The cloning of single genes is usually best carried out using plasmids, since the insert will rarely be larger than about 2 kb. However, for cloning of larger pieces of DNA (e.g., during gene library construction) these plasmids are not suitable as larger inserts increase the plasmid size, making the transformation inefficient. Large DNA molecules can be injected in host bacterial cell by viral particles (bacteriophages). Commonly used bacteriophages are M13, f1, fd and lambda (λ) phage.
(i) Phage Lambda (λ) as a Vector:
A commonly used vector is that of the lambda (λ) phage. Bacteriophage λ, which infects E. coli cells, can be used as cloning vector. DNA of λ phage is 48.5 kb in length. At its ends are the cos (cohesive) sites, which consist of 12 bp cohesive ends. The cos ends allow the DNA to be circularized in the host cell.
For the cloning of large DNA fragments, up to about 20 kb, much of the nonessential lambda DNA is removed and replaced by the insert (desired or ta. The recombinant DNA is then packaged within viral particles in vitro, and these are allowed to infect bacterial cells (E. coli) which have been plated out on agar.
Once inside the bacterial cells, the recombinant viral DNA is replicated. All the genes needed for normal lytic growth are still present in the DNA and so multiplication of the virus takes place by cycles of cell lysis and infection of surrounding cells. It gives rise to plaques of lysed bacterial cells on a background, or lawn, of bacterial cells. Cloned DNA can be recovered from the viruses in these plaques.
There are following two types of lambda vectors phages which differ in size of DNA they accept:
(a) Lambda replacement vectors; and
(b) Lambda insertion vectors.
(a) Lambda Replacement Vectors:
Lambda replacement vectors contain a restriction site for phage propagation in suitable bacterial host. Remaining part of the lambda genome is removed and is replaced by foreign DNA. Ligation is performed at a ratio of arms to target DNA that favours the formation of very long concatemers. The concatemers are multiple length copies with multiple replication complexes and forks. In each of concatemers, vector and target molecules are interspersed.
A number of so-called replacement vectors have been developed from phage λ, examples include EMBL4, EMBL3, λ DASH, etc. In most replacement vectors, the internal region that is replaced by the target contains a gene that renders the phage inviable (dead) in an appropriate E. coli host Vector molecule containing target DNA can be selected by infecting host. Recombinant phage in which the internal region is replaced by target DNA is viable and are mostly used for cloning eukaryotic DNA fragments.
(b) Lambda Insertion Vectors:
When cloning into an insertion vector, the phage DNA is cleaved with a restriction enzyme that cuts it only once, and the target is inserted into this site No phage DNA is removed, therefore, much smaller sized target DNA can be inserted.
In commonly used vector phage λ gt 10, the Eco II cloning site is in a gene which is deleterious to phage replication in certain host strains. This allows selection against non-recombinant phage.
(ii) Filamentous Phages as Cloning Vector:
It includes Ff class of filamentous phages, including strains f1, fd and M13, which infect E. coli cells. These Ff virions are long and thin and contain a closed loop of single- stranded DNA. Because the phages readily accept inserts of foreign DNA and they supply one strand of that DNA in an easily isolated form, vectors based on Ff phages have become standard choice of biotechnology.
a. M13 Bacteriophage:
The M13 is a filamentous bacteriophage of E. coli. It is 870 nm long and 6 nm wide. It has a protein coat (the capsid) which is made up of three kinds of capsomeres. This filamentous single stranded DNA phage infects the bacterial cell by adsorbing to and entering through a pilius. M13 phages only infect F+ or Hfr cells, not F– cells.
The M 13 phage particles contain a 6.7 kb circular single stranded DNA. After infection of a sensitive E. coli host, the complementary strand is synthesized, and the DNA replicated as a double standard circle, the replicative form (RF), with about 100 copies per cell.
Further M13 phages do not lyse the host cells (to release progeny phage and bacterial DNA from the ruptured bacterial host cells) like lambda phage during the lytic cycle. Instead of this, the progeny M13 viruses/phages are extruded through the layers of the plasma membrane and cell wall without major interference with cell growth.
Infected bacterial cells will continue to grow and extrude thousands of progeny virus particles (i.e., up to 1000 per cell generation), each containing a single- stranded genome to the medium. Since the virus particles are very small in comparison of the host bacteria, the host cells can be removed by low-speed centrifugation.
The virus particles can then be collected from the supernatant suspension by high-speed centrifugation and their single-strand DNA molecules can be isolated by single phenol-chloroform extractions. The DNA strand of the virus is always packaged; it is called the “+” or plus strand (its complement is the “-” or minus strand).
