Cloning vectors based on bacteriophages and cosmids

In this article, I briefly describe the cloning vectors based on bacteriophages and cosmids.

Table of Contents

Cloning vectors

A small piece of DNA into which a foreign DNA fragment is inserted for cloning purposes is known as a cloning vector. The cloning vector may be the plasmid from a bacterium, a higher organism’s cell, or DNA taken from a virus. The vector contains restriction sites that help in the convenient insertion and removal of a DNA fragment. The cloning vector and the foreign DNA are treated with a restriction enzyme that creates the same overhang, then ligating the fragments together. Genetically engineered plasmids and bacteriophages (such as phage λ) are perhaps most commonly used. Other types of cloning vectors are also used, which include bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

Bacteriophage

A bacteriophage is a virus that infects bacteria and replicates within it. It has a simple or elaborate structure. Bacteriophages are composed of proteins that encapsulate a DNA or RNA genome. These are ubiquitous viruses, and it is estimated there are more than 10³¹ bacteriophages on the planet, more than every other organism on earth, including bacteria combined.

There are so many bacteriophage types of all shapes and sizes. They are all categorized into one of the two replication methods, i.e., lytic and lysogenic. Virulent bacteriophages use the lytic cycle for replication. The lytic cycle is a cytoplasmic viral replication process in which the bacteriophage injects its genetic material into a host cell. This genetic material replicates inside the host producing many new phages.

Temperate bacteriophages use the lysogenic cycle for replication. In this cycle, the phage infuses its genetic material into a host. However, instead of rapid replication, this generic material makes its way to the host’s genetic material and infuses itself with it. Thus, it becomes a prophage. As a part of the host’s genetic material, it divides along with the division of the host cell.

Cloning vectors of M13 bacteriophage

Phage DNA molecules carry essential genes for replication, including genes coding for components of the phage protein coat and phage-specific DNA replicative enzymes. Alteration or deletion of any of these genes can destroy the replicative ability of the resulting molecule. So, the M13 genome has a limited scope for modification. Thus, phage cloning vectors are only slightly different from parent molecules.

M13mp1 and M13mp2 cloning vectors

The construction of an M13 cloning vector is done by introducing the lacZ’ genes into the intergenic sequence. This results in the production of M13mp1, which forms blue plaques on the X-gal agar medium. M13mp1 (figure 1a) contains the hexanucleotide sequence GGATTC, close to the start of the lacZ’ gene. A single nucleotide change in this sequence would make this GAATTC, which is an EcoRI site. This alteration is carried out by in vitro mutagenesis, resulting in an M13mp2 cloning vector (figure 1b), which has a slightly altered lacZ’ gene. M13mp2 is the simplest M13 cloning vector.

Construction of M13mp7 cloning vector

Construction of an M13mp7 vector is carried out with the introduction of additional restriction sites into the lacZ’ gene of M13 vectors. A polylinker (figure 2(a)), which is a short oligonucleotide sequence, having a series of restriction sites and EcoRI sticky ends is synthesized in the test tube. It is inserted into the EcoRI site of M13mp2 to give M13mp7 (Figure 2(b)). The vector is more complex with four possible cloning sites, e.g., EcoRI, BamHI, SalI, and PstI. The polylinker is so designed that it doesn’t disrupt the lacZ’ gene. Throughout the polylinker, a reading frame is maintained, and an altered but functional β-galactosidase enzyme is still produced.

Complex M13 vectors

Insertion of more complex polylinkers into the lacz’ gene produces complex M13 vectors. M13mp8 is a complex vector, and it can take DNA fragments with two different sticky ends. M13mp9 is another complex vector having the same polylinker but in a reverse orientation. A DNA fragment cloned into M13mp8, if excised by double restriction, and then inserted into M13mp9 will now itself be in the reverse orientation. This is important in DNA sequencing, in which the nucleotide sequence is read from one end of the polylinker and inserted into the DNA fragment.

Cloning vectors of λ bacteriophage

The two types of cloning vectors of λbacteriophage are insertion vectors and replacement vectors.

Insertion vectors

There is the presence of at least one specific restriction site in an insertion vector into which a new DNA can be inserted. λgt10 and λZAPII are two popular insertion vectors. λgt10 can carry up to 8kb of new DNA, inserted into a unique EcoRI site located in the cI gene. Insertional activation of this gene distinguishes recombinants as clear rather than turbid plaques.

Replacement vectors

A λ replacement vector possesses two recognition sites for the restriction endonuclease used for cloning. These sites flank a segment of DNA that is replaced by the DNA to be cloned. The replaceable fragment often carries additional restriction sites that can be used to cut it up into small pieces. Replacement vectors are designed to carry larger pieces of DNA. The two common replacement vectors are λWES. λB’ and λEMBL4.

Cosmid

It is a medium-sized cloning vector and is developed by combining the features of a plasmid and a bacteriophage. Thus, a cosmid is a hybrid between a phage DNA molecule and a bacterial plasmid and is designed in such a way that the enzymes that package the λ DNA molecule into the phage protein coat need only the cos sites (cohesive sites) to function. A cosmid is a plasmid that carries a cos site, the sequence yielding cohesive ends (figure 3). A typical cosmid has replication origin, unique restriction sites, and selectable markers from the plasmid, and only the cos site is taken from the λ phage. Cosmid vectors are constructed using recombinant DNA techniques.

Cloning with a cosmid

pJB is a cosmid vector with a size of 5.4 kb. It consists of a ColE1 origin of replication, an ampicillin resistance gene (Amp ^R), a cos site, and a restriction site. A specific restriction enzyme opens the cosmid vector at a unique site. Then, these vectors are mixed with DNA inserts prepared by using the same enzyme and annealed. These DNA fragments are produced usually by partial digestion with a restriction endonuclease. Because total digestion produces too small fragments to be cloned with a cosmid. Ligation is carried out, and it produces concatemers. (Figure 4).

**Figure 4: A cosmid vector is used to clone large fragments of DNA**

The concatemers are the monomers repeated in a chain. A part of a concatemer involves two cos sequences. There is a cleavage point in each cos site and a terminus-generating activity gene (ter gene) at the cos sequence. The ‘ter’ gene produces endonuclease recognizing cleavage site in the cos sequence and makes a staggered cut. This results in the formation of correct complementary sticky ends. Then, it starts the packaging of the chromosomes in phage heads. In vitro, packaging will cleave the cos sites and place the recombinant cosmids in mature phage particles. These λ phages are then used to infect E.coli culture. The infected cells are then plated onto a selective medium, and the grown antibiotic-resistant colonies are observed.

Conclusion

The cloning vector may be the plasmid from a bacterium, a higher organism’s cell, or DNA taken from a virus. A bacteriophage is a virus that infects bacteria and replicates within it. These are composed of proteins that encapsulate a DNA or RNA genome.

The construction of an M13 cloning vector is done by introducing the lacZ’ genes into the intergenic sequence of a bacteriophage. Three types of M13 cloning vectors can be constructed, i.e., M13mp1, M13mp2 and M13mp7. Insertion of more complex polylinkers into the lacz’ gene produces complex M13 vectors.

The two types of cloning vectors of λ bacteriophage are insertion vectors and replacement vectors. There is the presence of at least one specific restriction site in an insertion vector, whereas a λ replacement vector possesses two recognition sites for the restriction endonuclease used for cloning.

A cosmid is a hybrid between a phage DNA molecule and a bacterial plasmid. A cosmid is a plasmid that carries a cos site, the sequence yielding cohesive ends. Cloning with a cosmid is done by a specific restriction enzyme.