Plasmids

Question 1

Plasmid

Answer 1

• Extrachromosomal DNA molecule
• Circular or linear
• Autonomous replication
• Range in size from kilobases to megabases
• Control their copy number
• Ensure inheritance at each cell division by process called partitioning

Question 2

incompatibility (Inc.)

Answer 2

Plasmids with same replication mechanism can’t co-exist in the same cell – known as incompatibility
• Incompatibility can be due to similarities in
the replication machinery
• Incompatible plasmids are in the same
incompatible (Inc) group

Question 3

• Small high-copy –

* Large low-copy –

Answer 3

• Small high-copy – random plasmid partitioning
• Large low-copy – 1-2 copies per cell – directed
plasmid partitioning - replication linked to chromosome replication

Question 4

• Narrow-host-range –

* Broad-host range –

Answer 4

• Narrow-host-range – can only replicate in
related species
• Broad-host range – can replicate in a variety of
hosts – e.g. RP4 (IncP group plasmid) can
replicate in Gram-positive and Gram-negative
bacteria.
– Can also be considered as promiscuous due to
their ability to transfer by conjugation.

Question 5

Some characteristics that plasmids

encode

Answer 5

• Antibiotic resistance
• Metal/metalloid resistance
• Virulence determinants – animal and plant
pathogenicity
• Bacteriocin production
• Biodegradation

Question 6

To add after watching:
Antibiotic resistance RK2,
Virulence determinants Shigella flexneri

Answer 6

.

Question 7

Bacillus anthracis – virulence dependent on two

plasmids

Answer 7

• pXO1 – anthrax toxin
• pXO2 – capsule production – protection against immune system
• pXO1 loss = loss of virulence
• pXO2 used as live attenuated vaccine for some Bacillus strains

Question 8

Ti Plasmid

Answer 8

Plant pathogenicity

Question 9

Bacteriocin production

pMRC01

Answer 9

• Bacteriocin = antimicrobial agent used to kill bacteria not harbouring plasmid that confers immunity/resistance to the compound

Question 10

Aromatic degradation in Achromobacter xylosoxidans

Answer 10

? Located within transposon.

Question 11

Transposon

Answer 11

• DNA sequence with ability to move = “jumping genes”
– Example Tn3
• IR = inverted repeat e.g.
CTGCAG———————————GACGTC
GACGTC———————————CTGCAG
• bla encodes β-lactamase which confers resistance to ampicillin

Question 12

Plasmid vectors – what do you need?

Answer 12

• Origin of replication (oriV) – must replicate in your host – understanding host range important
• Selection e.g.
– antibiotic resistance – β-lacatamase gene –
confers resistance to Ap
– Blue/white colour selection – insertion of
fragment into β-galactosidase (lacZ) gene results
in gene inactivation = white colonies
• Multiple cloning site (MCS)/polylinker
• Promoter – inducible or constitutive?

Question 13

Suicide vector

Answer 13

• A plasmid which is used with an oriV that is
unable to replicate in host of interest
– E.g. pUC19 can be used as a suicide plasmid for
Vibrio cholerae

Question 14

Shuttle vectors

Answer 14

• A plasmid that contains two origins of replication allowing for replication in two hosts
eg. Yeast shuttle vector (conferred ability module)

Question 15

Bacillus coagulans shuttle vector

Answer 15

pMSR10

Question 16

Other important considerations

Answer 16

• Size of plasmid will determine how to transfer
into your host - transformation vs
conjugation?