Plasmids

Question 1

What are plasmids?

Answer 1

Extrachromosomal, circular dsDNA molecules generally found in archaea, but can also be found in archaea and eukaryotic organisms.

Question 2

Outline some basic characteristics of plasmids.

Answer 2

• Range in sizes from kb- mb.
• Can be circular/ linear, but mostly in circular conformation.
• Inheritance of the plasmids is controlled by partitioning systems.
• Plasmids are placed into incompatibility groups based on their replication machinery. Plasmids in the same Inc group cannot co-inhabit a host.
• Used as vehicles of communication of genetic information between bacteria.
• Represent a metabolic burden to the host.

Question 3

Describe the different conformations that can be adopted by plasmids.

Answer 3

• Supercoiled… compact.
• Circular… less compact, nicked strand.
• Linear… nicked with restriction enzymes, cleaving phosphodiester bonds of both strands at a particular sequence…
• Can identify these conformations on an electrophoresis gel.

Question 4

What is the purpose of plasmid partitioning?

Answer 4

To ensure stable inheritance of plasmids during cell division.

Question 5

What does a higher copy number mean for a plasmid?

Answer 5

This means that there is more chance that the daughter cells will receive plasmids after replication. Generally rely on passive diffusion to distribute the plasmids. However, these plasmids pose a metabolic burden on the host.

Question 6

Is copy number a fixed characteristic for plasmids?

Answer 6

Yes, under constant conditions.

Question 7

Where does replication begin in plasmids?

Answer 7

At the origin of vegetative replication (OriV).

Question 8

What are the characteristics of small high-copy plasmids?

Answer 8

Random plasmid partitioning, low chance that any of the daughter cells will not have a plasmid.
Relies on passive diffusion for plasmid maintenance.
Problem in the large increase in metabolic burden on the host system.

Question 9

What are the characteristics of large low-copy plasmids?

Answer 9

Direct plasmid partitioning, linked to chromosome replication. 
Par systems (partition systems), carried on plasmid DNA for their retention. 
Mechanisms to ensure even distribution of the plasmids.

Question 10

What is plasmid incompatibility?

Answer 10

The failure of two plasmids of the same incompatibility group to co-exist in a host, and be stably inherited together in the absence of external selection.

Question 11

How is incompatibility classified?

Answer 11

Based on plasmids that have the same/ similar replication machinery.
I.e. their replication strategy.

Question 12

What governs the number of plasmids in a cell?

Answer 12

Elements encoded within the origin of replication (ori).

Question 13

Why are plasmids incompatible with each other in the same inc group?

Answer 13

Due to competition between plasmids for replication factors.
This competition leads to growth advantages (i.e faster replication due to smaller size, less toxicity etc).

Question 14

What is classification into an Inc group always based on?

Answer 14

The amino acid sequence of the replication initiation protein (Rep).

Question 15

What is plasmid host range?

Answer 15

The range of hosts a plasmid can inhabit.
Can be narrow and replicate in related species or broad host range replicating in a variety of different hosts (NHR/ BHR).

Question 16

What Inc group is RP4/ RK2 in?

Answer 16

IncP

Question 17

What hosts can RP4/RK2 replicate in?

Answer 17

Gram-positive and Gram-negative bacteria.

Question 18

What property of RP4/ RK2 plasmids is thought to give a broad host range?

Answer 18

Multiple origins of replication.

Question 19

What other factor is thought to be important for determining host range in a plasmid?

Answer 19

GC content of the plasmid.

Question 20

What are some characteristics a plasmid can encode?

Answer 20

Antibiotic resistance, virulence determinants (plant and animal), bacteriocin production, metalloid resistance(Hg and As), symbiotic determinants (ability to fix nitrogen), biodegrative capabilities.

Question 21

Give an example of a plasmid which can convey antibiotic resistance, and give some of its characteristics.

Answer 21

RK2… broad host range plasmid, IncP group, 60Kbp long.
Genes for replication, maintenance, conjugation and antibiotic resistance (kanamycin, ampicillin and tetracycline).
kil (lethal to cell) and kor (complementary transcriptional repressor) genes, play a role in the broad-host range of RK2.

Question 22

What are virulence plasmids?

Answer 22

These are plasmids that determine some level of virulence in the bacterium, turning it into a pathogen.

Question 23

Give two examples of a plasmid that determines virulence in animals.

Answer 23

pWR501 in Shigella flexneri. 221851bp, causes shigellosis. Conjugative transfer abilities… and encodes various mobile elements too.

