Lab 3

Question 1

What is a plasmid

Answer 1

Molecules of DNA that are autonomously replicated independently of chromosomes

Question 2

Conjugation of plasmids

Answer 2

A pathway for acquisitiong o antibiotic resistance in genes - antibiotic resistance gene code binds to plasmid, allowing it to move from cell to cell

Question 3

What are the four ways plasmids move across bacterial cells

Answer 3

• Natural transformation
• Vesiduction
• Phage transduction
• Conjugation

Question 4

What are two possible reasons (of many) that plasmids are maintained in bacteria (the plasmid paradox)?

Answer 4

• Piggyback adaptation: adaptations to environments can have pleiotropic effect on plasmid carriage
• Compensatory evolution: Plasmid or bacteria may evolve to lower their fitness cost

Question 5

4 important features of a plasmid

Answer 5

• Multiple origins of replication (initiation of DNA rep)
• Partition mechanism
• Stability
• Mobilization factors (for conjugation)

Question 6

Introducing gene of interest into a plasmid

Answer 6

1. Make sure restriction sites are only present once (in right location)
2. Fragment must be digested, gene inserted
3. Use generate overhangs for pairing the insert (sticky ends)
4. DNA ligase will covalently link the fragments to be cloned

Question 7

What happens if you use a single restriction site?

Answer 7

50% of the plasmids will have one of the two possible orientations

Question 8

What are 5 features of a cloning vector?

Answer 8

• High # of origin of rep.
• Antibiotic resistance
• Multicloning site with choice of restriction sites only once in plasmid
• Contain lacZ
• Contain annealing sites

Question 9

Features of an expression vector

Answer 9

• Contain and inducible promoter
• Include a purification tag
• Orientation of the insertion and maintenance of the reading frame are important

Question 10

Golden gate cloning

Answer 10

Special REs are used to cut few base pairs

Question 11

Gibson assembly

Answer 11

Alternative to REs - DNA recombination
Exonuclease generates single strand regions that can anneal to vecotr fragments
Allows many genes to be cloned at once

Question 12

Why use cells?

Answer 12

• Can make many copies of themselves
• Carry machinery to perform complex tasks
• More ecofriendly (has to be)

Question 13

What are 4 required traits of biological parts

Answer 13

1. Standardization
2. Modularity (works autonomously)
3. Orthogonality (function separate from its part)
4. Predictive behaviour