Recombinant DNA Technology- Study of cloning vectors.pptx

Recombinant DNA Technology- Study of cloning vectors.
Third ear B. Pharmacy, sem-VI
Pharmaceutical biotechnology

1. RECOMBINANT DNA TECHNOLOGY PART -I STUDY OF CLONING VECTORS Mrs. POONAM NIKAM ASSISTANT PROFESSOR

2. Vectors are the DNA molecules, which can carry a foreign DNA fragment to be cloned. They are self- replicating in an appropriate host cell. The most important vectors are plasmids, bacteriophages , cosmids and phagemid ( phasmids ). Characteristics of an ideal vector : An ideal vector should be small in size. single restriction endonuclease site. origin of replication. 1-2 genetic markers (to identify recipient cells carrying vectors). It should be compatible to transfer (Prokaryotic and Eukaryotic) Naturally occurring plasmids rarely possess all these characteristics. VECTORS _THE CLONING VEHICLE

3. Plasmids are extrachromosomal , double- stranded, circular, self-replicating DNA molecules. Construction: Origin of Replication Genetic marker ( antibiotic resistance -AMP R & TET R and Lac Z gene -the production of the β- galactosidase enzyme ) Multiple cloning sites( MCS ) /Restriction sites for RE Plasmids can carry insert DNA that is less than 20 kb   ( upto 15 kb) The size of plasmids ranges from a few thousand base pairs to more than 100 kilobases PLASMIDS (Note : A few bacteria contain linear plasmids e.g. Streptomyces sp ., Borella burgdorferi ).

4. Types of plasmids They are categorized as conjugative if they carry a set of transfer genes ( tra genes ) that facilitates bacterial conjugation, and non-conjugative , if they do not possess such genes. Based on the copy number : Stringent plasmids (1-2 per cell) Relaxed plasmids occur in large number in each cell. F-plasmids possess genes for their own transfer from one cell to another, while R-plasmids carry genes resistance to antibiotics. In general, the conjugative plasmids are large, show stringent control of DNA replication, and are present in low numbers. On the other hand, non- conjugative plasmids are small, show relaxed control of DNA replication, and are present in high numbers.

5. It is a common practice to designate plasmid by a lower case p, followed by the first letter(s) of researcher(s) names and the numerical number given by the workers. Thus , pBR322 is a_ p plasmid BR discovered by Bolivar and Rodriguez who designated it as 322 , Some _ plasmids are given names of the places where they are discovered e.g . pUC is plasmid from University of California . Nomenclature of plasmids

6. pBR322 of E.coli is the most popular and widely used plasmid vector, pBR322 has a DNA sequence of 4,361 bp. It carries genes resistance for ampicillin (Amp‘) and tetracycline (Tel) that serve as markers for the identification of clones carrying plasmids. The plasmid has unique recognition sites for the action of restriction endonucleases such as EcoRI , HindlII , BamHI , Sall and Pstl . pBR322 — the most common plasmid vector Other plasmid cloning vectors: The other plasmids employed as cloning vectors include pUC19 (2,686 bp , with ampicillin resistance gene), and derivatives of pBR322-— pBR325, pBR328 and pBR329.

7. Advantages of plasmid: Small Size ( Easy to manipulate and isolate) Circular (more Stable) Replication Independent on host cell. Several copies may present (facilitates replication) Frequently have antibiotic resistance (detection assay) Disadvantages of Plasmid: Can not accept large fragment Size range 0-10 kb Standard method of transformation are inefficient.

8. BACTERIOPHAGES Bacteriophages or simply phages are the viruses that infect & replicate within the bacteria. The advantage with phages is that they can take up larger DNA segments than plasmids ( upto 25 kb) . Hence phage vectors are preferred for working with genomes of human cells .

9. Bacteriophage λ Construction: Phage λ consists of a head and a tail. The DNA, located in the head, is a linear molecule of about 50 kb . At each end of the DNA, there are single-stranded extensions of 12 base length each, which have cohesive ( cos ) ends/ sticky ends .

