipid bilayer.

Question 14

insulin

Answer 14

hormone responsible for regulating blood glucose levels.

Question 15

making recombinant human insulin: process of creating the recombinant plasmid

Answer 15

1. creating the recombinant plasmid.
   - plasmid vectors prepared with ampR for antibiotic resistance and lacZ which acts as a reporter gene. (lacZ contains B-galactosidase enzyme which converts x-gal from colourless to blue)
   - two plasmid vectors used: one for insulin A and one for insulin B. using a restriction endonuclease, both insulin samples are cut to form sticky ends. DNA ligase is used to re-establish the sugar-phosphate backbone and create 2 recombinant plasmids.

Question 16

making recombinant human insulin: process of creating transformed bacteria

Answer 16

2. creating transformed bacteria.
   - plasmids are added to e. coli solution and either heat shock or electroporation can be used to increase uptake of plasmids.
   - bacteria is spread and incubated on agar plates containing X-gal and antibiotic ampicillin. (colourless colonies can be determined to be transformed bacteria with the recombinant plasmid as their lacZ gene is dysfunctional since the gene of interest is located inside it.)
   - recombinant plasmids will produce insulin subunit with a B-galactosidase tail formed from half of the transcribed+translated lacZ gene

Question 17

making recombinant human insulin: process of protein production and extraction

Answer 17

• transformed bacteria that contain the recombinant plasmid are placed into conditions to exponentially reproduce before their membranes are broken down and the insulin produced is isolated and purified.
• two insulin chains have their B-galactosidase tails removed and are mixed together to form functional human insulin.