Vectors

vectors in rDNA technology

1. vectors SHIVA'S

2. phages Derivatives of phage have been developed as cloning vectors since the early days of gene technology. The phage derivatives are considered to be the most suitable cloning vehicles for cloning genomic eukaryotic DNA because of the following advantages over the plasmids. Thousands of phage plaques can be obtained in a single petri dish . Selection by DNA-DNA hybridization is possible. In vitro packaging into empty phage head is possible thus increasing phage infectivity Size selection of the packaged DNA is possible. Millions of independently cloned virus particle can be constituted to form a gene library . SHIVA'S

3. bacteriophages Bacteriophage is a genetically complex but very extensively studied virus of E.coli . The DNA of phage, is a linear duplex molecule of 48502 bp (~49kb) in length . The DNA isolated from virus particles is a double stranded linear molecule with short complementary single stranded projections of 12 nucleotides at its 5’ ends. These cohesive termini, also referred to as cos sites, allow the DNA to be circularized after infection of the host cell . SHIVA'S

4. General structure of bacteriophage The genetic map of phage comprises approximately 40 genes which are organized in functional clusters. Genes coding for head and tail are proteins (genes A-J ) are on the left of the linear map. The central region contains genes, such as int , xis , exo etc ., which are responsible for lysogenisation i.e the process leading to the integration of viral DNA and other recombination events. Much of this central region is not essential for lytic growth. Genes to the right of the central region comprise six regulatory genes , two genes ( O and P ) which are essential for DNA replication during lytic growth and two more genes ( S and R ) which are required for the lysis of the cellular membranes. SHIVA'S

5. In the phage DNA, larger central region is not essential for phage growth and replication . This region of phage can be deleted or replaced without seriously impairing the phage growth cycle. Using this non-essential region of phage, several phage vector derivatives have been constructed for efficient gene cloning . SHIVA'S

6. Types of phage vectors Wild type phage DNA itself cannot be used as a vector since it contains too many restriction sites. Further , these sites are often located within the essential regions for phage's growth and development. From these wild phages, derivatives with single target sites and two target sites have been synthesized. Phage vectors which contain single site for the insertion of foreign DNA have been designated as Insertional vectors ; vectors with two cleavage sites , which allow foreign DNA to be substituted for the DNA sequences between those sites, are known as replacement vectors . SHIVA'S

7. Insertional vectors A large segment of the non-essential region has been deleted, and the two arms ligated together. An insertion vector possesses at least one unique restriction site into which new DNA can be inserted . Two popular insertion vectors are: Egt10 : which can carry up to 8 kb of new DNA, inserted into a unique Eco RI site located in the c I gene . Insertional inactivation of this gene means that recombinants are distinguished as clear rather than turbid plaques. EZAPII : insertion of up to 10 kb DNA into any of 6 restriction sites within a polylinker inactivates the lacZ ′ gene carried by the vector. Recombinants give clear rather than blue plaques on X-gal agar. SHIVA'S

8. E.coli/ Replacement vectors Examples : EMBL3 and  DASH. A representative scheme for cloning: 1. The  vector DNA is cleaved with BamH1 and the long (19 kb) and short (9 kb) arms are purified; 2. The target fragments are prepared by digestion, also with Bam H1 or a compatible enzyme ( Sau 3A); 3. The target fragments are treated with alkaline phosphatase to prevent them ligating to each other; 4. The  arms and the target fragments are ligated together at relatively high concentration to form long linear products. B B  20kb B Can not Parking infect E.coli Long arm Short arm Replace. 48.5 kb SHIVA'S

9. Packaging and infection The Recombinants that can not be packaged: 1. Ligated  ends which do not contain an insert; 2. The insert is much smaller or larger than the 20 kb; 3. The recombinants with two left or right arms. in vivo B Replication  concata-mers cleave individual  genomes in vitro A mixture of phage coat proteins and the phage DNA-processing enzymes Packaging : Packaging phage particles Infection of E. coli 10 9 recombinants per mg of vector DNA. SHIVA'S

10. Formation of plaques Plaques are the analogs of single bacterial colonies. Formation : The infected E.coli cells from a packaging reaction are spread on an agar plate, The plate has been pre-spread with uninfected cells, which will grow to form a continuous lawn. After incubation, phage-infected cells result in clear areas, that are plaques, where cycles of lysis and re-infection have prevented the cells from growing. Recombinant  DNA may be purified : from phage particles isolated from plaques or from the supernatant of a culture infected with a specific recombinant plaque. E.coli lawn Plaques SHIVA'S

