Lecture 2: Introduction to Plasmids

Question 1

What is a plasmid?

Answer 1

A plasmid is a small, circular, double-stranded DNA molecule that is distinct and separate from a cell’s chromosomal DNA.

Question 2

What is special about the genes carried by plasmids?

Answer 2

Plasmids often carry genes that provide bacteria with genetic advantages, such as antibiotic resistance.

Question 3

What is the disadvantage to having plasmids during reproduction?

Answer 3

When a bacterium replicates and divides, all the plasmids contained within the cell are copied such that each daughter cell receives a copy of the plasmids. There is a fitness cost to the presence of plasmids; replicating multiple plasmids has an energy cost to the cell.

Question 4

What are the 4 key sites on a plasmid?

Answer 4

1. Restriction Site
2. Origin of Replication
3. Selectable Marker
4. Multiple Cloning Region

Question 5

What are restriction sites?

Answer 5

Locations on a DNA molecule containing specific (4-8 base pairs in length) sequences of nucleotides, which are recognized by restriction enzymes.

Question 6

What are restriction enzymes?

Answer 6

. Restriction enzymes/ endonucleases cleave DNA into fragments at or near specific recognition sites. Allow for the insertion or removal of genes in the plasmid.

Question 7

What is the ‘Origin of Replication’?

Answer 7

A sequence of DNA at which replication is initiated on a chromosome, plasmid or virus. For small DNAs, including bacterial plasmids and small viruses, a single origin is enough.

Question 8

What is the role of a selectable marker?

Answer 8

Used to enable identification of successful insertion of a gen into a plasmid.

Question 9

What is a ‘Multiple Cloning Region’?

Answer 9

DNA region within a plasmid that contains multiple restriction enzyme cut sites, allowing the plasmid to be cut and cloned at multiple different sites.

Question 10

What are the 3 stages of plasmid replication?

Answer 10

1. Initiation
2. Elongation
3. Termination

Question 11

What happens during the initiation stage of plasmid replication?

Answer 11

Introduction of a specific single strand break (called nicking) in the replication origin by a Rep protein, which is encoded by the plasmid.
Catalysed by a plasmid-encoded initiation protein that recognises plasmid-specific DNA sequences and determines the point from which replication starts.

Question 12

What happens during the elongation stage of plasmid replication?

Answer 12

Addition of amino acids to the newly synthesised strand by DNA polymerase III holoenzyme.

Question 13

What happens during the termination stage of plasmid replication?

Answer 13

Nicking and re-joining of the displaced single-strand at the termination site.

Question 14

What is a replisome?

Answer 14

A large protein complex that facilitates DNA replication, starting at the replication origin.

Question 15

How are replisomes adapted to their function?

Answer 15

Replisomes contain several enzyme activities, such as helicases, primases and DNA polymerase. Replisomes form a replication fork to duplicate both the leading and lagging strand.

Question 16

Why is control of replication and plasmid duplication needed?

Answer 16

To maintain a fixed concentration of plasmid molecule in the growing bacterial population. High levels of plasmid in the bacterial population has an energy cost associated.

Question 17

How is plasmid duplication controlled?

Answer 17

RNA (antisense RNA), DNA sequences (iterons), or antisense RNA and proteins acting in concert, can be used to control plasmid replication.

Question 18

How do restriction enzymes protect bacterial cells?

Answer 18

Restriction enzymes are thought to protect bacteria cells as phage and bacteria endonucleases have different degrees of methylation; this difference in methylation flags the phage and gives the bacteria the opportunity to destroy the phage.

Question 19

How does methylation of the bacterial chromosome protect it from phage attack?

Answer 19

Methylation of bacterial chromosomal DNA prevents the activity of viral endonucleases. Plasmid cannot undergo methylation so it is the plasmid that is attacked during infection.

Question 20

What are endonucleases?

Answer 20

Endonucleases are proteins that cleave the phosphodiester bond within the DNA.

Question 21

What are exonucleases?

Answer 21

Exonucleases are proteins that cleave the phosphodiester bond at the terminal points of the DNA.

Question 22

What is the role of endonucleases?

Answer 22

Endonucleases catalyse the hydrolysis of ester linkages within nucleic acids by creating internal breaks. Endonuclease rely on specific recognition sequences (restriction sites) in order to cut the DNA molecule.

Question 23

How can endonucleases be used in plasmid engineering?

Answer 23

Endonucleases form sticky end breaks, depending on the endonuclease used, and can be used in plasmid engineering; the same endonuclease in needed to cut the desired gene, to allow for insertion into the plasmid.

Question 24

What are the 2 responses to phage infection of bacteria?

Answer 24

1. The bacterial endonucleases will destroy (inactivate) the invading viral DNA, thus preventing phage replication.
2. The viral (phage) endonuclease will bind to the host DNA and destroy it.