## BACTERIOPHAGES

### BACTERIOPHAGES

2.      BACTERIOPHAGES

Bacteriophages are basically viruses that infect bacteria. Phages are very simple in the structure like other viruses and consist of DNA or rarely RNA as genetic material. The genetic material contains the information about replication and formation of a protective coat i.e., capsid which is made up of proteins.
There are 2 main types of phage structures
(a)    head and tail (e.g.,$\lambda$ phage)
(b)    filamentous (e.g., M13 phage).
2.1.    The phage infection cycle
The infection of phage in a bacterium is a three-step process-
•    Firstly, the phage attaches itself to the outer surface of the bacterium and injects its genetic molecule (DNA or RNA) into the cell.
•    The phage chromosome consists of genes which encode for specific enzymes required for its replication. The phage chromosome replicates within the host cell.
•    Other phage genes encode for the protein components of the capsid. Phage chromosome and capsid protein assemble to form new phage particles which are then released from the bacterium. For some bacteriophages, this cycle takes some minutes, and the phages are released with lysis of host bacterium. This type of infection cycle is known as lytic cycles.
The lytic cycle can be characterized by the immediate formation of capsid after infection and phage DNA is never maintained stably within bacterial chromosome.
On the contrary, some phage infection remains unaffected and no difference can be observed between infected and unaffected cell. The phage chromosome is inserted into bacterial genome and retained in stable form for many hundreds or thousands of cell division. This type of infection cycle is known as the lysogenic cycle. This type of insertion is similar to episomal insertion. This integrated form of phage DNA is known as a prophage, which remains quiescent and the bacterium is known as lysogen. However, the prophage may revert back to its lytic mode and cause the lysis of the host cell.
2.2.    The  phages use as cloning vector :
2.2.1.    Lambda ($\lambda$) phage
The  Size is approximately 42.5 kb.
However, 45% of l genome is non-essential. That means it can be replaced with desired DNA. Approximately insert size could be up to 25 kb.

Packing of DNA size is between 40 to 52 kb long. It follows the lysogenic cycle of infection. Lambda phage has a peculiar feature of clustering in which genes related in function are clustered together in the genome. The genes coding for the components of the capsid is grouped together in the left-hand side of the molecule. Which the genes responsible for the integration of phage general in the host are grouped in the middle of the molecule. This mechanism of the grouping of genes along the genes to turn on and off together rather than individually.
The DNA molecule in the l vector remains in linear conformation with two free ends. Which represents the phage head structure.
A single-stranded short stretch of 12 nucleotides is present at both the free ends. These two single-stranded stretches are complementary to each other and can join to form a circular DNA molecule. The single-stranded complementary ends are also known as sticky or cohesive ends as they can base pair or stick together to form a circular DNA molecule. These cohesive ends are known as cos sites and are very important during the infection cycle as it allows the linear DNA to circularize in the host cell. Only this circularized DNA molecule can be inserted into the bacterial host cell. Lambda phage follows rolling circle replication which produces copies of new linear l DNA molecules. These synthesized DNA molecules are then packaged into phage heads which from new l phages.
$\lambda$ genome vector is two types:
2.2.1.1.     Insertion vectors:
Insertion vectors are designed by deleting a large segment of the non-essential region and the remaining two arms are ligated together. It possesses at least one restriction site so that new DNA can be inserted. The possible length of new DNA that can be inserted depends on the length of the deleted non-essential region.
The common insertion vectors are :

$\lambda$ gt 10: This insertion vector has the capacity to carry new DNA of around 8kb in size. The ligation can be performed at unique EcoRI site in CI gene and recombinants can be distinguished as insertional inactivation. The recombinants would appear as clear plaque while other would be turbid.

$\lambda$ZAPII: The exogenous DNA of around 10 kb can be inserted into this vector. There are six restriction sites present in the vector and insertion of foreign DNA can inactivate lac gene. Thus, recombinants can be observed as clear rather than blue of colonies on X-gal agar. l gt 10 and Charon 16A also represent insertion vector.
2.2.1.2.    Replacement phage vectors :
1.    Insert DNA size is approximately 10 - 25 kb.
2.    Non-essential is replaced with interest DNA.
It generally consist of two recognition sites that can be used for insertion. The flanking segment of DNA to these sites are replaced by the foreign DNA. These replaced fragment of DNA is known as stuffer fragment. This replaced sequence usually carries one restriction site which allow it to be cut in smaller pieces so that reinsertion of this DNA for a cloning experiment become difficult. Replacement vectors has a large carrying capacity than insertion vectors. Recombinants can be distinguished by size as non recombinants are smaller in size and cannot be packaged in phage capsid.
The popular examples of replacement vectors are :
Charon 40 : The carrying capacity of charon 40 is similar to EMBL4. The stuffer fragment in this vector is composed of multiple repeats of a short stretch of DNA. This is known as polystuffer. This is advantageous for Charon 40 as the restriction enzyme. EcoRI can be used to cut the polystuffer into its component parts. This increases the probability of surviving phages to be recombinant and efficient removal of polystuffer during vector preparation. There is more choice of restriction enzyme in Charon 40 than EMBL4 as the polystuffer region is flanked by an extensive range of restriction sites.
EMBL4 : The exogenous DNA can be inserted by removing any of flanking site of ECORI, Bam HI and Sal1 sites. Thus a variety of genes can be cloned to these sites. It can carry DNA molecule up to 20 kb in length. Recombination selection can be done by utilizing the special phenotype or on the basis of size.