Lecture 10

Question 1

What is transformation?

Answer 1

Uptake of naked DNA into a competent recipient cell

Question 2

In Griffith’s transformation experiment what did the smooth strain cause?

Answer 2

Damage

Question 3

In Griffith’s transformation experiment what did the rough strain cause?

Answer 3

Didn’t cause any damage

Question 4

In Griffith’s transformation experiment what happened when heat was applied to the smooth strain?

Answer 4

The smooth strain would no longer cause damage

Question 5

Heated smooth + rough = ? Which strain was found in the mouse

Answer 5

Damage

Only the smooth strain was found in the mouse, rough had been converted by Griffith’s theory of the ‘transforming principles’

Question 6

Transformation occurs naturally in how many bacteria species?

Answer 6

80 species of bacteria

Question 7

What is transformation dependent on?

Answer 7

The specialised cell state (competence)

Question 8

Describe the early stages of the Pnuemoccal transformation process

Answer 8

1. Environmental signals trigger onset of competence
2. Once competent, foreign dsDNA is captured by the pillus
3. An endonuclease degrades one DNA strand
4. The ssDNA is transferred across the membrane into the cytoplasm and has two fates

Question 9

What are the two fates the ssDNA may encounter in the early stages of the Pnuemoccal transformation process?

Answer 9

1. Bound by a single strand binding protein which protects against nucleases, holds for later use as nucleotides
2. DNA protein loads recombination protein onto the DNA

Question 10

Describe the late stages of the Pnuemoccal transformation process

Answer 10

5. ssDNA can be integrated via homologous recombination, forming a transformation heterodupluex
6. After integration, ssDNA is covalently joined to flanking DNA strands
7. Replication then cell division produces 1x wild type and 1x transformed cell that has acquired the transformed DNA sequence

Question 11

What is quorum sensing?

Answer 11

Ability of bacterium to sense its own density

Question 12

What can quorum sensing be induced by?

Answer 12

Stress e.g. antibiotics

Question 13

What happens in quorum sensing?

Answer 13

Small peptide is secreted and allows sensing of population density to know when more potential donor cells are available

Question 14

What does competence depend on?

Answer 14

The extracellular concentration of the secreted small peptide

Question 15

What does induction of competence trigger?

Answer 15

Lysis and DNA release from a fraction of the cells in the population (5-20%)
FRACTICIDE = some cells die and aid genetic recombination

Question 16

Where is the transformation apparatus in the cell for Strep?

Answer 16

At mid-cell

Question 17

Is E.Coli naturally transformable?

Answer 17

No

Question 18

How can E.Coli become competent?

Answer 18

Treatment with CaCl in the cold

Question 19

What are other methods to introduce naked DNA?

Answer 19

1. Electroporation

2. Biolistic transformation

Question 20

What is electroporation?

Answer 20

Creating transient pores in the membrane using an electrical pulse

Question 21

What is Biolistic transformation?

Answer 21

A gene gun