The packaged “+” strand of the phase has the same “sense” as the mRNA, i.e., nucleotide triplets of + DNA strand of M13 phage correspond to the mRNA codons, but with T in place of U.
Packaging of single strands of phage DNA in progeny phage provides a neat biological purification of single-strand DNA. Importantly, this statement holds true for a foreign gene cloned in the viral chromosome just as for the phage genes themselves. This property of M13 phase has been exploited for its use as a vector.
Lastly, phage M13 is not used as a primary vector to clone new DNA targets, but fragments are normally sub-cloned into M13RF using standard plasmid methods when the single-stranded form of a fragment is required.
1. Genetic organisation of wild type M13 bacteriophage:
The DNA molecule of M13 phage is single-stranded and circular. It is 6407 bases long having 10 closely packed genes. All these genes are essential for the replication of the phages. There is a segment of 507 base-long intergenic sequence (IS) which contains origin of replication (OR).
The IS containing region of viral genome is manipulated for cloning without disrupting the origin of replication. Hence, the wild M13 phage has limited use in gene cloning experiments. The size of the phage particle is decided by the size of the phage DNA. Upto six times the normal length of M13 phage DNA can be packaged.
2. Construction of M13 based vectors:
In M13 phage DNA, the intergenic sequence is the only region which can be manipulated for gene cloning. As this region has only two restriction sites (Asa I and Ava II), wild type phage is not an efficient vector. However, the intergenic sequence can be modified to introduce additional restriction sites.
A few such vectors are discussed below:
M13mp1 and M13mp2:
The gene lac Z is introduced into the wild type intergenic sequence to get the M13 mpl phages. This step produces blue plaques on X-gal agar plates. The lac Z gene does not have any restriction site. However, it has a hexanucleotide sequence, GGATTC near the start. If the second G residue is substituted by an A residue this sequence becomes an Eco R1 site, i.e., GAATTC. This type of conversion is done by in vitro mutagenesis.
Now this phage is called M13 mp2. The lac Z’ gene now has a slightly altered base sequence. Due to this change, the β-galactosidase enzyme which is encoded by lacZ gene has amino acid asparagine instead of aspartic acid (amino acid) in the fifth position. Such a change (due to mutation) does not affect the activity of the β-galactosidase enzyme. This can be tested by X-gal agar where the plaque gives a blue colour.
Gene lac Z of E.coli encodes the N-terminal a-peptide of the enzyme (3-galactosidase which is responsible for hydrolysis of lactose into galactose and glucose.
The M13 mp2 is the simplest cloning vector derived from M13 phage. In the unique Eco RI site of M13 mp2, any foreign DNA with Eco RI sticky ends can be inserted. This insertion inactivates lac Z gene and is called insertional inactivation. Such recombinant phages fail to produce blue plaques on X-gal agar, instead, they produce clear plaques.
M13 mp7:
The M13 mp7 is a derivative of MB mp2. When a polylinker is inserted into the Eco RI site of lac Z’ gene, the M13 mp7 becomes M13 mp7. The polylinker is designed in such a way that it does not inactivate the lac Z’ gene.
When the vector phage M13 is cut with Eco RI, Bam HI, Sal I or Pst I, the entire polylinker or a part of it is excised and form sticky ends. Foreign DNA with the corresponding sticky ends is inserted to produce recombinant M13 mp7. Thus, M13 mp7 is a more complex vector having four possible insertional sites.
Insertion of foreign DNA tends to inactivate the lac Z gene and the production of galactosidase enzyme is prevented. This is shown by the formation of clear plaques on X-gal agar by the recombinant phage DNA.
3. M13-Plasmid Hybrid Vectors:
The hybrid vectors contain components from both plasmids and phage chromosomes. These vectors replicate in E.coli as normal double-stranded plasmids until a helper phage is provided. After the addition of the “helper” phage, they switch to the phage mode of replication (i.e., rolling circle replication) like that observed for phage ф×l- and package single strands of DNA in phage particles.
The helper phage is a mutant that replicates its own DNA inefficiently, but provides viral replication enzymes and structural proteins for the production of plasmid DNA molecules that are packaged in phage coats.
(b) pUC 118 and pUC 119 Cloning Vectors:
The phage-plasmid “hybrid” vectors pUC118 and pUC119 are a pair of vectors that are essentially identical except that the regions into which foreign DNAs are inserted are present in opposite orientation (i.e., turned end-to-end relative to the rest of the genes of the vector.
