Plasmid DNA

Question 1

What are bacterial plasmids?

Answer 1

• Loops of DNA found in bacteria

- Contain ‘special genes’ (antibiotic resistance)

Question 2

Where do plasmids exist?

Answer 2

Outside chromosome but within bacterial cytoplasm

Question 3

How do plasmids replicate?

Answer 3

Bidirectionally until they meet (replicating enzyme complexes disengage)

Question 4

What is stringent replication control?

Answer 4

Both chromosome and host (natural) plasmid are replicated simultaneously

Question 5

What does chloramphenicol?

Answer 5

-Can amplify small plasmid copies (relaxed replication control)

Question 6

What does the origin of replication allow for?

Answer 6

The engineered plasmids to be copied independently of the bacterial host chromosome

Question 7

What does a low copy number of host plasmids indicate?

Answer 7

Physiological burden to maintain the large plasmids in the host bacterium

Question 8

What are the defined parameters for engineered cloning vectors?

Answer 8

• Antibiotic resistance marker genes

- Unique restriction enzyme cloning sites

Question 9

What 3 resistant genes does the cloning vector pBR325 have?

Answer 9

• Chloramphenicol
• Ampicillin
• Tetracycline

Question 10

What does the intertion of a DNA fragment at a cloning site do?

Answer 10

Create an additional/ duplicate site at the insertion point.

Question 11

What does a smaller universal cloning vector do?

Answer 11

Generate a higher copy number per bacterial cell (higher yield of plasmid DNA)

Question 12

What occurs in the process of transformation?

Answer 12

-ice-cold calcium chloride treatment
-addition of plasmids
-transient passage of a plasmid into a competent cell
-Only transformed cells grow in antibiotic presence
(only occurs in 3% of cells)

Question 13

Why are the cells kept ice-cold in transformation?

Answer 13

• Allows Ca2+ ions and DNA to remain bound to the cell envelope
• Plasmid is than adsorbed to the calcium ions by electrostatic interaction

Question 14

What does the short high temperature cause in transformation?

Answer 14

Cells are temporarily permeable to the plasmid DNA (high temp dislodges the Ca-DNA interaction)
-Plasmid can cross the cell envelope

Question 15

How do you amplify plasmid DNA?

Answer 15

• Adding chloramphenicol / spectinomycin (protein synthesis inhibitors) to early-log phase of culture
• # of cells doesn’t increase
• DNA keeps copying

Question 16

What are the steps in plasmid DNA isolation?

Answer 16

1. Alkaline lysis
2. Neutralize w/ potassium acetate
3. Centrifuge to remove denatured chr DNA, protein, lipid, SDS, KOAc
4. Digest RNA with RNase
5. purify plasmid DNA w/ precipitation

Question 17

When does alkaline lysis work well?

Answer 17

• Not for large plasmids

- Method of choice for isolation of plasmid DNA from bacterial cultures