Cloning Vectors

Vectors are those DNA molecules that can carry a foreign DNA fragment when inserted into it. Vectors are also known as vehicle DNAs. Based on the nature and sources, the vectors are grouped into bacterial plasmids, bacteriophages, and cosmids and phasmid.

Plasmids
Plasmids are the extrachromosomal, self-replicating, and double stranded closed and circular DNA molecules present in the bacterial cell. Plasmids contain sufficient genetic informations for their own replication. A number of host properties are specified by plasmids, such as antibiotic and heavy metal resistance, nitrogen fixation, pollutant degradation, bacteriocin and toxin piroductioivcolicin factors and phages (Dahl et al, 1981). Naturally occurring plasmids can be modified by in vitro techniques. Cohen et al. (1973) for the first time reported the cloning DNA by using plasmid as vector.

A plasmid can be considered a suitable cloning vehicle if it possesses the following features:

1. It can be really isolated from the cells,
2. It possesses a single restriction site for one or more restriction enzyme(s),
3. Insertion of a linear molecule at one of these sites does not alter its replication properties,
4. It can be reintroduced into a bacterial cell and cells carrying the plasmid with or without the insert can be selected or identified (Bernard and Helinski, 1980),
5. They do not occur free in nature but are found in bacterial cells.

In many cases the principal objective of cloning experiment is the insertion of a particular restriction fragment into a suitable plasmid vector and its amplification. Amplification is a process of increasing the number of plasmid in a bacterial cell. In this process, a cell containing a relaxed plasmid is treated with a drug to inhibit protein synthesis. Consequently, cells stop replicating. The relaxed plasmid pBR322, continues to replicate despite drug treatment. Replication of relaxed plasmid neither depends on cell replication nor requires protein synthesis. For example, addition of chlorumphenicol causes pBR322 to get increased about 3000 per cell. Finally the ratio of plasmid DNA is increased to chromosomal DNA which makes easy to isolate the plasmid DNA. In order to replicate and clone a fragment of foreign DNA, it is necessary to possess a sequence of nucleotides which are recognized by the host bacterium as an origin of replication. The origin of replication occurs naturally in plasmid and transferred to onward progenies. During cell division these genes are transferred resulting in gradual decrease in number of such genes. However, due to continuous exchange of genetic materials between plasmid and chromosomal DNA, new genes originate with respect to environmental conditions. Therefore, plasmids have been artificially developed from naturally occurring form and only special features that help in cloning have been preserved. Some of the plasmids are given in Table 3.3.

Table 3.3. Some cloning vectors.
*, 1 Kb (Kilobase pairs) = 1,000 base pairs = 0.66 mega dalton;**,

Resistance to ampicillin (Amp^r), tetracycline (Tet¹), erythrdmycine (EryO* streptomycine (Strr),
Kanamycin (Kan^r), rifampicin (Rif^r), and colicin production (EL imm.) A physical map of plasmid pBR 322 is shown in ( Fig. 3.4.) The pBR 322 is constructed from the plasmids of E. coli, pBR318 and pBR320. It contains origin of replication (ori) that was derived from a plasmid related to naturally occur ring plasmid Col El. Therefore, its replication may be more faster than bacterial DNA. It also possesses genes conferring resistance to antibiotics e.g. ampicillin (amp¹) and tetracycline (tef), and unique recognition sites for 20 restriction endonucleases. Certain restriction sites for example, Bam HI in the tet^r genes of the plasmid are present within the gene in such a way that the insertion of foreign segment of DNA will inactivate the tef gene. The recombinant plasmid will allow the cells to grow only in the presence of ampicillin but will not protect them against tetra-cycline. The presence of an antibiotic resistant gene in a plasmid of bacterium will confer resistance to that antibiotic.

Therefore, the antibiotic resistant cells can be selected by culturing the cells on nutrient medium supplemented with ampicillin or tetracycline.

In addition to E. coli, some of the Gram-negative and Gram positive-bacteria have also been investigated and used as cloning vector for medical and agricultural studies. Recently, a broad host range cloning vehicle has been developed from RK2 which is a plasmid of P-1 group of Gram negative bacteria. RK2 plasmid contains a single restriction site for EcoRl, Hind III, BamHIand BglII. However, a broad host range cloning system has been developed from it by incorporating the transfer and replication regions of this plasmid into two different plasmids. The three regions of RK2 plasmid essential for replication (e.g. OriV, trfA and trfB) have been incorporated into the plasmid cloning vector, the pRK290 (Bernard and Helinski, 1980).

Species of Pseudomonas consist of a variety of plasmids which encode enzymes capable of degrading an enormous range of natural and synthetic organic compounds. The most studied plasmid among them is pwwo from P. putida. This is one member of a set of plasmids which have required the generic name TOL plasmids. It carries the genes that utilize toluene, m-and p-xylene, 3-ethyltoluene and 2,4- trimethyl benzene as well as their alcohol, aldehyde and carboxylic acid derivatives (Glover, 1984).