11. RF Bacteriophage M13 Genome features: Size is small (6.7 kb); Single-stranded; Circular genome; DNA; Positive-sense. g3p g6p g7p g8p g9p Host enzymes end ini Infection: M13 particles attach specifically to E.coli sex pili (encoded by a plasmid called F factor), through a minor coat protein (g3p). Binding of g3p induces a structural change in the major capsid protein. This causes the whole particle to shorten, injecting the viral DNA into the host cell. SHIVA'S

12. E.coli /M13 phage vectors Structure :  The phage particles contain a 6.7 kb circular ssDNA .  After infection of a sensitive E. coli host, the complementary strand is synthesized, like a plasmid , and the DNA replicated as a dsDNA , the replicative form (RF). Features :  The host cells can continue to grow slowly. ssDNA : The single-stranded forms are continuously packaged and released from the cells as new phage particles. ssDNA has a number of applications, including  DNA sequencing and  site-directed mutagenesis. dsDNA : The RF (dsDNA) can be purified in vitro and manipulated exactly like a plasmid. SHIVA'S

13. Cloning in M13 Purpose : When the single-stranded DNA of a fragment is required, a M 13 vector can be used as a common cloning tool. Preparation of ssDNA : 1. Cloning : standard plasmid cloning method can be used to incorporate recombinant DNA into M13 vectors; 2. Transformation : the M13 then infects sensitive E. coli cells; 3. Plating : the host cells grow to form the plaques; 4. Isolation : the ssDNA may then be isolated from phage particles in the growth medium of the plate. Screening : Blue-white screening using MCSs and lacZ ' has been engineered into M13 vectors. Examples : The M13mpl8 and M13mp19 , which are a pair of vectors in which the MCS are in opposite orientations relative to the M13 origin of replication. SHIVA'S

14. Hybrid plasmid-M13 vectors Definition : A number of small plasmid vectors, for example pBlue -script, have been developed to incorporate M13 functionality. Structure : They contain both plasmid and M13 origins of replication, but do not possess the genes required for the full phage life cycle. Working ways : 1. Plasmid way : they normally propagate as true plasmids, and have the advantages of rapid growth and easy manipulation of plasmid vectors; 2. Phage way : they can be induced to produce single-stranded phage particles by co-infection with a fully functional helper phage , which provides the gene products required for single-strand production and packaging. SHIVA'S

15. cosmids Cosmids are hybrids between a phage DNA molecule and a bacterial plasmid , and their design centers on the fact that the enzymes that package the  DNA molecule into the phage protein coat need only the cos sites in order to function. The in vitro packaging reaction works not only with  genomes, but also with any molecule that carries cos sites separated by 37–52 kb of DNA. A cosmid is basically a plasmid that carries a cos site . It also needs a selectable marker , such as the ampicillin resistance gene, and a plasmid origin of replication , as cosmids lack all the  genes and so do not produce plaques . Instead colonies are formed on selective media, just as with a plasmid vector. SHIVA'S

16. The following table provides a list cosmid vectors and their structural features. Cosmid Size(kb) Cleavage sites Size of insertion (kb) MUA3 4.76 EcoRI / PstI / PvuII / PvuI 40 – 48 pJB8 5.40 BamHI 32 – 45 Homer I 5.40 EcoRI / ClaI 30 – 47 Homer II 6.38 SstI 32 – 44 pJC79 6.40 EcoRI / ClaI / BamH I 32 – 44 SHIVA'S

17. Bacterial Artificial Chromosomes (BAC) BACs are based on bacterial mini-F plasmids, which are small pieces of episomal bacterial DNA that give the bacteria the ability to initiate conjugation with adjacent bacteria . They have a cloning limit of 75-300 kb . SHIVA'S

18. Yeast Artificial Chromosomes (YAC) YACs are artificial chromosomes that replicate in yeast cells. They consist of Telomeres , which are ends of chromosomes involved in the replication and stability of linear DNA. Origin of replication sequences necessary for the replication in yeast cells. A yeast centromere , which is a specialized chromosomal region where spindle fibers attach during mitosis. A selectable marker for identification in yeast cells. Ampicillin resistance gene for selective amplification . Recognition sites for restriction enzymes. SHIVA'S

