In Vivo Gene Cloning - the use of vectors 2

Question 1

What is a vector in genetic engineering?

Answer 1

A carrying unit that transfers a DNA fragment into a host cell

Question 2

What is the most commonly used vector in gene cloning?

Answer 2

A plasmid

Question 2

Why are plasmids commonly used as vectors?

Answer 2

They naturally contain genes for antibiotic resistance and can replicate independently within bacteria

Question 3

How is a plasmid prepared for insertion of the DNA fragment?

Answer 3

A restriction endonuclease isn used to cut the plasmid at one of its antibiotic resistance genes, breaking the loop

Question 4

Why must the same restriction endonuclease be used ot cut both the DNA fragment and the plasmid?

Answer 4

To ensure the sticky ends of the plasmid and DNA fragment are complementary and can join together

Question 5

What enzyme is used to permanently join the DNA fragment to the plasmid?

Answer 5

DNA ligase

Question 5

What happens when the DNA fragments are mixed with the opened up plasmids?

Answer 5

Some DNA fragments become incorporated into the plasmids

Question 6

What is a plasmid called once it contains the inserted DNA fragment?

Answer 6

A recombinant plasmid

Question 7

What is recombinant DNA?

Answer 7

DNA that has been artificially combined from different sources

Question 8

What is the process of introducing recombinant plasmids into bacterial cells called?

Answer 8

Transformation

Question 9

What conditions are required for transformation to occur?

Answer 9

The bacterial cells and plasmids have to be mixed into a medium containing calcium ions, with temperature changes to make the bacterial membrane permeable

Question 10

How do calcium ions and temperature change help in transformation?

Answer 10

They make the bacterial cell membrane permeable, allowing plasmids to pass into the cytoplasm

Question 11

Why do not all bacterial cells take up recombinant plasmids? (3 reasons)

Answer 11

1. Only small percentage of bacterial cells take up plasmids
2. Some plasmids close without incorporating DNA fragment
3. Some DNA fragments join together forming their own plasmid

Question 12

How can scientists identify bacterial cells that have taken up a plasmid?

Answer 12

By using antibiotic resistance genes as markers to identify transformed bacteria

Question 12

How can antibiotic resistance be used to identify transformed bacteria?

Answer 12

Bacteria that has taken up plasmids with an antibiotic resistance gene can survive on a medium containing that antibiotic, while non - transformed bacteria die

Question 13

What is first step in identifying bacteria that has taken up plasmids?

Answer 13

Grow all bacterial cells on a medium containing ampicillin

Question 14

What happens to bacteria that have successfully taken up the plasmid the antibiotic resistant gene?

Answer 14

They survive because they can break down the antibiotic ampicillin

Question 15

What happens to bacteria that have not taken up the plasmid?

Answer 15

They lack ampicillin resistance and die

Question 16

What is the next step after selecting for ampicillin resistance?

Answer 16

Use marker genes to identify and eliminate bacteria that do not contain the new gene

Question 16

Why is this method not completely reliable for identifying bacteria with the desired gene?

Answer 16

Some bacteria take up plasmids that have close without incorporating the new gene, so they survive but do not contain the desired gene