Vectors

Vector is an agent that can carry a DNA fragment into a host cell in which it is capable of replication. If it is used only for reproducing the DNA fragment, it is called a cloning vector. If it is used for expression of foreign gene, it is called an expression vector. Properties of a good vector: 1) It should be autonomously replicating i.e. it should have ori region. 2) It should contain at least one selectable marker e. g. gene for antibiotic resistance. 3) It should have unique restriction enzyme site (only one site for one RE) for different REs to insert foreign DNA. 4) It should be preferably small in size for easy handling. 5) It should have relaxed control of replication so that multiple copies can be obtained. Vectors are of different types depending on the host. These are as follows: 1) 2) 3) 4) Bacterial vectors Yeast vectors Plant vectors Animal vectors

Bacterial vectors
E.coli is the most commonly used bacterium for gene cloning though other bacteria such as Bacillus are also used. Vectors for cloning in these bacteria are described below: Vectors for cloning in E.coli A number of vectors are used for cloning in E.coli. These are categorized as plasmids, phages, cosmids, phagemids and bacterial artificial chromosomes. I. Plasmid vectors Plasmids are autonomously replicating circular, double stranded DNA molecules found in bacteria. They have their own origin of replication (ori region), and can replicate independently of the host chromosome. The size of plasmids ranges from a few kb to 200 kb. Plasmid vectors are often used for cloning DNA segments of small size (upto 10 kilobases). Some of the commonly used plasmid vectors are described below: a. pBR322 The first plasmid vector that has been constructed artificially is pBR322. It is named after the scientists Bolivar and Rodriguiz who constructed it in 1977. It is 4362bp in size. It has an origin of replication derived

from a colicin-resistance plasmid (ColE1). This origin allows a fairly high copy number, about 100 copies of the plasmid per cell. Plasmid pBR322 carries two selectable markers viz. genes for resistance to ampicillin (Apr) and tetracycline (Tcr). Several unique RE sites are present within these genes for insertion of foreign DNA (Fig 1). When a foreign DNA segment is inserted in any of these genes, the antibiotic resistance by that particular gene is lost. This is called insertional inactivation. For instance, insertion of a restriction fragment in the SalI site of the Tcr gene inactivates that gene. One can still select for Apr colonies, and then screen to see which ones have lost Tcr. b. pUC A series of small plasmids (about 2.7 kb) have been developed at the University of California and hence the name pUC e.g. pUC7, 8, 18 and 19 etc. (Fig 2). These are high copy number plasmids that carry an ampicillin resistance gene and an origin of replication, both from pBR322. They also have a multiple cloning site (MCS) a sequence of DNA that carries unique sites for many REs. The MCS contains a portion of lacZ gene that codes for the enzyme -galactosidase. When such plasmids are introduced into E. coli, the colonies are blue on plates containing X-gal (substrate for - galactosidase) and IPTG (isopropyl thiogalactoside, an inducer). When a foreign DNA is introduced in MCS, the -galactosidase activity is lost. Thus cells containing recombinant plasmids form white (not blue) colonies. II. Phage vectors Bacteriophages or phages are viruses that specifically infect bacteria. The phage particle attaches to the outer surface of bacterium and injects its DNA into the cell. The phage DNA is then replicated inside the host and its genes are expressed to make phage capsid proteins and new phage particles are assembled and released from the bacterium. Phage vectors can accommodate more DNA (upto 25 kb) than plasmids and are often used for preparation of genomic libraries. They also have higher transformation efficiency as compared to plasmids. Two bacteriophages namely, Lambda () and M13 have been commonly used for construction of vectors for cloning in E. coli. a. Lambda () phage vectors Lambda is a temperate bacteriophage with a genome size of 48.5 kb. Its entire DNA sequence is known. The lambda genome is a linear, double-stranded molecule with single-stranded, complementary ends. These ends can hybridize with each other (and do so when the DNA is within an infected cell) and are thus termed cohesive (cos) sites. The phage can have two modes of life cycles i.e. lytic and lysogenic. During lytic cycle, it replicates independently in the host cell and produces a large number of phage particles which are released by lysis of the host. Alternatively, it can take up lysogenic growth, meaning that it integrates its DNA into the bacterial chromosome and multiplies along with it. Two types of vectors have been constructed from

lambda phage. These are insertional and replacement vectors (Fig 3). Insertional vectors have one unique restriction site for a particular restriction enzyme and can accommodate 6-7 kb DNA. Examples of insertional vectors are gt10, gt11 and ZAP II. On the other hand, replacement vectors have two cleavage sites for a restriction enzyme and can accommodate up to 25 kb DNA. When vector is cut with a restriction endonuclease, a stuffer fragment is removed and replaced with a foreign DNA. Some examples of replacement vectors are EMBL3, EMBL3A, EMBL4, DASH, FIX, GEM11 and GEM12. Bacteriophage lambda can be reconstituted in a test tube by simply mixing phage DNA with a mixture of phage proteins, an infective viral particle with the DNA inside the phage head can be produced. This process is called in vitro packaging. There is a strict size requirement for the piece of DNA that goes into the phage head. That is, it should not be more than 52 kb and less than 38 kb. This feature allows only the recombinants to be packaged inside the phage head. In addition, some lambda phage vectors have a stuffer fragment that carries the -galactosidase gene. When it is removed or when foreign DNA is cloned within the gene, -galactosidase activity may be abolished. The accompanying loss of activity may be used to select recombinant clones. b. M13 Phage vectors M13 is a filamentous bacteriophage of E. coli and contains a single stranded circular DNA of 7.2 kb. A series of vectors (M13 mp series) have been developed from this phage. These vectors have a polylinker with unique restriction enzyme sites in lac Z gene that complements host (e.g. JM 103 or JM 104). Screening of recombinants is done based on formation of blue/white plaques. M13 vectors are used for obtaining sufficient quantity of DNA for sequencing by Sanger's dideoxy chain termination method. III. Phagemids Phagemids are hybrid vectors derived from plasmids and phages e.g. pBluescript. They contain origin of replication from M13 or F1 phage and remaining features of the vector from plasmids. 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 singlestranded copies of the phagemid can be obtained. IV. Cosmids The cosmid vector is a combination of the plasmid and bacteriophage lambda. It is small (5-7 kb) circular DNA containing an origin for DNA replication (ori), selectable markers and restriction sites from plasmid plus a sequence from lambda needed for packaging the DNA (cos site). Cosmids may be used to clone large DNA molecules of up to 45 kb. They also have high