19. The procedure for making YAC vectors is as follows 1. The target DNA is partially digested by a restriction endonuclease, and the YAC vector is cleaved by restriction enzymes. 2. The cleaved vector segments are ligated with a digested DNA fragment to form an artificial chromosome. 3. Yeast cells are transformed to make a large number of copies. They are the largest of the cloning vectors, with a cloning limit of 100-1000 kb , however they have very low efficiency . SHIVA'S

20. Yeast/YAC vectors CEN4 is the centromere of chromosome 4 of Yeast . The centromere will segregate the daughter chromosomes. ARS is autonomously replicating sequence, its function is as a yeast origin of replication. TRP1 and URA3 are yeast selectable markers, one for each end, to ensure the right reconstituted YACs survive in the yeast cells. TEL is the telomeric DNA sequence, which is extended by the telomerase enzyme inside the yeast cell. SUP4 is a gene, which is insertionally inactivated, for a red-white color test, like blue-white screening in E. coli . Function : YAC vectors can accept genomic DNA fragments of more than 1 Mb, and hence can be used to clone entire human genes. B B S pYAC3 Sna BI Bam HI SHIVA'S

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22. Shuttle vectors Definition : They are the vectors that can shuttle between more than one host, for example, one is E. coli and the other is yeast. Structure and function : Most of the vectors for use in eukaryotic cells are constructed as shuttle vectors. In E. coli : This means that they can survive and have the genes ( ori and amp r ) required for replication and selection in E. coli . In the desired eukaryotic cells : They can also survive in the desired host cells , and let the target insert sequences take effects. E.coli Yeast SHIVA'S

23. Yeast episomal plasmids Structure of YEps a ori : for replication in E.coli a amp r : for selection in E . coli a 2  origin: for replication in yest LEU2 : is homologous gene and a selectable marker in yeast, involved in leucine synthesis. X gene : a shuttle sequence. ori amp r 2  origin LEU2 X gene Function of YEps It replicates as plasmids It integrates into a yeast chromosome by homologous recombination. YEps SHIVA'S

24. Expression vectors In DNA cloning experiments all the genes cloned are not expressed fully because of weak promoters in vector DNA. This can be dramatically improved by placing such genes downstream of strong promoters. An additional problem in maximizing expression of cloned genes in E. coli which is frequently encountered with genes from a heterologous source is that the gene carries no translation start signal which can be efficiently recognized by the E. coli translation system . This problem may arise for heterologous genes cloned into any host. Thus, even though the gene can be transcribed from a promoter within the vector, the resulting mRNA is poorly translated and little or no protein product will be synthesized . In such cases alternative strategies available are fusing the gene to amino terminal region of vector gene that is efficiently translated in the host or coupling the gene to a DNA fragment carrying both strong promoter and a ribosomal binding site. Vectors with this additional feature are called expression vectors . SHIVA'S

25. SHIVAS T7 expressional vector E.coli /T7 expression vectors Definition of expression vectors : Cloned gene expression vector hostfusion protein . Structure T7 promoter : a strong promoter; RBS : ribosome binding site; ATG : translation initiation condon MCS : Multiple cloning sites TT : transcription terminator. amp r , . ori , His-tag : Some expression vectors are designed to have six histidine codons that encode a hexahistidine tag at the N terminus of the expressed protein, which allows one-step purification on an affinity column containing Ni 2+ . T7 RBS MCS TT ATG

26. Insect cell/ Baculovirus Definition : Baculovirus is an  insect virus which can be used for the overexpression of  animal proteins  in insect cell culture . Mechanism : Viral promoter : This viral gene has an extremely active promoter. Insect cell culture : The same promoter can be used to drive the over-expression of a foreign gene engineered into the baculovirus genome. Function : This method is being used increasingly for large-scale culture of proteins of animal origin , since the insect cells can produce many of the post-translational modifications of animal proteins, which a bacterial expression system cannot . Baculovirus -infected SF21 cells SHIVA'S

27. SHIVAS Mammalian cell/viral vectors SV40 : This virus can infect a number of mammalian species. The SV40 genome is only 5.2 kb in size. Since it has packaging constraints similar to phage  , so it can be not used for transferring large fragments.

28. SHIVAS Mammalian cell/viral vectors Retroviruses : They have a ssRNA genome, which is copied into dsDNA after infection. The DNA is then stably integrated into the host genome by a transposition mechanism. They have some strong promoters, and they have been considered as vectors for gene therapy , since the foreign DNA will be incorporated into the host genome in a stable manner .