In Bacillus anthracis (causing anthrax)… virulence is dependent on two plasmids… two circular extrachromosomal dsDNA plasmids… pXO1 and pXO2, both required for full virulence.
pXO1- encodes the anthrax toxin (AtxA)… allowing the organisms to be virulent.
pXO2- capsule production, to protect the bacterium from the immune system. Also used as a live attenuated vaccine for some strains of Bacillus anthracis.

Question 24

Give an example of a plasmid that determines virulence in plants.

Answer 24

Ti plasmid… tumour inducing plasmid. Allows agrobacterium to cause disease of plants.
Can transfer between agrobacterium species by conjugation. Can also transfer the T-DNA region into the plant genome.
Then T-region integrates into the plant genome and those genes encoded by the T-DNA help to make the plant cell proliferate (auxins, cytokines etc). Produces tumours.
Also get production of Opine, as well as Opine catabolism through the plasmid. Allows the bacterium to use opine as a food source.

Question 25

What is the significance of bacteriocin production.

Answer 25

Bacteriocin is an antimicrobial agent, produced by bacteria to kill other bacteria not harbouring the plasmid that confers immunity/ resistance to the compound.

Question 26

Give two examples of plasmids that confer bacteriocin production.

Answer 26

pMRC01- 60.3 Kbp, conjugative transfer functions, can move by conjugation. Replication region, so can replicate. Phage resistance (CRISPR-Cas).

Coli1. 6.6 Kbp, original plasmid for pUC19.

Question 27

Give an example of a plasmid with biodegradative capabilities.

Answer 27

pA81- 98192bp, Aromatic hydrocarbon degradation in Achromobacter xylosoxidans.
Encodes benzene and toluene degradation.
Can transfer by conjugation, due to tra genes involved in transfer/ conjugative transfer.
Ability to degrade benzene and toluene is located in the transposon, in the transposable element (Tn).

Question 28

Why are plasmids that provide fitness benefits stably maintained in the host?

Answer 28

I.e. conferring antibiotic resistance… because provide fitness benefits to the bacterium.

Question 29

What is the problem with conjugative plasmids?

Answer 29

Require energy in ATP to move from one cell to the other, so slow down the cells’ generation time if conjugative plasmids are present… i.e. lower replication rate.

Question 30

What factors do you need to consider when making a vector?

Answer 30

Origin of replication.
- Plasmid must replicate in the host you are working on.

Selective markers present.
-I.e. antibiotic resistance, blue white colour selection.

Promoters present.

• Need a promoter in order to express the gene of interest, unless you are cloning in a promoter.
• Can be constitutive (on all the time), or inducible (need to add something for promoter to express gene).

Method of introducing the plasmid into the host.
-Dependent on size of plasmid.

Question 31

By what methods can the plasmid be introduced into the host?

Answer 31

Method chosen depends on the size of the plasmid.

• > Transformation (i.e. some organisms are naturally transformable and naturally take up the DNA, but if not need to make them take it up i.e. treatment with divalent cations, electroporation etc. makes micropores in membrane to allow the plasmids to enter.
• > Conjugation, if transformation doesn’t work. This is the way conjugation happens naturally, with direct contact between the cells via a pilus.

Question 32

How does blue/ white screening work?

Answer 32

Add the bacteria onto an agar plate with antibiotic on it. So cells that have not taken up a plasmid die, those that have taken up a plasmid survive and appear as blue colonies, a s they still synthesis beta-galactosidase from the plasmid, which interacts with X-gal, producing a blue colour. Those that have taken up recombinant plasmids appear white, as the beta-galactosidase gene is disrupted, so no functional beta-galactosidase is produced, so no interaction with X-gal, and no colour produced.

Question 33

What is a suicide vector?

Answer 33

A plasmid which is replication incompetent i.e. cannot reproduce in a host. It can be used to knock out/ mutate a gene of interest.
Clone the homologous gene to the one you are interested in, into the plasmid MCS… and into the host, where the plasmid will incorporate itself into the chromosome where the gene is found.

Question 34

Give an example of a suicide vector.

Answer 34

pUC19 can be used as a suicide vector for vibrio cholerae.

Question 35

What is a shuttle vector?

Answer 35

A plasmid with two origins of replication, meaning it can replicate in two or more different hosts.

Question 36

What does a shuttle vector require?

Answer 36

DIfferent origins of replication for the different organisms, and different selectable markers, so that it can be selected in the different organisms.

Question 37

Do shuttle vectors occur in nature?

Answer 37

No, they are man made.

Question 38

Give an example of a shuttle vector.

Answer 38

pMSR10… E.coli and Bacillus coagulans species shuttle vector… can shuttle between eukaryotic and bacterial cell…
Has ColiE1 and Rep1 origins of replication.
Also has two antibiotic resistance genes… ampicillin and chloramphenicol.