10. On attachment with tail to E.coli , phage A injects its DNA into the cell. Inside E.coli , the phage linear DNA cyclizes and gets ligated through cos ends to form a circular DNA. The phage DNA has two fates-lytic cycle and lysogenic cycle. Lytic cycle : The circular DNA replicates and it also directs the synthesis of many proteins necessary for the head, tail etc , of the phage. The circular DNA is then cleaved (to form cos ends) and packed into the head of the phage. About 100 phage particles are produced within 20 minutes after the entry of phage into F.coli . The host cell is then subjected to lysis and the phages are released . Lysogenic cycle : In this case, the phage DNA (instead of independently replicating) becomes integrated into the E.coli chromosome and replicates along with the host genome. No phage particles are synthesized in this pathway.

11. Figure: The phage lytic cycle and lysogenic cycle

12. Use of phage λ as a vector Only about 50% of phage λ DNA is necessary for its multiplication and other functions. Thus, as much as 50% ( i.e.up to 25kb ) of the phage DNA can be replaced by a donor DNA for use in cloning experiments. However, several restriction sites are present on phage A which is not by itself a suitable vector. The λ -based phage vectors are modifications of the natural phage with much reduced number of restriction sites. Some of them are discussed hereunder . Advantage of using phage vectors No transformation is required. More capacity of accommodation of foreign DNA ( 24kb ) as compare to plasmid ( 15kb ) Easy to screen phage plaques than bacterial colonies to identify recombinant vectors.

13. Phage M13 vectors Phage M13 (bacteriophage M13) is a single- stranded DNA phage of E.coli . Inside the host cell, M13 synthesizes the complementary strand to form a double-stranded DNA ( Replicative form DNA; RF DNA ). For use as a vector, RF DNA is isolated and a foreign DNA can be inserted on it. This is then returned to the host cell as a plasmid. Single- stranded DNAs are recovered from the phage particles .

14. COSMIDS Cosmids ( Cos + Plasmid ) are the vectors possessing the characteristics of both plasmid and bacteriophage λ . A foreign DNA ( about 40 kb ) can be inserted into cosmid DNA. The recombinant DNA so formed can be packed in phages. Cosmids behave just like plasmids and replicate. The advantage with cosmids is that they can carry larger fragments of foreign DNA compared to plasmids . cosmid pHC79 which is a cos -containing derivative of the vector pBR322.

15. Phagemid combination of ( M13 Bacteriophage and Plasmid ) Construction: Origin of replication of both Lac-Z gene Ampicillin resistant gene can be propagated (as plasmid or phage) in appropriate E.coli . Use: As cloning vector, expression vector, sequence in vehicle. Eg . pBluescript II Phagemid vectors ( Phasmid )

16. ARTIFICIAL CHROMOSOME VECTORS 1) Human artificial chromosome (HAC ) Developed in 1997 (by H. Willard ) synthetically produced vector DNA, possessing the characteristics of human chromosome. HAC may be considered as a self-replicating microchromosome with a size ranging from 1/10th to 1/5th of a human chromosome. The advantage with HAC is that it can carry human genes that are too long. Further, HAC can carry genes to be introduced into the cells in gene therapy.

17. 2) Yeast artificial chromosomes (YACs ) Introduced in 1987 (by M. Olson), ( YAC) synthetic DNA that can accept large fragments of foreign DNA (particularly human DNA). YACs are the largest capacity vectors (200-2000 KB) available. They possess centromeric and telomeric regions, and therefore the recombinant DNA can be maintained like a yeast chromosome . 3) Bacterial artificial chromosomes (BACs ) The construction of BACs is based on one F-plasmid which is larger than the other plasmids used as cloning vectors. BACs can accept DNA inserts of around 100-300 kb . The advantage with bacterial artificial chromosome is that the instability problems of YACs can be avoided . In fact, a major part of the sequencing of human genome has been accomplished by using a library of BAC recombinant.

