PLASMID VECTOR

Dr. Ramandeep Kaur

Asstt. Prof. Botany

CONTENTS:

• Cloning vectors :-
• Types of cloning vectors
• Bacterial vectors
• Plasmids – Conjugative plasmids
• Plasmid vectors : pBR322 and pUC8
• Ti- Plasmid
• Phage vectors : lambda phage vectors and M13 phage vectors
• Cosmid vectors
• Yeast vectors
• Retroviruses

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Cloning Vectors

• Cloning describes the processes used to create an exact genetic replica of another cell, tissue or organism. The copied material, which has the same genetic makeup as the original, is referred to as a clone.
• “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”.
• Vectors are ships for carrying the target DNA into a host cell.
• Cloning vector :– used for obtaining millions of copies of cloned DNA segment. Used for creating genomic library or preparing the probes or genetic engineering experiments or other basic studies.
• Expression vector :– allows expression of cloned gene, to give the product (protein). This can be achieved through the use of promoters and expression cassettes and regulatory genes. Used for transformation to generate transgenic plants, animals or microbes where cloned gene expresses to give the product.

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Properties of a good vector:

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(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 (tet^r for tetracycline resistance).

(3) It should have unique restriction enzyme site (only one site for one RE) for different REs (preferably in one of the marker genes) 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.

(6) It should contain specific control systems like promoters, terminators, ribosome binding sites etc. so that the cloned DNA should express properly.

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Uses of vectors��Two primary uses of vectors are:�1. To isolate, identify and archive fragments of larger genome.�2. To selectively express proteins encoded by specific genes. ��Types�

• Vectors are of different types depending on the host. These are as follows:
• 1. Bacterial vectors
• 2. Yeast vectors
• 3. Plant vectors
• 4. 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.

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Bacterial plasmid�

PLASMIDS

A plasmid is a DNA molecule, other than bacterial chromosome, that is capable of independent replication and transmission. Plasmids are circular and may exist either independent or may become integrated into the bacterial chromosome; the latter are called episomes. An E. coli cell may contain upto 7 different kinds of plasmids.

• Plasmids are classified: ��1. By their ability to be transferred to other bacteria
• Conjugative�The sexual transfer of plasmids to another bacterium through a pilus. Those plasmids possess the 25 genes required for transfer.

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• Non-conjugative �Non-conjugative plasmids don’t initiate conjugation.
• They can only be transferred with the help of conjugative plasmids.
• Mobilisable�An intermediate class of plasmids are mobilisable, and carry only a subset of the genes required for transfer. These plasmids can 'parasitise' another plasmid, transferring at high frequency in the presence of a conjugative plasmid.

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2. By function

1. Fertility-(F) plasmids,

They are capable of conjugation (they contains the genes for the pili).

2. Resistance-(R) plasmids,

contain gene(s) that can build resistance against one or several antibiotics or poisons.

3. Col-plasmids,

contain genes coding for colicines, proteins that can kill other bacteria.

4. Degradative plasmids,

able to digest unusual substances, e.g., toluene or salicylic acid.

5. Virulence plasmids,

turn a bacterium into a pathogen.

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Conjugative plasmids�

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The sexual transfer of plasmids to another bacterium through a pilus. Those F plasmids, possess the 25 genes required for transfer.

Plasmid forms: 1. Covalently closed circles –both strands of DNA intact

2. Open circles-only one of the two strand is intact

3. Linear

4. Supercoiled circles

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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).
• Single copy plasmid - maintained as single copy per cell
• Multicopy plasmid - maintained as 10-20 copies per cell
• Plasmids under relaxed control of replication – over 1000 copies per cell – used as cloning vectors.
• In each bacterial cell about 20-25 plasmids are maintained under normal growth condition.
• Some of the commonly used plasmid vectors are described below:

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pBR322

• The first plasmid vector that has been constructed artificially is pBR322.
• It is named after the scientists Bolivar and Rodriguez who constructed it in 1977.
• It is 4363 bp in size and most widely used cloning vector.
• It has an origin of replication derived from a colicine-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 (amp^r ) and tetracycline (tet^r ).