transformation efficiency. Some examples of cosmid vectors include pJB, PWE and SuperCos series (Fig 4). V. Bacterial artificial chromosomes (BAC) BACs are based on bacterial mini-F plasmids, which are small pieces of episomal bacterial DNA that give the bacteria the ability to initiate conjugation with adjacent bacteria. They have a cloning capacity of 75300 kb. These have a lower copy number (like F) but are stable and relatively easy to work with. BACs have become one of the most frequently used vectors for preparation of genomic libraries. i. Vectors for cloning in Bacillus Most strains of Bacillus contain extra chromosomal DNA molecules called plasmids. However, plasmids present in Bacillus are cryptic which are devoid of any selectable markers. Ehrlich (1977) observed that plasmids of Staphylococcus aureus were able to replicate in Bacillus subtilis. So most of the plasmid vectors used for cloning in B.subtilis are derived from S.aureus plasmids. As none of the natural S.aureus plasmids carries more than one selectable marker, so improved vectors have been constructed by gene manipulation, e.g. PHV11.It has been derived from pC194 and carries TcR gene of pT127. Because of difficulties in direct cloning in B.subtilis hybrid plasmids called shuttle vectors (vectors that can multiply in two different hosts) have been constructed so that they can be used for cloning in E.coli as well as B. subtilis. E.coli replicon required for development of shuttle vectors has been obtained from pBR322. Additional antibiotic resistance genes or selectable markers derived from other S.aureus plasmids, chromosomal DNA of other strains of Bacilli or from pBR322 have also been incorporated. Some examples of shuttle vectors that can multiply both in Bacillus and E.coli are pHV 33, pLB5 and pPL603. ii. Vectors for cloning in yeast The discovery of a 2m plasmid in most strains of Saccharomyces cerevisiae led to the development of cloning vectors in yeast. The 2m plasmid is 6 kb in size. It is present in 50-100 copies per cell. A number of shuttle vectors based on 2m plasmid and bacterial plasmids have been constructed which can replicate either in E.coli or yeast. Yeast plasmid vectors are of four types, yeast episomal plasmids (YEps), yeast integrative plasmids (YIps) yeast replicative plasmids (YRps) and yeast centromeric plasmids (Ycps). In addition to plasmid vectors, yeast artificial chromosomes

(YACs) are also used as vectors for cloning large pieces of DNA. A brief description of these vectors is given below: a. Yeast episomal plasmids (YEps) These are derived from 2m plasmid. Some YEps contain the entire 2m plasmid; others include just the 2m origin of replication. An example of latter type is YEp13 (Fig 5). It is a shuttle vector and can be replicated both in E.coli and yeast. It contains 2m origin of replication, yeast gene leu2 as selectable marker and entire sequence of pBR322.The leu2 gene codes for an enzyme involved in biosynthesis of amino acid leucine. YEps may replicate autonomously or integrate in one of the yeast chromosomes by homologous recombination. They have high transformation frequency of 10,000 to 100,000 transformants/ g DNA. b. Yeast integrative plasmids (YIps) These are basically bacterial plasmids carrying a yeast gene. YIp5 is an example of yeast integrative plasmid. It has ura3 gene inserted in pBR322. The gene ura3 codes for an enzyme involved in biosynthesis of pyrimidine nucleotides and acts as selectable marker. The plasmid cannot replicate autonomously as it lacks 2m origin of replication and survives by integrating in yeast chromosomal DNA. They have very low transformation frequency, less than 100 transformants/ g DNA. c. Yeast replicative plasmids (YRps) They carry a part of chromosomal DNA with an origin of replication and one or two selectable markers and are capable of independent replication. They have transformation frequency between 1000 and 10,000 transformants/ g DNA. d. Yeast centromeric plasmids (YCps) These are shuttle vectors that behave as small chromosomes and replicate only once during each cell divison. They contain; i) Origin of replication called ARS sequence ii) CEN sequence (for proper segregation of chromosomes) iii) A selectable marker such as leu2 from yeast and sequences from bacterial plasmid having ori region and selectable marker (Apr). They are stably maintained at one copy per cell. e. Yeast Artificial Chromosomes (YACs) YACs are artificial chromosomes that replicate in yeast cells. Main features of these vectors are: 1. Autonomously replicating sequence (ARS) necessary for the replication in yeast cells (Fig 6). 2. Telomeres (TEL), which are ends of chromosomes involved in the replication and stability of chromosomes. 3. A yeast centromere (CEN), required for proper segregation of chromosomes 4. Selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome.

5. Unique RE sites. YACs are capable of carrying a large DNA fragment (up to 3000 kb), but their transformation efficiency is very low.