18. 1. Vectors for Bacteria: These are special bacterial origin of replication and antibiotic resistance selectable markers. Bac t eria support different kinds of vectors, e.g.; plasmid vectors , bacteriophages vectors, cosmids , phasmids , phagemids , etc . 2. Vectors for Yeast: They have special origin of replication called as autonomously replicating sequences (ARS), e.g., yeast replicative plasmid vectors ( YRp ) etc . 3. Vectors for Animals: These vectors are needed in biotechnology for the synthesis of recombinant protein from genes that are not expressed correctly when cloned in E. coli or yeast , and methods for cloning in humans are being sought by clinical molecular biologists attempting to devise techniques for gene therapy, in which a disease is treated by introduction of a cloned gene into the patient, e.g., P-element , SV40 etc . 4. Vectors for Plants: The production of genetically modified plants has become possible due to successful use of plant vectors . e.g., Ti-plasmid, Ri -plasmid etc .

19. SHUTTLE VECTORS The plasmid vectors that are specifically designed to replicate in two different hosts (say in E.coli and Streptomyces sp ) are referred to as shuttle vectors. The origins of replication for two hosts are combined in one plasmid. Therefore, any foreign DNA fragment introduced into the vector can be expressed in either host. Further, shuttle vectors can be grown in one host and then shifted to another host (hence the name shuttle). A good number of eukaryotic vectors are shuttle vectors.

20. METHODS OF GENE TRANSFER TRANSFORMATION: Transformation is the method of introducing Foreign DNA into bacterial cells (e.g. E.coli ). The uptake of plasmid DNA by E.coli is carried out in Icecold CaCl2(0-5°C ), and a subsequent heat shock (37-45°C for about 90 sec) . By this technique , the transformation frequency , which refers to the fraction of cell population that can be transferred , is reasonably good. Transformation efficiency It refers to the number of transformants per microgram of added DNA . It is believed that the CaCl2 affects the cell wall, breaks at localized regions, and is also responsible for binding of DNA to cell surface. A brief heat shock (i.e. the sudden increase in temperature from 5°C to 40°C) stimulates DNA uptake. In general, large-sized DNAs are less efficient in transforming . Other chemicals: Calcium Phosphate, Diethyl aminoethyl dextran (DEAE -dextran)

21. 2) CONJUGATION Conjugation is a natural — microbial recombination process. During conjugation, two live bacteria (a donor and a recipient) come together, join by cytoplasmic bridges and transfer single-stranded DNA (from donor to recipient). Inside the recipient cell, the new DNA may integrate with the chromosome (rather rare) or may remain free (as is the case with plasmids ). Conjugation can occur among the cells from different genera of bacteria ( e.g Salmonella and Shigella cells ). APPLICABLE FOR ONLY CONJUGATIVE PLASMID

22. 3) ELECTROPORATION: Thus , electroporation is a technique involving electric field mediated membrane permeabilization . Electric shocks can also induce cellular uptake of exogenous DNA (believed to be via the pores formed by high electric voltage/pulses ) from the suspending solution. Electroporation is a simple and rapid technique for introducing genes into the cells from various organisms (microorganisms, plants and animals).

23. 4) LIPOSOME MEDIATED GENE TRANSFER: Liposome are circular lipid molecule which have an aqueous interior that can carry nucleic acids . Several techniques have been developed to encapsulate DNA in liposomes. The liposome- DNA mediated gene transfer, referred to as lipofection

24. 4) TRANSDUCTION Sometimes, the foreign DNA can be packed inside animal viruses. These viruses can naturally infect the cells and introduce the DNA into host cells. The transfer of DNA by this approach is referred to as transduction. 5) DIRECT TRANSFER OF DNA Microinjection and particle bombardment are the two techniques commonly used for this purpose . useful to introduce DNA into large cells such as oocytes, eggs and the cells of early embryos. The term transfection is used for the transfer DNA into eukaryotic cells, by various physical or chemical means.