Prof. Anand Mohan Chakrabarty and coworkers (1979) at the University of Illinois, USA. have developed strains of Pseudomonas which can be applied for degradation of synthetic organic compounds and pesticides. They are also making efforts to isolate bacterial strains that could reduce the viscosity of heavy oils by degrading the constituents of oils, waxes and paraffins. Thus, plasmids are an excellent vector for small fragment of DNA and, therefore, a necessary tool for establishing the cDNA bank.

Isolation of bacterial plasmid
Following are the steps for isolation of bacterial plasmids:

1. Treat the prokaryotic cells with detergent. Cell membrane is solubilized. Plasmid DNA, chromosomal DNA and the other molecules are released. This mixture is known as lysate.
2. Treat the lysate with potassium acetate/acetic acid solution. Chromosomal DNA with some protein molecule is precipitated.
3. Remove the precipitate from lysate by centrifugation at high speed. Clear lysate is left. It contains plasmid DNA along with contaminating RNA, protein and a small amount of chromosomal DNA debris.
4. Treat the lysate with-RNAase. Consequently, RNA is digested,
5. Mix the lysate with phenol. Phenol and water is separated into layers. Phenol layer contains the contaminating proteins and RNAase, and water based lysate layer contains plasmid DNA.
6. Remove the phenol layer. Precipitate the water based layer with alcohol as the genomic DNA is removed. Make use of it as desired.

Bacteriophages
A bacteriophage is virus which eats upon bacteria. A number of such viruses of different genetic material have been reported, for example ΦX174, phage λ, M_I3, Fd¹¹, R209, etc. Unlike plasmid vectors, the phage vectors are required for cloning of large DNA fragment and, therefore, the gene bank or genomic libraries can be constructed. They are an alternative to bacterial plasmids, and related cosmids. The process of infection and replication of phage in E.coli cell are shown in (Fig. 3.5).

Phage l contains a proteinaceous head and a long tail attached to the head. In the head it possesses 50 genes in its 49kb (kilobase pairs) genome of which about half of genes are essential. On attachment with tail to cell wall of E. coli it injects its linear DNA into the cell The linear double stranded DNA molecule cyclizes through the single strand of 12 nucleotides commonly known as cos sited at its end. The cos sites are the key feature of the DNA. Replication cycle of phage λ is accomplished into two pathways: the lytic and lysogenic pathways (Wu and Taylor, 1971). In the lytic pathway, early in the infection sites the circular DNA replicates as theta (θ) forms. By a rolling circle mechanism it produces the long concatemeric molecules joined end to end, and composed of several linearly arranged genomes. At the same time phage DNA directs the synthesis of many proteins required to produce empty heads where DNA is packed after the cleavage of concatemeric DNA at its cos site to yield fragments of such sizes as to fit in their heads. Eventually, a tail is attached to the head and finally the mature phage particles are released out the bacterial cell.

The lysogenic pathway for replication is another alternative mode of propagation where it becomes stably integrated into the host chromosome and replicated along with the bacterial chromosome. Phage genome integrates by an attachment site (att) with a partially homologous site on the E. coli chromosome, where it replicates as a chromosomal DNA segment. In this case a protein is produced by cl genes which represses all the genes responsible for lytic pathways. In this pathway no phage structural proteins are synthesized. The interactions of two proteins, the cl genes expressed protein (by phage genome) and cro gene expressed protein (by E.coli chromosomes) decide between the events of the lytic and lysogenic pathways. Phage lhas about 50 genes where only about 50% of these are necessary for growth and plaque formation.
Non-essential DNA sequence, therefore, is replaced by donor DNA and the inserted DNA propagated as phage lrecombinant with no detrimental to its replication or packaging.

Following are the advantages of phage cloning system over the plasmids: (i) DNA can be packed in vitro into phage particles and transduced into E. coli with high efficiency, (ii) foreign DNA upto 25 Kb in length can be inserted into phage vector, and (iii) screening and storage of recombinant DNA is easier (Dahl et al, 1981). Before using the phage λ as vector, it is essential to remove the genome from the restriction sites for the enzymes commonly used for cloning. The restriction sites are eliminated by mutation or deletion before obtaining an useful cloning vector. Two types of phage cloning vectors have been constructed : the insertion vector and the replacement vector. Cloning procedures of both the vectors are shown in Pigs. 3.6 and 3.7.

Insertion vector
Insertion vectors have unique cleavage site into which relatively small piece of foreign DNA is inserted. Foreign DNA fragment does not affect the function of phage. The upper and lower limits of size of DNA that may be packed into phage particles is between 35-53 Kb. Therefore, the minimum size of vector must be above 35 Kb. It means the maximum size of a foreign DNA to be inserted is about 18 Kb. Cloning of an insertion vector is shown in Fig. 3.6.