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• Several (over 40 enzymes) unique RE sites are present within these genes for insertion of foreign DNA.
• EcoRIV, BamHI, SphI, SalI, XmaIII, and NruI are present within the gene coding for tetracycline resistance, two sites (HindIII and ClaI) within the promoter of the tetracycline resistance gene and the three sites (PstI, PvuI and ScaI) within the β lactamase gene that provide resistance to ampicillin.
• 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 tet^r gene inactivates that gene.
• One can still select for amp^r colonies, and then screen to see which ones have lost tet^r .

Fig. pBR322

The map shows the positions of the ampicillin-resistance gene (amp^R), the tetracycline-resistance gene (tet^R), the origin of replication (ori) and the recognition sequences for seven restriction endonucleases.

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Fig. Recombinant selection with pBR322

pUC

• A series of small plasmids (about 2.7 kb) have been developed (by Messings and co-workers in1983) at the University of California and hence, the name pUC. e.g. pUC7, 8,9,12,13, 18 and 19 etc.
• 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 β-galactosidase.
• When such plasmids are introduced into E. coli, the colonies are blue on plates containing X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside, the substrate for β- galactosidase) and IPTG (isopropyl thiogalactoside, an inducer).

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• Recombinants and non-recombinants can therefore, be distinguished simply by plating the transformed cells onto agar containing ampicillin and X-gal.
• All colonies that grow on this medium are made up of transformed cells because only transformants are ampicillin resistant.
• Blue colonies contain cells with functional β-galactosidase enzymes and hence with undisrupted lacZ′ genes these colonies are therefore, non-recombinants.
• The white colonies comprise cells without β-galactosidase activity and hence with disrupted lacZ′ genes; these are recombinants.
• Thus, cells containing recombinant plasmids form white (not blue) colonies.

Fig. Recombinant selection with pUC8

������� Must have:�1) Ori region�2) A dominant Selectable�Marker�3) Cleavage sites for cloning�4) High copy no.�

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• The plasmid cloning vector pUC19:- This plasmid has an origin of replication (ori), an amp^R selectable marker, and a polylinker located within part of the β-galactosidase gene lacZ+.

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Ti- Plasmid

Ti-plasmid, or tumour-inducing plasmid, is an extra chromosomal molecule of DNA found commonly in the plant pathogen Agrobacterium tumefaciens. It is also found in other species of Agrobacterium such as A. rubi, A. vitis and A. rhizogenes.

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History

Agrobacterium is a gram negative bacteria that belongs to the class Alphaproteo bacteria. It is one of the pathogenic species belonging to this class. Other non-pathogenic and plant symbiotic species include Caulobacter, Rhodobacter and Rhizobium.

The Ti-plasmid in the bacteria is known to induce crown gall disease in plants by transferring crucial regions from the plasmid. These crucial regions were seen to modify the plant cells into a tumour to produce synthetic plant hormones and cause crown gall.

Features of Ti Plasmid

• Virulence Region: The virulence region codes for virulence genes that are responsible for the transfer of T-DNA to the plant cells and also recruiting various effector proteins for infecting the plant cells.
• T-DNA: The T-DNA region is the crucial region that gets transferred to the plant cell for infection. It is approximately 15-20 kbp in length and is transferred to the plant cell via means of genetic recombination.

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• Opine Catabolism: The opine catabolism region is the region from where the bacteria sources its nutrients for the whole process. Opines are derivatives of amino acid or sugar phosphates that can be catabolized to use in the form of nutrients. The types of opines found in Ti-plasmid are nopaline and octopine types.
• Origin of Replication: The origin of replication is the region where replication of the plasmid is initiated.

Uses in Bioengineering

• The ability of the Ti-plasmid to modify the plant cells has been taken advantage of for the production of transgenic plants.
• The plasmids have been modified into a cloning vector now which are no more pathogenic to plants.
• They are being used to transfer genes of our interest into the plant and produce plants with better quality and quantity.
• It is therefore known as ‘nature’s genetic engineer’