Vehicles for gene cloning

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Vehicles for gene cloning

1)Introduction 2)Properties expected 3)Plasmids and bacteriophages as vehicles 3.1)Plasmids 3.1.1)Integrative and non integrative plasmids 3.1.2)Size and copy numbers 3.1.3)Plasmid classification 3.2)Baceriophages 3.2.1)Basic features of bacteriophages 3.2.2)Bacteriophage types 3.2.3)Vectors based on bacteriophages 3.2.3.1).Bacteriophage lamda 3.2.3.2)M13 vector 4)Cosmids 5)Fosmids 6)Artificial chromosomes 6.1)Bacterial artificial chromosome 6.2)Yeast artificial chromosome 6.3)Human artificial chromosome

A vehicle also known as vector is basically an intentionally designed DNA molecule that helps in the replication and/or expression of a DNA molecule of interest which has been inserted in it. It does so by carrying along the attached DNA molecule into a system where replication and/or expression of this assembly(recombinant DNA molecule) can take place.

Minimal properties expected: • The vehicle must be able to autonomously replicate within the host cell so that numerous copies of the recombinant DNA molecule can be produced and passed on to the daughter cells. • A cloning vehicle must be relatively small( ideally less than 10kb in size) as large molecules present problems during purification and manipulation. • It must have multiple restriction sites where the gene fragment of interest can be readily inserted. • It must also have a couple of marker genes that can help in the identification of the cells containing this plasmid and more importantly those that do not contain it

PLASMIDS AND BACTERIOPHAGES as vehicles:

plasmids and BActeriophages are two types of DNA entities that that satisfy the above criteria and hence are widely used as vehicles in gene cloning.

PLASMIDS: plasmids are extra chromosomal circular DNA molecules separated from chromosomal DNA and capable of independent transmission and replication.

Plasmid size varies from 1 to over 400 kilobase pairs (kbp). All plasmids possess at least one DNA sequence that can act as an origin of replication, so that they are able to multiply within the cell quite independently of the main bacterial chromosome. The smaller plasmids make use of host cells own DNA replicative enzymes in order to make copies of themselves whereas some of the larger ones carry genes that code for special enzymes that are specific for plasmid replication.

Integrative and non integrative plasmids:

Integrative plasmids:some plasmids replicate themselves by inserting themselves into the bacterial chromosome these are called integrative plasmids or Episomes.

NON integrative plasmids: These plasmids bring about their replication independent of the bacterial chromosome without getting integrated into it.

SIZE AND COPY NUMBER OF PLASMIDS:

The size and copy number of plasmids are particularly important as far as cloning is concerned. Plasmid size of less than 10kb is desirable for a cloning vehicle. Plasmids in the range from 1.0kb to 250kb have been frequently used as cloning vehicles.

The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell.each plasmid has a characteristic value that may be as low as one ( especially for large molecules ) or as many as 50 or more. To be a useful cloning vector the plasmid needs to have a high copy number so that large quantities of Recombinant DNA molecule can be obtained.

Plasmid classification

Plasmids are classified on the basis of the main characteristics coded by plasmid genes. Five main types of plasmid according to this classification are: 1)Fertility or F plasmids : carry only 'tra' genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids, eg- F plasmid of E.coli 2) Resistance or R Plasmids- carry genes conferring on the host bacterium resistance to one or more antibacterial agents like chloramphenicol, ampicillin etc. 3)col plasmids- code for colicins, proteins that kill other bacteria, eg- coLE1 of E.coli 4) Degradative plasmids - allow host bacterium to metabolize unusual molecules such as toluene and salicylic acid eg-TOL of pseudomonas putida 5) Virulence plasmids- confer pathogenecity to the host bacterium eg-Ti plasmids of Agrobacterium tumefacians

Bacteriophages

Basic features of Bacteriophages. bateriophages are viruses that specifically infect bacteria. phages are very simple in structure and consist merely of a DNA ( or occasionally Ribonucleic acid(RNA)) molecule carrying a number of genes, including several for replication of the phage surrounded by a protective coat called capsid made up of protein molecules.

Bacteriophage types: There are two main types of bacterial viruses: Lytic or virulent and temperate or avirulent. LYTIC PHAGE: When lytic phage infects cells, the cells respond by producing large number of new viruses which at the end of the incubation period lyse the cell causing it to burst releasing new phages to infect other host cells. This is called lytic cycle.

