Wright Lecture 3: Recombinant DNA Technology

Question 1

DNA ligase

Answer 1

Enzyme that catalyzes the formation of phosphodiester bonds between nucleotides
Covalently joins together two complementary ends of DNA molecules

Question 2

Cloning vectors

Answer 2

Fooling bacteria host into replicating foreign DNA fragment
Commonly E. Coli
Can put human gene in expression vector and make bacteria make human protein

Question 3

Plasmids

Answer 3

Used as cloning vector
Replicate autonomously in bacteria cells (independent of host chromosome)
Presence of plasmid in bacterium detected by bacterium’s resistance to an antibiotic
20-40 copies per cell: only contains a few genes
Occur naturally in various micro-organisms
Some constructed for recombination technology
Posses unique restriction endonuclease recognition sequences employed as cloning (insertion) sites for foreign DNA
Cloning vectors are very small

Question 4

Naming plasmids

Answer 4

p: plasmid
Initials: developers
Number: number of tries

Question 5

Antibiotics

Answer 5

Drugs that specifically inhibit a metabolic process to impede growth or kill bacteria cells but not human cells

Question 6

Ampicillin

Answer 6

Derivative of penicillin
Inhibits production of cell wall around bacteria
Bacteria cells break open and lyse when treated with this antibiotic

Question 7

Tetracycline

Answer 7

Inhibits protein synthesis of bacterial cells by interrupting the function of the bacterial ribosome

Question 8

Rifampicin

Answer 8

Inhibits bacterial RNA polymerase: transcription of bacterial genes is prevented

Question 9

pBR322

Answer 9

Can be cleaved as Pst1 site to add foreign DNA with complementary overhand
Will no longer be resistant to ampicillin as DNA was inserted within ampicillin gene
Without recombination, resistant to both tetracycline and ampicillin

Question 10

Cells with no plasmid

Answer 10

Sensitive to tetracycline and ampicillin

Question 11

Recombinant plasmid

Answer 11

Resistant to tetracycline and not resistant to ampicillin as foreign DNA was added within ampicillin gene

Question 12

Detecting plasmid recombination

Answer 12

Grow colonies on medium
All cells will grow on one plate of medium containing antibiotic
Replica plate onto medium containing other antibiotic: colonies containing recombinant plasmid will not grow on on this medium