Replacement vector
The replacement vectors have cleavage sites present on either side of a length of non-essential DNA of phage. As a result of cleavage left and right arms are formed, each arm has a terminal cos site and longer a stuffer region, the non-essential region, which can be substituted by foreign DNA fragment Fig. 3.7.

The maximum size of inserted DNA fragment depends on how much of the phage DNA is non-essential. It has been found that about 25-30 Kb of genome codes for essential products for lytic cycle. The remaining 20-25 Kb of genome could be replaced with the foreign DNA fragments of known essential products. The substituted vectors are gt, WES and λ 1059 (Dahl et al., 1981).

Non-essential part of the λ genome can be separated from the arms by electrophoresis or velocity gradient ultracentrifugation that facilitates to make size differences. Formation of multiple inserts can be checked by using alkaline phosphatase before ligation with insert fragment. Recombinant DNA formed by multiple inserts has too large genome to be packed in viral head. However, after ligation the recombinant DNA molecules have left arm plus large insert plus right arm linked by their cos sites at the arms.
Optimum distance from cos sites governs efficiency for packaging in E. coli. As a result of ligation the size of recombinant DNA fragment may be less or more than the required size or may have more than two cos sites of many small fragments of foreign DNA. Those having the range of viral head can be packed in vitro using a preparation of head and tail proteins. The viruses thus constructed are allowed to multiply in E. coli. Development of plaque turbidity is a useful criteria for the selection of recombinant phages. Plaque turbidity is determined by the presence of nonlysed bacteria. The recombinant phages give clear plaques due to inactivation of cl gene. By using these methods the clones containing recombinant DNA can be isolated from the wild type clones i.e. turbid plaques. The constructed genome has all the informations required for DNA replication and synthesis of the viral protein. The inserts can be identified by colony hybridization technique as described in Techniques of Genetic Engineering.

Till now, phage DNA has been most widely used as phage cloning vector because of ease in handling and screening of large number of recombinant DNA containing phages. That is why it is used as a tool in the building of gene bank, for example, rat, yeast, mouse and human gene banks (Dahl et al, 1981).

Cosmids
Based on the properties of DNA and Col El plasmid DNA, a group of Japanese workers (Fukumaki et al., 1976) showed that the presence of a small segment of phage lDNA containing cohesive end on the plasmid molecule is a sufficient prerequisite for in vitro packaging of this DNA into infectious particles. The cosmids can be defined as the hybrid vectors derived from plasmids which contain cos site of phage λ. For the first time it was developed by Collins and Hohn (1978).

Cosmids lack genes encoding viral proteins; therefore, neither viral particles are formed with the host cell nor cell lysis occurs. Special features of cosmids similar to plasmids are the presence of (i) Wigin of replication, (ii) a marker gene coding for antibiotic resistance, (iii) a special cleavage site for the insertion of foreign DNA, and (iv) the small size.

Character dissimilar to plasmid is the presence of extra phage DNA, the cos site, which has about 12 bases. It helps the whole genome in circularization and ligation.

The cosmids have a length of about 5 Kb, the upper size limit of the foreign DNA fragment that may be inserted in cosmids and packed into phage particles is, therefore, approximately 45 Kb, much larger than it would be possible to clone in phage X or plasmid vector (Dahl et al., 1981). According to the size of cos sites and upper size limit in the head of phage, the recombinant DNA molecules can be packed into bacteriophage particles in in vitro packaging system consisting of packaging enzymes, head and tail proteins.

Procedure of DNA cloning by using cosmid vector is shown in Fig. 3.8. Upon infection of E. coli by bacteriophage, the recombinant DNA cyclizes through cos sites and then replicates as a plasmid and expresses the drug resistance marker. Recently, based on cosmid vectors, a number of cosmid vectors have been determined from E. coli, yeast, and mammalian cells, and gene bank has been constructed (Dahl et al., 1981; Grosveld et al. 1982). Moreover, other vectors which have been used in cloning are bacteriophage M13, virus SV40, Ti-plasmid, etc. but no one of them are useful for building gene bank. Ti -plasmid and SV40 have been described elsewhere (see section on Animal viruses).

Phasmids
The plasmids may be inserted into a phage λ genome, as a phage λ genome into the bacterial chromosome, during lysogenic cycle. Insertion of plasmid into phage λ is done with a view to have a specific site responsible for recombinational insertion of the phage into bacterial chromosome during lysogenic cycle. This insertion of plasmid into phage λ genome is reversible and referred to as 'lifting' the plasmid. It generates a phage genome containing att site and one or more plasmid molecule(s). These new genetic recombinations are called as phasmids (Brenner et al, 1982). The phasmids contain functional ori genes of plasmids and of phage λ. Moreover, they may be allowed to propagate as plasmid or phage in appropriate E. coli strains. Plasmids are released when reversal of lifting processes takes place.

Phasmids may be used in multifarious ways. They may be used as a phage cloning vector from which a recombinant plasmid may be released. Also phage particles are easy to store for a long time.