Temperate or avirulent phage:

In the temperate type of infection the result is not so apparent. The viral nucleic acid is carried and replicated  in the host bacterial cells from one generation to another without any cell lysis. However temperate phages may spontaneously become virulent at some subsequent generation and  lyse the cells. 

Vectors based on bacteriophages

Bacteriophages are viruses that infect bacterial cells by injecting their DNA into these cells. The injected DNA is selectively replicated and expressed in the host bacterial cell resulting in a number of phages which burs out and of the cell ( lytic pathway) and reinfect other neighbouring cells. This ability to transfer DNA from the phage genome to the specific bacterial hosts during the process of bacterial infection is the underlying basis for using bacteriophages as useful vectors for gene cloning experiments.

Phages being used as vectors: Two phages namely Lamda and M13 have been extensively modified for the development of cloning vectors. Bactreiophage Lamda : Has a double stranded, linear DNA genome of 48,514 bp, in which the 12 bases at each end are unpaired but complimentary. These ends are sticky or cohesive ends and are referred to as cos sites (cohesive end sites). These sites are important for packaging DNA into phage heads. An important feature of of the Y genome is that a large fragment in the central region of its genome is not essential for lytic infection of E.coli cells, and hence it can be replaced by a foreign DNA. Foreign DNA upto 23kb in size can be inserted in the place and hence cloned.

M13 phage: this is a filamentous phage which infects E.coli having F pilli, as it enters E.coli through the F.pillus. Its genome is a single stranded circular DNA of size 6047bp. Foreign DNA can be inserted into it without disrupting any of the essential genes. After the M13 phage enters the bacterial cell, it is converted to a double stranded molecule known as the replicative form, which replicates until there are about 100 copies in the cell. At this point DNA replication becomes asymmetric and single stranded copies of the genome are produced and extruded out of the cells as M13 particles. The major advantages for developing vectors based on M13 are that :

• Its genome is less that 10kb in size . • The replicative form can be purified and manipulated exactly like a plasmid. • Genes cloned in M13 vectors can be obtained in the form of single stranded DNA. Single stranded forms of cloned genes are useful for various techniques , including DNA sequencing and site directed mutagenesis. Cosmids: Cosmids have been constructed by combining certain features of plasmid and the ‘cos’ sites of phage lambda. The simplest cosmid vector contains a plasmid origin of replication, a selectable marker, suitable restriction enzyme sites and the lamda ‘cos’ site. Cosmids can be used to clone DNA fragments upto 45kb in length.

FOSMIDS: Fosmids are similar to cosmids but are based on the bacterial F-plasmid. The cloning vector is limited, as a host (usually E. coli) can only contain one fosmid molecule. Low copy number offers higher stability than comparable high copy number cosmids. Fosmid system may be useful for constructing stable libraries from complex genomes vector containing the single copy E.coli F-factor replicon, developed as an improved method for constructing libraries of cosmid-sized (approximately 40 Kb) clones. The stability of inserts cloned into fosmid vectors has been shown to be substantially greater than in high copy vectors. Copy control fosmids, e.g. pCC1fos, contain both the E. coli F-factor replicon and the oriV high copy origin of replication, thus providing the user the clone stability afforded by single-copy fosmid cloning and the high yields of DNA that can be realized from cosmid clones.

Artificial chromosomes as vectors: Bacterial artificial chromosome:

Bacterial artificial chromosomes : are vectors based on the natural, Extra chromosomal plasmid of E.coli, the Fertility or F-plasmid. A bacterial artificial chromosome vector contains genes for replication and maintenance of the F factor, a selectable marker and cloning sites. The BAC vectors can accommodate unto 300-350kb of foreign DNA and are also being used in genome sequencing projects.

Yeast artificial chromosomes:

A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb). It is an artificially constructed chromosome and contains the telomeric, centromeric, and replication origin sequences needed for replication and preservation in yeast cells. Built using an initial circular plasmid, they are linearised by using restriction enzymes, and then DNA ligase can add a sequence or gene of interest within the linear molecule by the use of cohesive ends.

Human artificial chromosome: A human artificial chromosome (HAC) is a microchromosome that can act as a new chromosome in a population of human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10 megabases in size, and able to carry new genes introduced externally.