Cosmid vectors

The cosmid vectors consist of modified plasmids carrying cos sites. A DNA sequence required for packaging the DNA or the genome into a phage head during the assembly is known as a cos site. A cosmid vector consists of an origin of replication belonging to E coli (ColE1). It also consists of the ampicillin resistance gene (Amp^R). The cosmid is also known as a plasmid carrying the λ (lambda) cos site. The substrates for in vitro packaging of phage DNA involve concatemers of cosmids, linked at their cos sites. The cos sites act as recognizing elements for a λ genome. Hence, the proteins getting packaged into a λ phage particle solely depend on the cos sites.
 A specialty of a cosmid is that it does not direct the synthesis of the new phage particle after entering the cell. Hence, a cosmid replicates as a plasmid. Instead of forming plaques (like phage infection), we obtain colonies. They help in identifying the recombinants. The size of the cosmid is 8kb or less. The new DNA insert size is up to 40 kb. A cosmid vector helps to reduce the size of the human genome library to one-quarter of a million clones. Cloning large segments of the foreign DNA is possible with cosmid.

Image: Cosmid vector

Examples of cosmid vectors:

1.     pJB8: It has a size of 5.4 kb. It is possible to clone the DNA fragments up to 46kb using a pJB8 cosmid vector. The pJB8 cosmid constructs consist of a ColE1 origin of replication, an ampicillin resistance gene (Amp^R), a cos site, and a restriction site. The derivation of the cos site involves Charon 4A. Charon phage or Charon 4A is a lambda bacteriophage derivative. It is useful for cloning the DNA fragments of interest. The four restriction sites in pJB8 vector include Bam HI, Eco RI, Cla I, and Hind III. However, there is a disadvantage of the pJB8 vector. It consists of only one cos site. Hence, it causes hindrances in concatenation (linking in a chain). Cloning experiments require two cos sites. The concatemers are the monomers repeated in a chain. A concatemer serves in the generation of λ chromosome. For example, a part of a concatemer involves two cos sequences. There is a cleavage point at each cos site and a ter gene at the cos sequence. The ter gene or a terminus-generating activity gene produces endonuclease recognizing a cleavage site in the cos sequence and makes a staggered cut. It results in the formation of correct complementary sticky ends or single-stranded ends. Then, it results in the packaging of the chromosomes in phage heads.

2.     C2RB Cosmid: The size of a C2RB vector is 6.8 kb. It is a vector consisting of two cos sites. A typical C2RB cosmid construct consists of an E coli CoE1 origin of replication, ampicillin, and kanamycin resistance genes (Amp^R and Kan^R). PKCT plasmid vectors help in the deriving ColE1 origin of replication. MUA-3 vectors help in deriving two cos sites. The restriction sites present in C2RB include Bam HI, Eco RI, Cla I, and Hind III. During packaging, the kanamycin resistance gene of 1.7 kb gets skipped. Hence, there is a loss in the related character. MUA-3 is a high capacity E coli cosmid vector. It consists of major parts of Pbr322 pSTI endonuclease recognition site. It is possible to prepare a cosmid library using MUA-3 vectors. After deriving two cos sites from the MUA-3 vector, the C2RB cosmid gets involved in a cloning experiment. The first step of cloning involves digestion of the restriction site with Sma I and Bam HI. It is a type of sequential digestion. It results in the derivation of two types of fragments. Each fragment consists of a cos sequence with a cohesive end and a blunt end. Next step involves deriving a foreign DNA.

The derivation of the foreign DNA involves getting the DNA sequence of interest for ligation purpose. The foreign DNA must have termini capable of binding with the vector. Hence, the fragments get a phosphatase treatment. The phosphatase enzymes get involved in removing the 5’ phosphate groups. Hence, they play a crucial role in ligation. The 10X Antarctic phosphatase buffer also crucial along with the use of foreign DNA and phosphatase enzymes. The procedure involves the addition of phosphatase buffer to the foreign DNA sample followed by the addition of one microlitre of Antarctic phosphatase enzyme. After incubation of the above components at 60 minutes and 37⁰C, the procedure follows heat inactivation for 5 minutes at 65⁰C. Now, the phosphate groups get removed from the 5’ ends of the DNA fragments. The digester vector DNA gets ligated to the foreign DNA (with phosphatase treatment).

Construction of a cosmid library:

The first step involves the digestion of the cosmid DNA vector with restriction enzymes such as Bam HI. The dephosphorylating enzymes help in the prevention of the vector re-ligation. Partial ligation of chromosomal DNA with another set of restriction enzymes such as MBOI and Sau 3A1 produce fragments. Agarose gel electrophoresis helps in separation of the fragments and visualizing them with the appropriate sizes. The insert DNA gets ligated into the vector to produce concatemeric hybrids. Next step involves mixing the ligated product with phage packaging extract. A phage packaging extract consists of phage lysate, empty phage heads, unattached phage tails, and phage proteins. 
The following table describes the phage proteins and their functions:

Phage proteins	Functions
Nu1, A	The packaging of cosmid DNA at the cos site
B,C,D,E,Nu3	Synthesis and assembly of phage heads
G,H,I,M,T,U,V,Z	Assembly of phage tails

Table: Bacteriophage proteins and their functions 

Hence, it necessary to have a vector with two cos sites. If there are two cos sites, it becomes easy to get packaged into the phage heads. The E coli strains get mixed with the packaged phages and propagate the recombinant plasmid in the cells. It is known as a cosmid library. The cosmid gets injected into the bacterial cells via connector present on the bacteriophage. It acts as an orifice for injecting the linearized cosmid DNA. Then, the cosmid DNA uses host ligating enzymes for joining the cohesive sticky ends to form a circularized structure. The process ensures no loss of genes.

It is easy to prepare genomic libraries using cosmid vectors. Cosmids get involved in the genome mapping. It is advantageous due to the insert size ranging from 35-46 kb. Apart from serving as vectors, cosmids also serve as probes in chromosome painting and FISH. 

References:
[1] Recombinant DNA Technology, Sardul Singh Sandhu

[2] Genetics, 9th Edition (Multicolour Edition), Verma P.S. & Agarwal V.K.

[3] Gene Cloning and Manipulation, Christopher Howe

[4] Genetic Engineering, Verma P.S. & Agarwal V.K.
[5] Biotechnology-4: Including Recombinant DNA Technology, Environmental,  S. Mahesh

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