VECTORS: TYPES AND CHARACTERISTICS E.g. PLASMIDS, PHAGES, HYBRID VECTORS, ARTIFICIAL CHROMOSOMES

VECTORS Small DNA molecule capable of self replication. Cloning vehicle or Cloning DNA. Carrier of DNA fragment. E.g. Plasmid, Phage, Hybrid vector, Artificial Chromosomes. a vector is a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and/or expressed. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes.

CHARACTERISTICS Self replication, multiple copies. Replication origin site. Cloning site. Selectable marker gene. Small size. Low molecular weight. Easily isolated & purified. Easily introduced into host cell. Control elements – promoter, operator, ribosome binding site.

TYPES Cloning Vectors Two types :- Propagation or cloning of DNA insert inside a suitable host cells. Examples: Plasmids, Phage or Virus Obtaining millions of copies. Uses :- Genomic library. Preparing probes. Genetic Engineering Experiments. Selection of cloning vector depends on :- (a) Objective of cloning experiment (b) Ease of working. (c) Existing Knowledge about the vector. (d) Suitability. (e) Reliability.

Express the DNA insert producing specific protein. Expression Vectors Express the DNA insert producing specific protein. They have prokaryotic promoter. Ribosome binding site. Origin of replication. Antibiotic resistance gene. Expression vectors with strong promoters. Inducible Expression Vectors. Eukaryotic expression vectors. Expression vectors produce proteins through the transcription of the vector's insert followed by translation of the mRNA produced, they therefore require more components than the simpler transcription-only vectors. Expression in different host organism would require different elements, although they share similar requirements, for example a promoter for initiation of transcription, a ribosomal binding site for translation initiation, and termination signals.

VECTOR TARGET HOST CELL Plasmid Bacteriophages Cosmid Yeast Cloning Vectors Ti & Ri Plasmids Bacteria, Streptomyces Bacteria Yeasts Transformation of cloned gene in higher plants. Ti: tumor induced plasmid Ri: Roots induced plasmid

AGENTS USED AS VECTORS PLASMIDS BACTERIOPHAGES COSMID ARTIFICIAL CHROMOSOME VECTORS In 1973, Cohen described first successful construction of recombinant vector. Plasmid PSC101 - Ecoli

PLASMID Extra chromosomal DNA molecules. Self replicating. Double stranded. Short sequence of DNA. Circular DNA molecules. Found mainly in prokaryotes. CHARACTERISTICS a. Minimum amount of DNA. b. Two suitable markers for identification . c. Single restriction site. d. More restriction enzyme. e. Size range 1kbp – 1000kbp. f. Relaxed replication control. g. Restriction endonuclease enzyme. Plasmids are double-stranded and generally circular DNA sequences that are capable of automatically replicating in a host cell. Plasmid vectors minimalistically consist of an origin of replication that allows for semi-independent replication of the plasmid in the host. Plasmids are found widely in many bacteria, for example in Escherichia coli, but may also be found in a few eukaryotes, for example in yeast such as Saccharomyces cerevisiae

PLASMID classification There are five main classes: 1.Fertility F-plasmids, which contain tra genes. They are capable of conjugation. 2.Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or poisons. 3.Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria. 4.Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid. 5.Virulence plasmids, which turn the bacterium into a pathogen. Another way to classify plasmids is by function. There are five main classes: Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pilli. Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood. Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria. Degradative plasmids, which enable the digestion of unusual substances, e.g. toluene and salicylic acid. Virulence plasmids, which turn the bacterium into a pathogen. Plasmids can belong to more than one of these functional groups

EXAMPLES OF PLASMID VECTORS pBR322 pBR327 pBR325 pBR328 pUC8 pUC9 pUC12 pUC13 pGEM3Z

Structure of E.Coli plasmid cloning vector pBR322

PBR322 Cloning Vector. 15 copies. Reconstructed plasmid. Derived from Ecoli plasmid- ColE1. PBR322 – 4362 base pairs P – denotes Plasmid B – Scientific Boliver R - Rodriguez 322 – number given to distinguish. Ampilicin resistance gene derived from RSF2124. Tetracycline resistance gene from PSC101. Origin of replication from PMB1. Two selectable markers ampr tetr

pUC8 Popular Ecoli cloning vector. Derivative of pBR322. Two parts derived:- Ampicillin resistance gene. ColEI – origin of replication. 2686 base pairs. lac Z gene derived from Ecoli. Polylinker sequence having unique restriction sites lies in lac region. lac operon (lactose operon) is an operon required for the transport and metabolism of lactose in Escherichia coli and many other enteric bacteria. Although glucose is the preferred carbon source for most bacteria, the lac operon allows for the effective digestion of lactose when glucose is not available. Gene regulation of the lac operon was the first genetic regulatory mechanism to be understood clearly, so it has become a foremost example of prokaryotic gene regulation. when lactose is required as a sugar source for the bacterium, the three genes of the lac operon can be expressed and their subsequent proteins translated: lacZ, lacY, and lacA. The gene product of lacZ is β-galactosidase which cleaves lactose, a disaccharide, into glucose and galactose. LacY encodes lactose permease, a protein which becomes embedded in the cytoplasmic membrane to enable transport of lactose into the cell. Finally, lacA encodes galactoside O-acetyltransferase.

