Lecture 18

Question 1

What is gene cloning?

Answer 1

Using bacterial cells to make identical copies of a specific gene

Question 2

Where did the lux operon (insert DNA) come from?

Answer 2

pineapplefish to bioluminescent bacteria to lux operon

Question 3

What is the overall process of transforming a bacterial cell?

Answer 3

insert DNA into vector DNA

ligate together to make a recombinant DNA

transform (put recombinant DNA in a bacterial cell)

Question 4

Is the lux operon an insert or vector DNA?

Answer 4

insert DNA

Question 5

Is E. coli plasmids insert or vector DNA?

Answer 5

vector DNA

may confer antibiotic resistance

Question 6

What are the two common lab plasmids?

Answer 6

pUC18 (2686 bp) and pBluescript (2961 bp)

Question 7

What are the two enzymes needed?

Answer 7

restriction enzymes: cut DNA at specific recognition sites to create compatible ends (makes sticky ends)

DNA ligase: joins DNA fragments by forming phosphodiester bonds between them

Question 8

What type of enzyme is EcoRI?

Answer 8

RE

Question 9

What type of enzyme is DNA ligase?

Answer 9

T4 DNA ligase

Question 10

Bacterial cells could be natural strains and lab strains of E. coli. What are the two natural strains of E. coli?

Answer 10

O157:H7 - pathogenic

RY13 - harmless but not suitable

Question 11

Bacterial cells could be natural strains and lab strains of E. coli. What is the lab strains of E. coli? What is it made by?

Answer 11

DH5𝛂 made by Invitrogen

Question 12

What are characteristics of DH5𝛂 that help in the cloning process?

Answer 12

mutations remove RE (RE usually acts as a defense system that cuts up foreign DNA that enters the cell, but in cloning, we WANT foreign DNA (recombinant plasmid) to survive and replicate): cell won’t destroy the plasmid your introduce

mutations in thiamine synthesis: auxotrophic (requires thiamin supplementation): bacteria can’t survive well outside a nutrient-rich lab environment

made competent (temporary holes in cell wall) to allow DNA uptake during transformation): allows plasmid DNA to enter the cell (ex. treat with CaCl2 or heat shock/electoporation)

Question 13

What is the plasmid procedure?

Answer 13

1) digest insert DNA (i.e. cut 4 kb YFG with EcoRI)

2) digest vector DNA (cut with same enzyme to produce compatible ends)

3) mix and ligate DNA (add T4 DNA ligase to form recombinant DNA)

4) transform competent E coli DH5a cells (only small fraction transform. Each transformed cell replicates plasmids, amplifying recombinant DNA)

5) plate cells on ampicillin containing agar (only cells with plasmid (carrying ampicillin resist gene) survive

6) isolate and test colonies (verify recombinant plasmid using EcoRI digest and agarose gel electrophoresis)

Question 14

Where is ampR located?

Answer 14

plasmid