Cloning Vectors

 

Genetic engineering can be defined as transfer of DNA between host or species by in vitro enzymatic manipulations. This implies that the DNA to be transferred will be duplicated in the new host. Since, most DNA fragments are incapable of self-replications in host cell, an additional segment of DNA, capable of autonomous replication, must be linked to the fragment to be cloned. 

DNA molecule that has ability to replicate in an appropriate host cell, and into which the desired gene or  DNA insert is integrated are called molecular cloning vector or simply vector. These form a very important part of the tools of recombinant DNA technology as they are the ultimate vehicles that carry forward the desired gene into the host organism.

 Plasmids and bacteriophages are the most common vectors in recombinant DNA technology that are used as they have very high copy number. 

Vectors are of two types: 

Cloning vector is used increasing the number of copies of a cloned DNA fragment. 

Expression vector is used for expression of foreign gene into a protein. 

If a vector is designed to perform equally in two different hosts, it is called a shuttle vector. 

Properties of an ideal vector: 

- Low molecular weight

- Be easy to isolate and purify

- Be easily introduced into the host cells

- Be able to replicate autonomously i.e. should have ori (origin of replication) region. This is a sequence which is recognized by the host cell’s replication machinery and from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA.

- Contain unique target sites for restriction enzymes into which the DNA insert can be integrated

- Selectable marker : In addition to ‘ori’, the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the recombinant. E.g., gene for antibiotic resistance.

- When expression of the DNA insert is desired, the vector should contain suitable regulatory elements – promoter, operator, ribosome binding sites

- Have multiple cloning sites - the sites recognized by the restriction enzymes where desired DNAs are inserted.

- High copy number The selection of a suitable vector system depends mainly on the size limit of                 insert DNA and the type of host intended for cloning or expression of foreign DNA.

Plasmid DNA as a vector

Plasmids are naturally occurring extrachromosomal double-stranded circular DNA molecules that carry

an origin of replication and replicate autonomously within bacterial cells. The plasmid vector pBR322, constructed in 1974, was one of the first genetically engineered plasmids to be used in recombinant DNA. 

Cutting the circular plasmid vector with restriction enzymes results in a single cut, creating a linear DNA molecule.

A foreign DNA molecule, cut with the same enzyme can now be inserted, by joining the ends of broken circular DNA molecule to the two ends of foreign DNA, thus regenerating a bigger circular DNA molecule.

 Ligations of the insert to vector are not 100% productive, because the two ends of a plasmid vector can be readily ligated together, which is called self-ligation. The degree of self-ligation can be reduced by treatment of the vector with the enzyme phosphatase, which removes the terminal 5′-phosphate. When the 5′-phosphate is removed from the plasmid it cannot be recircularized by ligase, since there is nothing with which to make a phosphodiester bond. 

But, if the vector is joined with a foreign insert, the 5′-phosphate is provided by the foreign DNA.

Plasmids are named with a system of uppercase letters and numbers, where the lowercase“p” stands for “plasmid.” In the case of pBR322, the BR identifies the original constructors of the vector (Bolivar and Rodriquez), and 322 is the identification number of the specific plasmid.

 These early vectors were often of low copy number, meaning that they replicate to yield only one or two copies in each cell. pUC18 is a derivative of pBR322. This is a “high copy number” plasmid (500 copies per bacterial cell).

Plasmid vectors are modified to contain a specific antibiotic resistance genes, which plasmid carry against one or more antibiotics. If a plasmid has two such genes conferring resistance against two antibiotics and if the foreign DNA insertion site lies within one of these two genes, the chimeric or recombinant vector loses resistance against one antibiotic, the gene for which has foreign DNA inserted within its structure.

In such a situation, the parent vector in bacterial cells can be selected by resistance against two antibiotics and the chimeric DNA can be selected by retention of resistance against only one of the two antibiotics.

They have multiple cloning site (also called the polylinker region) which has a number of unique target sites for restriction endonucleases.

pBR322 and pBR327 vectors

The naturally occurring plasmids may not possess all the above and other essential properties of a suitable cloning vector. Therefore, one may have to restructure them by inserting genes of relaxed replication and genes for antibiotic resistance. One of the standard cloning vectors widely used in gene cloning experiments is pBR322 (derived from E. coli plasmid ColE1), which is 4,362 bp DNA and was derived by several alterations in earlier cloning vectors (pBR322 was named after Bolivar and Rodriguez, who prepared this vector).

pBR322 is an artificial plasmid,which is regarded as the  ‘work house’ of gene cloning laboratory. It was constructed by using three different naturally occurring plasmids. The ampicillin resistance gene was derived from RSF 2124. The tetracycline resistance gene was taken from pSC 101. The origin of replication was obtained from pMB1 which is related to plasmid CoIE.

