Bacterial cloning vectors (for cloning in ecoli).pptx

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1. Bacterial cloning vectors BY PRAISE KHULANI MOYO DEPT OF APPLIED BIOSCIENCES AND BIOTECHNOLOGY MIDLANDS STATE UNIVERSITY

2. Introduction A vector is an agent that can carry a DNA fragment into a host cell in which it is capable of replication. Cloning vector – used for obtaining millions of copies of cloned DNA segment and for creating genomic library or preparing the probes or genetic engineering experiments or other basic studies. (c) 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.

3. Properties of an ideal 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 (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.

4. Classification of vectors according to host 1. Bacterial vectors 2. Yeast vectors 3. Plant vectors 4. Animal vectors

5. Bacterial Vectors (for cloning in E.coli ) (a) E.coli is the most commonly used bacterium for gene cloning though other bacteria such as Bacillus are also used. (b) Vectors used for cloning in E.coli include plasmids, phages, cosmids , phagemids and bacterial artificial chromosomes.

6. Plasmids

7. Classification of plasmids Plasmids are classified by either their ability to be transferred to other bacteria or their function

8. Classification according to transfer ability (a) Conjugative plasmids can be sexually transfered to another bacterium through a pilus thus possessing the 25 genes required for transfer. (b) Non- conjugative plasmids don’t initiate conjugation , they can only be transferred with the help of conjugative plasmids . (c) Mobilisable plasmids are an intermediate class of plasmids which are mobilisable , and carry only a subset of the genes required for transfer.

9. Classification by function 1. Fertility -(F) plasmids , They are capable of conjugation ( they contains the genes for the pili ). Resistance -(R) plasmids , contain gene (s) that can build resistance against one or several antibiotics or poisons . Col- plasmids , contain genes coding for colicines , proteins that can kill other bacteria. Degradative plasmids , able to digest unusual substances , e.g ., toluene or salicylic acid Virulence plasmids , turn a bacterium into a pathogen . Addiction system . These plasmids produce both a long-lived poison and a short- lived antidote .

10. pBR322 (a) The first plasmid vector that has been constructed artificially is pBR322. (b) It is named after the scientists Bolivar and Rodriguiz who constructed it in 1977. (c) It is 4362bp in size and most widely used cloning vector. (d) It has an origin of replication derived from a colicin-resistance plasmid ( ColE1 ). (e) This origin allows a fairly high copy number, about 100 copies of the plasmid per cell. (f) Plasmid pBR322 carries two selectable markers viz. genes for resistance to ampicillin (Ap r) and tetracycline ( Tc r ).

11. pBR322 (g) Several (over 40 enzymes) unique RE sites are present within these genes for insertion of foreign DNA (h) Eco RIV , Bam HI, Sph I , Sal I, Xma III , and Nru I are present within the gene coding for tetracycline resistance, two sites ( Hin dIII and Cla I ) within the promoter of the tetracycline resistance gene and the three sites ( Pst I , Pvu I and Sca I ) within the β lactamase gene that provide resistance to ampicillin ( i ) 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.

12. 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.

13. Phage vectors (g)The first attempts to develop vectors able to handle larger fragments of DNA centered on bacteriophage λ. (h) Two bacteriophages namely, Lambda (λ) and M13 have been commonly used for construction of vectors for cloning in E. coli. ( i ) The phage can have two modes of life cycles i.e. lytic and lysogenic

14. Cont’d (a) During lytic cycle, a phage replicates independently in the host cell and produces a large number of phage particles which are released by lysis of the host. (b) Alternatively, it can take up lysogenic growth, meaning that it integrates its DNA into the bacterial chromosome and multiplies along with it.

15. M13 Phage vectors (a) M13 is a filamentous bacteriophage of E. coli and contains a single stranded circular DNA of 7.2 kb. (b) A series of vectors (M13 mp series) have been developed from this phage. (c) These vectors have a polylinker with unique restriction enzyme sites in lac Z gene that complements host (e.g. JM 103 or JM 104). (d) Screening of recombinants is done based on formation of blue/white plaques. (e) M13 vectors are used for obtaining sufficient quantity of DNA for sequencing by Sanger's dideoxy chain termination method.

16. Vector system Host cell Insert capacity (kb) Plasmid E. coli 0.1-10 Bacteriophage l E. coli 10-20 Cosmid E. coli 35-45 Bacteriophage P1 E. coli 80-100 BAC (bacterial artificial chromosome) E. coli 50-300 P1 bacteriophage-derived AC E. coli 100-300 YAC Yeast 100-2,000 Human AC Cultured human cells >2,000 Cloning vectors and their insert capacities

17. A typical cosmid pJB8 is 5.4 kb in size and carries the ampicillin -resistance gene ( amp R ), a segment of λ DNA containing the cos site, and an Escherichia coli origin of replication ( ori ).

18. 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.

19. Bacterial artificial chromosomes (BACs) A bacterial artificial chromosome (BAC) is an engineered DNA molecule used to clone DNA sequences in bacterial cells (for example, E. coli). BACs are often used in connection with DNA sequencing. Segments of an organism's DNA, ranging from 100,000 to about 300,000 base pairs, can be inserted into BACs. The BACs, with their inserted DNA, are then taken up by bacterial cells. As the bacterial cells grow and divide, they amplify the BAC DNA, which can then be isolated and used in sequencing DNA.

20. Phagemids (a) A phagemid (plasmid + phage) is a plasmid that contains an f1 origin of replication from an f1 phage. (b) It can be used as a type of cloning vector in combination with filamentous phage M13. (c) A phagemid can be replicated as a plasmid, and also be packaged as single stranded DNA in viral particles.

21. fosmids Fosmids are DNA vectors that use the F-plasmid origin of replication and partitioning mechanisms to allow cloning of large DNA fragments. A library that provides 20–70-fold redundant coverage of the genome can easily be prepared