Bacteriophage cloning

Question 1

How many nucleotides is M13 made up of?

Answer 1

6407 nucleotides, a bit bigger than plasmids.

Question 2

What is the infective form of M13?

Answer 2

Contains single stranded DNA and is packed in its protein coat outside the E.coli cell.

Question 3

What happens when it is injected into an E.coli cell?

Answer 3

A second strand is synthesised and it becomes double stranded to form the replicative form.

Question 4

What happens when the threshold level of the replicative form is reached?

Answer 4

The mode of replication changes a linear single stranded infective form DNA is produced by the rolling circle mechanism.

Question 5

What happens to these single stranded infective form DNA produced by the rolling circle mechanism?

Answer 5

They circularise and are packaged into the protein coat and are extruded from the E.coli cell into the medium.

Question 6

What is now left in the medium? (after rolling circle mechanism)

Answer 6

Mature bacteriophage particles in the medium and double stranded replicative form inside the cells.

Question 7

How can the life cycle of the M13 bacteriophage be exploited for use in cloning?

Answer 7

The replicative form can be manipulated with restriction enzymes and ligases and transfected into the E.coli cell. When inside the cell, it will be replicated and eventually will be extruded as the infective form after the rolling circle mechanism.

Question 8

How is the infective form useful in cloning?

Answer 8

It is an ideal template for DNA polymerases and can be used in DNA sequencing.

Question 9

What use do the lacZ’ and restriction sites from pUC vectors in M13?

Answer 9

They can be put into the M13 and enable blue/white selection, along with shuttling DNA from pUC to M13 vectors.

Question 10

What is the first step in a typical cloning experiment using M13 vectors?

Answer 10

Restriction fragments are ligated into the double stranded replicative form M13 vecor.

Question 11

What is the second step in a typical cloning experiment using M13 vectors?

Answer 11

The ligated DNA is transfected (transformed) into an E.coli cell

Question 12

What is the third step in a typical cloning experiment using M13 vectors?

Answer 12

The transfected cells are plated out onto an E.coli lawn on an agar medium containing IPTG and X-gal (but not antibiotic).

Question 13

What will be seen on the lawn in the cloning experiment with M13 vectors?

Answer 13

E.coli containing M13 will produce plaques - blue plaques are non-recombinant whereas white plaques are recombinant.

Question 14

What will be done with the white plaques in the M13 cloning experiment?

Answer 14

They can be used to infect E.coli and purify replicative form (double stranded) from the cell and infective form (single stranded) from the medium. The infective form can be used for DNA sequencing.

Question 15

How does transfection differ from transformation?

Answer 15

They are the same process (DNA into a bacterial cell) but transfection is when naked bacteriophage is used. It has a different name as it is a virus when in the E.coli cell.

Question 16

What is the difference between infection and transfection?

Answer 16

Infection is when a bacterial cell is infected by a complete bacteriophage, whereas transfection is the introduction of naked bacteriophage DNA. Infection is very efficient whereas transfection is not.

Question 17

What are plaques?

Answer 17

Small clearings on an E.coli lawn where M13 has inhibited E.coli growth. They contain slow-growing bacteria and M13 phage particles.

Question 18

How do lytic plaques differ from M13 plaques?

Answer 18

Lytic plaques have all of the bacteria lysed - there is a high titre of bacteriophages.

Question 19

Why are bacteriophages used intstead of plasmid vectors?

Answer 19

Transformation is not very efficient - there are not many clones per ug of plasmid DNA. Small fragments are also easier to clone than larger ones (there is a limit on size of clonable fragments).

Question 20

What are the two lifecycles of lambda?

Answer 20

The lytic life cycle which is of interest to a genetic engineer and a lysogenic lifecycle which is of interest to a geneticist.

Question 21

What happens in the lytic life cycle of lambda?