Thus, if a foreign DNA is inserted into a specific restriction site in both vectors, one vector will package the complementary strand of the gene. Therefore, both strands of the gene can be isolated, sequenced, subjected to site-specific mutagenesis, and so on.
The vectors were designated pUC for plasmid, University of California (where the first pUC vectors in the series were constructed by J. Messing and J. Vieira 1987) and 118, 119 to distinguish from earlier members (i.e., lower numbers) of the series.
Vectors pUC 118 and pUC 119 differ from earlier vectors in the pUC series by the addition of are origin of replication from phage M13. This permits pUC118 and pUC119 to replicate either (1) as a double standard plasmid in the absence of “helper” phage or (2) as a single-stranded DNA that is packaged in M13 phage coats and extruded from the cell in the presence of “helper” phage (most commonly an M13 derivative called K07).
In the absence of “helper” phage, replication is controlled by the plasmid origin of replication. The pUC vectors were derived from an early plasmid cloning vector called pBR322 by a series of direct modifications.
The pUC vector such as PBR322 contain an origin of replication initially present in plasmid Col E1. In the presence of helper” phage replication of pUC118 and pUC119 is directed by the M13 phage origin of replication that has been added to the plasmids.
Certain important features of the pUC11S and pUC119 cloning vectros are the following:
1. They are present as small, supercoiled, covalently closed circular DNA. Due to this feature they are easily isolated and manipulated in ‘ vitro.
2. They carry the amp” gene as a selectable marker. Thus, only bacteria harbouring a plasmid will grow on medium containing the antibiotic amplicillin.
3. They carry high copy number, up to 5000 copies per bacterium. Thus, there would be large yields of DNA from small cell cultures.
4. They carry a polycloning region which have a variety of restriction enzyme cleavage sites. Thus many different types of restriction fragments can be inserted without modification.
5. The polycloning region interrupts the coding region of the 5′ end of E. coli lac Z gene. Thus, colonies harbouring plasmids with foreign DNA inserts can be distinguished from those carrying plasmids with no insert by a simple colour test.
6. The lac Z gene is under the control of the lac promoter. Thus, genes inserted in frame (codons in proper reading frame) can be expressed to produce β-galactosidase- foreign protein fusion products.
7. They carry a plasmid origin of replication. Thus, replication of the vector DNA produces large numbers of double-stranded plasmid DNAs in the absence of “helper” phage.
8. They carry a phage M13 origin of replication. Thus production of single-stranded DNA and packaging of this DNA in phage coats take place in the presence of “helper” phage.
9. The polycloning regions are present in pUC118 and pUC119 in opposite orientations. Thus, if pUC118 packages one strand of a cloned gene, pUC119 will package the complement strand.
Different members of the pUC vectors series contain different, but related, sets of restriction enzyme cleavage sites. The polycloning regions of pUC118 and pUC119 contain 10 clustered restriction enzyme cleavage sites. Some of these sites are substrates for two or more different restriction enzymes.
The utility of pUC is greatly increased by a simple colour test that allows one of distinguish cells harbouring plasmids with foreign DNA inserts from those harbouring plasmid with no insert. The basis of this colour indicator test is the functional inactivation of the 5’ segment of the lac Z gene present in the vector by the insertion of foreign DNA into the polycloning region.
The pEMBL8 Cloning Vehicle:
The pEMBL8 cloning vehicle is constructed by transforming a 1300 bp fragment of the genome of M13 phage into the pUC8 plasmid. This piece of M13 genome has the signal sequence recognised by the enzymes that convert the dsM13 DNA into a ssDNA molecule. So the pEMBL 8 molecules are also converted into single-stranded DNA molecules and these will be released as infective phage particles.
When pEMBL 8 vector is used, the E. coli cells should also be infected with an intact M13 particle. This will act as a helper phage by providing necessary enzymes and phage coat proteins. Since pEMBL 8 is derived from pUC8 it has the polylinker at the necessary lac Z’ gene.
Hence this plasmid vector has the following advantages of pUC8 plasmids and that of the M13 phages:
1. Foreign DNA fragments with two different sticky ends can be inserted at the multiple cloning site on the lac Z’ gene.
2. Recombinant pEMBL 8 genome can be packaged into the capsid of M13 phage.
3. The phage particles containing the recombinant pEMBL 8 genome are as efficient as the wild type M13 phages in their ability to infect E. coli cells and then in expression of the foreign gene.
4. Recombinant pEMBL 8 genome can be easily screened using the medium containing X-gal.