Lab 6 - Bacteriophage Titre and DNA Isolation

Question 1

What is a phage titre?

Answer 1

It is a way of quantifying the number of bacteriophages. It is represented in PFU/ul usually.

Question 2

How do we calculate a phage titre?

Answer 2

We perform a plaque assay. We then count the number of plaques formed and determine the PFU/ul.

Question 3

Why do we use top-agar solution?

Answer 3

To keep the bacterial cells stable and limit the spread of the virus. Also, the plaques are all on one layer then and they are easier to see.

Question 4

What are some examples of virulence factors that phages carry?

Answer 4

Cholera, diptheria, and scarlet fever

Question 5

Describe the phage-bacterium relationship.

Answer 5

It can range from the simple predator-prey model to a complex symbiotic relationship. This would help promote survival and evolutionary success of both.

Question 6

What are some examples of “damage” phages can cause?

Answer 6

They can infect bacteria used in the fermentation industry, leading to financial losses.

Question 7

How can phages be used for good?

Answer 7

Use phages and their gene products as potential therapeutic agents.
Use phage to combat the growing menace of antibiotic-resistant infections.

Question 8

Who was the lambda phage discovered by?

Answer 8

Esther Lederberg in 1950

Question 9

What is a lambda phage?

Answer 9

It is bacteriophage that specifically infects E. coli

Question 10

How can lambda phages be used?

Answer 10

Great model organism and tool in molecular biology.

It can be used as a vector for the cloning of recombinant DNA.

Question 11

What is a model organism?

Answer 11

It is a non-human species that is extensively studied to understand biological phenomena, under the assumption that the discoveries can be applied to more organisms.

Question 12

What is horizontal gene transfer?

Answer 12

The transfer of genes between organisms besides normal reproduction.

Question 13

How does the lambda phage cause horizontal gene transfer?

Answer 13

Specialized transduction.
It incorporates its DNA into the host DNA. Then, when it is going to lyse, it takes some of the host’s DNA with it. Now when it infects another bacteria, the bacteria will have some new DNA from another bacteria.

Question 14

How has the phage lambda been manipulated today?

Answer 14

1) foreign DNA can be incorporated into its chromosome in a site specific manner
2) it cannot enter the lysogenic cycle, resulting in the ability to specifically clone, express, and isolate foreign DNA from a host bacterium

Question 15

How many bacteriophages are there in the world?

Answer 15

10^31

Question 16

What is the difference between how we titred the phage and collected it?

Answer 16

In the collection, we know how many viruses there are (it has already been titres). Also, there are extra steps at the end involving chloroform.

Question 17

What is the function of DNase 1 and RNase A?

Answer 17

Deoxyribonuclease 1 - endonuclease (from humans) that cleaves phosphodiester linkages

Ribonuclease A - pancreatic ribonuclease that cleaves single stranded RNA

Question 18

What is used to precipitate DNA first?

Answer 18

Polyethylene glycol (8000) and NaCl

Question 19

What is the TE used for?

Answer 19

It is a buffer that helps protect and stabilize DNA and RNA. It was used when we microfuged tube and were concentrating the DNA.

Question 20

What is SDS used for?

Answer 20

It is a strong anionic detergent that removes -ve ions from proteins, destroying their conformations.

Question 21

What does isopropanol do? How?

Answer 21

It precipitates out the DNA. Because DNA is insoluble in isopropanol (and ethanol).