PBR327 Derived from PBR322. 30 – 40 copies. Expression plasmid. Ampilicin resistance gene. Tetracycline resistance gene. 4.3 Kbp

PHAGE Cloning large DNA fragmance. Linear Phage molecule. Efficient than plasmid. Used in storage of recombinant DNA. Commonly used Ecoli phages :- λ phage M13 Phage

BACTERIOPHAGE VECTORS Cloning Vectors. It infects bacteria. Commonly used Ecoli phages :- λ phage M13 Phage Lambda phage vector Genome size is 48,502 bp. High transformation efficiency. 1000 times more efficient than the plasmid vector. Origin of replication. Genome linear in head. Single- stranded protruding cohesive ends of 12 bases. Cos site – site of cleavage of phage DNA. The genome contains linear DNA, with 12-base single-strand segments at both 5' ends. These two single-stranded segments are the "sticky ends" of what is called the cos site. The cos site circularizes the DNA in the host cytoplasm. In its circular form, the phage genome, therefore, is 48,502 base pairs in length. The lambda genome can be inserted into the E. coli chromosome and is then called a prophage

Phage M13 Vectors Filamentous bacteriophage of Ecoli Phage M13 Vectors Filamentous bacteriophage of Ecoli. Used for obtaining single stranded copies. DNA sequencing. Single stranded. Inside host cell become double stranded.

HYBRID VECTOR Component from both plasmid & phage chromosomes. Helper phage provided. Developed in 1978 by Barbara Hohn & John Collins. 30 – 40 Kb Origin of replication, cloning site, marker gene, DNA cos site. Smaller than plasmid. Use – construction of genomic libraries of eukaryotes. e.g. Cosmid

COSMIDS Structure of Cosmid Combine parts of the lambda chromosome with parts of plasmids. Contain the cos sites of λ and plasmid origin of replication. Behave both as plasmids and as phages. Cosmids can carry up to 50 kb of inserted DNA. Structure of Cosmid Origin of replication (ori). Restriction sites for cleavage and insertion of foreign DNA. Selectable marker from plasmid. A cos site - a sequence yield cohesive end (12 bases). Ampicillin resistance gene (amp).

ARTIFICIAL CHROMOSOME Linear or Circular. 1 0r 2 copies per cell. Different types – Bacterial Artificial Chromosome (BAC) Yeast Artificial Chromosome (YAC) P1 derived artificial chromosome (PAC) Mammalian Artificial Chromosome (MAC) Human Artificial Chromosome. (HAC) YAC – Cloning in yeast BAC & PAC – Bacteria MAC & HAC – Mammalian & Human cells. A bacterial artificial chromosome (BAC) is a DNA construct, based on a functional fertility plasmid (or F-plasmid), used for transforming and cloning in bacteria, usually E. coli. F-plasmids play a crucial role because they contain partition genes that promote the even distribution of plasmids after bacterial cell division. A similar cloning vector called a PAC has also been produced from the bacterial P1-plasmid. Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid. 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 (Mb) in size instead of 50-250 Mb for natural chromosomes, and able to carry new genes introduced by human researchers. Ideally, researchers could integrate different genes that perform a variety of functions, including disease defense.

BACTERIAL ARTIFICIAL CHROMOSOME 1st BAC Vector – PBAC108L. Cloning of large regions of eukaryotic genome. Origin of replication from bacterium Ecoli F -factor. BAC vectors are pBACe3.6, pBeloBAC11. Used in analysis of genomes.

YEAST ARTIFICIAL CHROMOSOME Linear Plasmid Vector. Clone large DNA segment ( 100 – 1400kb). Occurring two forms:- Circular – grows in bacteria. Linear – multiplies in yeast cells. pYAC3 - first YAC developed. It contains :- ARS sequence – replication CEN4 sequence – centromeric function TRP1 & URA3 – 2 selectable markers Use – mapping complex eukaryotic chromosome .

PYAC3 VECTOR