Answer 21

It infects E.coli as a linear DNA molecule and replicates as a circular molecule. The replication method then changes to the rolling circular one and long concatamers of the lambda genome are produced. Each genome is separated by a cos site. Enzymes recognise the cos sites and snip the concatamers at them which package individual linear lambda genomes into their respective protein coats. The e.coli cell then lyses which releases the lambda particles.

Question 22

What happens in the lysogenic life cycle of lambda?

Answer 22

Lambda infects the E.coli but integrates into the E.coli genome and is passively replicated with the E.coli, instead of replicating its DNA.

Question 23

What can happen under certain circumstances in the lysogenic cycle?

Answer 23

The lysogenic lambda can de-integrate from the E.coli chromosome and enter the lytic cycle. The genetic engineer only exploits the former.

Question 24

What are cos ends?

Answer 24

12 base single stranded complementary ends to the lambda genome.

Question 25

Why are cos ends useful?

Answer 25

As they are complementary to the ends of the lambda genome, base pairing can facilitate circularisation on infection. Endonuclease enzymes also recognise cos sites and cleaves DNA which is packaged by other lambda proteins to form phage particles in late replication.

Question 26

What is an advantage of lambda over plasmids?

Answer 26

The process of infection of E.coli by lambda particles is highly efficient and you can get more clones for your DNA.

Question 27

What are some problems with lambda?

Answer 27

The maximum size of DNA that the lambda can package into its coat is 52kb, whereas a normal genome is 49kb so the maximum insert of DNA is less than 3kb. There are also multiple restriction sites that produce many fragments.

Question 28

How can the size issues with lambda be solved?

Answer 28

The genes for the lysogenic life cycle are found in the central region and can be removed, so as much of 18kb of new DNA can be inserted.

Question 29

How can the restriction sites problem with lambda be solved?

Answer 29

Selection in the lab can be used to select mutant phage in which particular restriction sites have mutated to sequences which are no longer recognised by the restriction enzyme.

Question 30

What are the two categories of lambda vectors and what are they used for?

Answer 30

Insertion vectors that are used for cloning smaller fragments (less than 5kb) and in cDNA cloning and replacement vectors that are used for cloning larger fragments (15-22kb) with high efficiency and producing genomic libraries (a collection of clones that together contain all the DNA of an organism).

Question 31

How is an insertion vector constructed?

Answer 31

Some of the non-essential regions of lambda are removed to create the vector by ligation. A convenient restriction site for cloning is inserted. There is a minimum and maximum size that can be packaged into the protein coat so not too much of the non-essential region should be removed.

Question 32

What are two types of insertion vectors and what selection methods do they use?

Answer 32

Lambdagt10 - clear plaques for recombinants and turbid for non-recombinants. lambdaZAPII has blue/white selection for recombinants.

Question 33

What is the general protocol for insertion vector cloning?

Answer 33

Cut the DNA and lambda with restriction enzymes, ligate the lambda arms and inserts, add packaging mix (enzymes and proteins required to assemble the particle) - the enzymes will recognise the cos sites and package DNA between them, infect the appropriate strain of E.coli, plate out onto a lawn and obtain millions of plaques.

Question 34

Why do replacement vectors sometimes need to be used instead of insertion vectors?

Answer 34

Not all the non-essential regions can be removed as there is a minimum size of the lambda genome that is capable of being packaged successfully. This means larger fragments cannot be cloned by using insertion vectors.

Question 35

What is a stuffer fragment?

Answer 35

A fragment that can replace non-essential regions so that the vector is of a size capable of replication. This allows the quantities of DNA neccessary for cloning experiments can be used. The stuffer fragment is bordered with convenient restriction sites.

Question 36

What is the process for cloning in a replacement vector?

Answer 36

The vector is cut with restriction enzymes and the left and right arms are purified from the stuffer fragment. It is ligated to the digested DNA and packaged in vitro and infects the E.coli cell. The size of the library can be assessed by plating onto an E.coli lawn and determining the titre in plaque-forming units per ml. Arms alone are too small to be packaged so all plaques should be recombinants.