 It has genes for resistance against two antibiotics (tetracycline and ampicillin), an origin of replication and a variety of restriction sites for cloning of restriction fragments obtained through cleavage with a specific restriction enzyme. 

The plasmid is much smaller than a natural plasmid, and this makes it more resistant to damage by shearing, and increases the efficiency of uptake by bacteria during transformation. Since this plasmid is small, foreign DNA upto 6kb in length can be inserted. 

Another vector pBR327 was derived from pBR322, by deletion of nucleotides between 1,427 to 2,516. 

These nucleotides are deleted to reduce the size of the vector and to eliminate sequences that were known to interfere with the expression of the cloned DNA in eukaryotic cells. pBR327 still contains genes for resistance against two antibiotics (tetracycline and ampicillin). Both pBR322 and pBR327 are very common plasmid vectors.

pUC vectors

 Another series of plasmids that are used as cloning vectors belong to pUC series (after the place of their initial preparation ie. University of California). These plasmids are 2,700 bp long and possess,(i) ampicillin resistance gene, (ii) the origin of replication derived from pBR322 and (iii) the lac Z gene derived from E. coli. 

Within the lac region is also found a polylinker sequence having unique restriction sites identical to those found in phage M13.

When DNA fragments are cloned in this region of pUC, the lac gene is inactivated. These plasmids when transformed into an appropriate E. coli Strain, which is lac(eg. JM103, JM109), and grown in the presence of PTG (isopropyl thiogalactoside, which behaves like lactose, and induces the syhthesis of f galactosidase enzyme) and X-gal (substrate for the enzyme), will give rise to white or clear colonies. 

On the other hand,pUC having no inserts and transformed into bacteria will have an active lac Z gene and therefore will produce blue colonies, thus permitting identification of colonies having pUC vector with cloned DNA segments.

 Cloning vectors belonging to pUC family are available in pairs reversed orders of restriction sites relative to lac Z promoter. pUC9 make one such pair. Other similar pairs include pUC12 and pUC13 or pUC18 and pUC19.

Another strategy involves using two different restriction endonuclease cutting sites with non- complementary sticky ends. This inhibits self-ligation and promotes annealing of the foreign DNA in the desired orientation within the vector.

Bacteriophages as Vectors

Bacteriophages provide another source of cloning vectors.  Phage vectors such as those derived from bacteriophage λ can carry larger inserts and can be introduced into bacteria more efficiently. Recombinant bacteriophage can be introduction into E. coli by infection.

 Bacteriophage lambda (λ) has been widely used in recombinant DNA since engineering of the first viral

cloning vector in 1974. λ phage has a duplex DNA genome of about 50 kb. The internal 20 kb can be replaced with foreign DNA and still retain the lytic functions.  Hence restriction fragments up to 20 kb can replace the λ sequences, allowing larger genomic DNA fragments to be cloned and useful for preparing genomic libraries.

Today many variations of λ vectors exist. Insertion vectors have unique restriction endonuclease sites that allow the cloning of small DNA fragments in addition to the phage genome. These are often used for preparing cDNA expression libraries.

Replacement vectors have paired cloning sites on either side of a central gene cluster. This central cluster contains genes for lysogeny and recombination, which are not essential for the lytic life cycle.

The central gene cluster can be removed and foreign DNA inserted between the “arms.” 

All phage vectors used as cloning vectors have been disarmed for safety and can only function in special laboratory conditions.

The recombinant viral particle infects bacterial host cells, in a process called “transduction.” The host cells lyse after phage reproduction, releasing progeny virus particles. The viral particles appear as a clear spot of lysed bacteria or “plaque” on an agar plate containing a lawn of bacteria. Each plaque represents progeny of a single recombinant phage and contains millions of recombinant phage particles. Most contemporary vectors carry a lacZ′gene allowing blue-white selection.

Some other bacteriphage vectors for cloning are derived from the virus M13. One can obtain single stranded DNA from M13 vectors and recombinants. M13 is a virus with a genome of single stranded DNA. It has a nonessential region into which foreign genes can be inserted. It has been modified to carry a gene for  galactosidase as a way to screen for recombinants. Introduction of recombinant M13 DNA into E. coli will lead to an infection of the host, and the progeny viral particles will contain single stranded DNA. The replicative form is duplex, allowing one to cleave with restriction enzymes and insert foreign DNA.

 Vectors designed to carry larger inserts

 Fragments even larger than those carried in λ vectors are useful for studies of longer segments of chromosomes or whole genomes. Several vectors have been designed for cloning these very large fragments, 50 to 400 kb.

