In Vivo Gene Cloning - the use of vectors 3

Question 1

What is the purpose of marker genes in genetic engineering?

Answer 1

To identify bacterial cells that have successfully taken up a desired gene

Question 2

How do marker genes help distinguish transformed bacteria?

Answer 2

They are separate genes on the plasmid that produce an easily identifiable trait

Question 3

What are three common types of marker genes?

Answer 3

1. Antibiotic - resistance marker genes
2. Fluorescent markers
3. Enzyme markers

Question 3

How does each of the three common types of marker genes (antibiotic - resistance marker genes, fluorescent markers + enzyme markers) identify the bacterial cells that have successfully taken up the desired genes?

Answer 3

1. Antibiotic - resistance genes = Allows bacteria to survive in the presence of an antibiotic
2. Fluorescent markers = Causes bacteria to glow under UV light
3. Enzyme markers = Causes a detectable colour change when exposed to a specific substrate

Question 4

How does replica plating work? (four stages)

Answer 4

1. Bacteria are first grown on a medium with the antibiotic
2. A replica of the bacterial colonies is transferred to a second plate containing the bacteria
3. Colonies that fail to grow on the antibiotic, contain the desired gene
4. These colonies are identified by comparing with the original plate and can be grown separately

Question 5

Why does cutting the antibiotic - resistance gene help identify transformed bacteria?

Answer 5

Bacteria that have incorporated the new gene will no longer be resistant to the antibiotic and will not grow on a antibiotic containing medium

Question 6

What is a disadvantage of using antibiotic resistance as a marker?

Answer 6

If bacteria are directly grown on an antibiotic, those with the desired gene will die, preventing identification

Question 6

How does replica plating solve the problem of identifying bacteria without killing them?

Answer 6

It allows identification by comparing colonies grown on different plates, preserving the original bacteria for further use

Question 7

What is a fluorescent marker gene used for in gene cloning?

Answer 7

To identify bacterial cells that have taken up the desired gene by using a gene that produces a fluorescent protein

Question 8

How does the GFP marker system work?

Answer 8

The gene to be cloned is inserted into the middle of the GFP gene, disrupting its function

Question 8

What fluorescent protein is commonly used as a marker in genetic engineering?

Answer 8

Green fluorescent protein (GFP) from jellyfish

Question 9

How can transformed bacteria be identified using GFP?

Answer 9

Bacteria that has taken up the plasmid with the desired gene do not produce GFP and do not fluoresce

Bacteria that has taken up the plasmid without the desired gene continue to produce GFP and fluoresce

Question 10

Why is the use of fluorescent markers an improvement over antibiotic resistance markers?

Answer 10

It does not require replica plating
Bacterial cells containing the desired gene are not killed
Results can be obtained quickly by viewing under a microscope

Question 11

What is an enzyme marker in gene cloning?

Answer 11

A gene that produces an enzyme, allowing identification of bacterial cells that have taken up the desired gene

Question 12

What enzyme is commonly used as a marker in genetic engineering?

Answer 12

Lactase

Question 13

How does lactase act as a marker enzyme?

Answer 13

Lactase breaks down a colourless substrate, turning it blue

Question 14

How is the lactase marker gene used to identify transformed bacteria?

Answer 14

1. Desired gene is inserted into the lactase gene, disrupting its function
2. Bacteria that has taken up the plasmid with the desired gene cannot produce lactase and does not turn substrate blue
3. Bacteria without the desired gene still produces lactase and turns the substrate blue