Cosmids are hybrid DNA molecules and can live a dual life. Cosmids are  plasmids that have the cohesive ends of λ phage.  They can be packaged in vitro into infective phage particles to give a more efficient delivery of the DNA into the cells.  They can carry about 35 to 45 kb inserts.

Their plasmid part enables them to replicate as it has origin of replication. Plasmid part also helps in selection due to the presence of marker gene. Cleavage site is located in the marker gene.

Their λ part or cos sequences allows them to be packaged in a phage coat and to be transduced to a recipient by the λ infection machinery. It has no genes for viral proteins, therefore, viral particles are not formed in the host. Host cell lysis is also absent. 

Yeast artificial chromosomes (YACs) are yeast vectors with  centromeres and telomeres. They can carry about 200 kb or larger fragments (in principle up to 1000 kb = 1 Mb).  Thus very large fragments of DNA can be cloned in yeast. In practice, chimeric clones with fragments from different regions of the genome are obtained fairly often, and some of the inserts are unstable.

Some vectors are hybrids between plasmids and single strand phage; these are called phagemids. One example is pBluescript. Phagemids are plasmids (with the modified, high-copy number ColE1 origin) that also have an M13 origin of replication. Infection of transformed bacteria (containing the phagemid) with a helper virus (e.g. derived from M13) will cause the M13 origin to be activated, and progeny viruses carrying single stranded copies of the phagemid can be obtained.  Hence one can easily obtain either double  or single stranded forms of thes plasmids. {The "blue" comes from the blue white screening for recombinants that can be done when the multiple cloning sites are in the  galactosidase gene. The "script" refers to the ability to make RNA copies of either strand in vitro with phage RNA polymerases.}

Artificial chromosome vectors

Bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs) are important tools for mapping and analysis of complex eukaryotic genomes. Much of the work on the Human Genome Project and other genome sequencing projects depends on the use of BACs and YACs, because they can hold greater than 300 kb of foreign DNA. BACs are constructed using the fertility factor plasmid (F factor) of E. coli as a starting point. The plasmid is naturally 100 kb in size and occurs at a very low copy number in the host.

 The engineered BAC vector is 7.4 kb (including a replication origin, cloning sites, and selectable markers) and thus can accommodate a large insert of foreign DNA. The characteristics of YAC vectors are discussed below.

Immediately after the construction of the first YAC in 1983, efforts were undertaken to develop a mammalian artificial chromosome (MAC). Like YACs, MACs rely on the presence of centromeric sequences, sequences that can initiate DNA replication, and telomeric sequences. Their development is considered an important advance in animal biotechnology and human gene therapy for two main reasons. First, they involve autonomous replication and segregation in mammalian cells, as opposed to random integration into chromosomes (as for other vectors).

 Second, they can be modified for their use as expression systems of large genes, including not only the coding region but all control elements. A major drawback limiting application at this time, however, is that they are difficult to handle due to their large size and can be recovered only in small quantities. Two principal procedures exist for the generation of MACs. In one method, telomere-directed fragmentation of natural chromosomes is used. For example, a human artificial chromosome (HAC) has been derived from chromosome 21 using this method. Another method involves de novo assembly of cloned centromeric, telomeric, and replication origins in vitro.

Yeast artificial chromosome (YAC) vectors

Yeast, although a eukaryote, is a small single cell that can be manipulated and grown in the lab much like bacteria. YAC vectors are designed to act like chromosomes. Their design would not have been possible without a detailed knowledge of the requirements for chromosome stability and replication, and genetic analysis of yeast mutants and biochemical pathways. YAC vectors include an origin of replication (autonomously replicating sequence, ARS), a centromere to ensure segregation into daughter cells, telomeres to seal the ends of the chromosomes and confer stability, and growth selectable markers in each arm.

 These markers allow for selection of molecules in which the arms are joined and which contain a foreign insert. For example, the yeast genes URA3 and TRP1 are often used as markers. Positive selection is carried out by auxotrophic complementation of a ura3-trp1 mutant yeast strain, required for the biosynthesis of the nitrogenous base uracil (orotidine-5′-phosphate decarboxylase).

 TRP1 encodes an enzyme that is required for biosynthesis of the amino acid tryptophan (phosphoribosylanthranilate isomerase). YAC vectors are maintained as a circle prior to inserting foreign DNA. After  cutting with restriction endonucleases BamHI and EcoRI, the left arm and right arm become linear, with the end sequences forming the telomeres. Foreign DNA is cleaved with EcoRI and the YAC arms and foreign DNA are ligated and then transferred into yeast host cells. 

The yeast host cells are maintained as spheroplasts (lacking yeast cell wall). Yeast cells are grown on selective nutrient regeneration plates that lack uracil and tryptophan, to select for molecules in which the arms are joined bringing together the URA3 and TRP1 genes.