A comprehensive study of shuttle vector & binary vector and its rules of in gene transfer

Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning

1. PRESENTATION ON A comprehensive study of shuttle vector & binary vector and its rules of in gene transfer SUMANT PRATAP SINGH A-9606/16 DEPARTMENT OF PMB&GE N.D. University of Agriculture & Technology Kumarganj , Faizabad

2. Vector: A vector is a DNA molecule that has the ability to replicate autonomously in an appropriate host cell and into which the DNA fragment to be cloned is integrated for cloning. E. coli supports several type of vector, some natural, some constructed, which can be grouped as e.g.1) Plasmid 2) B acteriophase (Both natural) 3) Cosmid 4 ) Phasmid 5) S huttle vector( Last three constructed by man ) 6)Artificial chromosome 7) P hagamid Plasmid : A plasmid is a double stranded circular DNA molecule, other then the bacterial chromosome, that is capable of independent replication and transmission. Three Widely studies type are as: F plasmid ( Responsible for conjugation)R plasmid (Carry gene for resistance to antibiotics) and Col plasmid ( Code for colicincs ). INTRODUCTION

3. SHUTTLE VECTOR A vector (e.g. a plasmid) constructed in such a way that it can replicate in at least two different host species ( e.g. a prokaryote and a eukaryote). A DNA recombined into such a vector can be tested or manipulated in several cell types.

4. DESINETED OF SHUTTLE VECTOR A shuttle vector designated to replicate in E. coli and Streptomyces has been constructed follows; The modules for DNA replication in Streptomyces and methylenomycin A resistance are derivated from a Streptomyces plasmid . Replication module for maintenance in E. coli and a gene for antibiotics resistance are taken from E. coli plasmid . Shuttle vector have been designated to specifically satisfy this need, i.e. the initial cloning of DNA insert in E. coli and subsequent test in the species to which the DNA insert belong. Most of the eukaryotic vector are, in fact shuttle vector

5. BINARY VECTOR The binary vector system consists of an Agrobacterium strain along with a disarmed plasmid called vir helper plasmid. B oth of them are not physically linked. The plasmid is said to be "disarmed", since its tumor-inducing genes located in the T-DNA have been removed. Along with T- DNA it can replicate in E . coli and Agrobacterium . Two different plasmids employed in binary vector system. A wide-host-range small replicon. A helper Ti plasmid.

6. A wide-host-range small replicon; has an origin of replication ( ori ) that permits the maintenance of the plasmid in a wide range of bacteria including E. coli and Agrobacterium . This plasmid typically contains : F oreign DNA in place of T-DNA, T he left and right T-DNA borders (or at least the right T-border), Markers for selection and maintenance in both E. coli and A. tumefaciens , A selectable marker for plants. A helper Ti plasmid , harbored in A. tumefaciens , which lacks the entire T-DNA region but contains an intact vir region .

7. CONSTRUCTION OF BINARY VECTOR Obtain plasmids and other DNA fragments necessary for constructions of vectors from appropriate sources. Combine the bacteria-selectable marker and the plasmid replication functions for E . coli . Insert the plasmid replication functions for A. tumefaciens , if necessary. Insert the plasmid mobilization functions, if necessary. Insert the RB, the LB, and the MCS to give the empty vector. Construct the expression unit of the selectable marker gene separately. Insert the unit into the empty vector to give the selection vector. Construct the expression unit of the reporter gene separately. Insert the unit into the selection vector to give the reporter vector .

8. BASIC STRUCTURE 0F BINARY VECTORE T-DNA borders Selectable marker genes for plants  Reporter genes Introduction of DNA fragments to T-DNA Plasmid replication functions Bacterial selection marker  Plasmid mobilization functions Promotars 3 Signals

9. T-DNA BORDERS The RB and the LB are imperfect, direct repeats of 25 bases to define and delimit T-DNA. The RB and the LB are integrated in binary vectors as DNA fragments cloned from well-known Ti plasmids, either octopine or nopaline type.

10. SELECTABLE MARKER GENES FOR PLANTS Choice of selectable marker genes is a key factor in plant transformation. Antibiotics or herbicides resistance genes, such as kanamycin, hygromycin , phosphinothricin , and glyphosate, are very popular. Kanamycin resistance has been most frequently employed in the transformation of many dicotyledonous plants. Hygromycin resistance is the most effective in rice ( Oryza sativa) transformation, whereas phosphinothricin resistance is the most effective in maize. Selectable marker genes are usually driven by constitutive promoters.

11. REPORTER GENES Some genes being trasferred produce enzyme whose activity can be easily detected or used as a basis of selection for the transformed cells, e.g. gene for herbicide resistance. However, most gene need to be tagged with another gene. called reporter gene An ideal reporter gene Scorable reporter gene Selectable reporter gene

12. INTRODUCTION OF DNA FRAGMENTS TO T-DNA Insertion of genes of interest into appropriate locations of a binary vector is traditionally carried out by standard subcloning techniques. Multiple cloning sites, which are similar or identical to those in pUC , pBluescript , and other standard vectors, are still very useful in this regard, but recently constructed vectors are more user friendly. Recognition sites for ‘‘rare cutters,’’ which are restriction enzymes with long recognition sequences, are very convenient in this respect because the DNA fragments that are to be inserted scarcely have such sites.

13. PLASMID REPLICATION FUNCTIONS Binary vectors need replication functions active in E. coli and A. tumefaciens . Replication functions active in a wide range of bacteria, such as ones of plasmid incompatibility group P or W may be conveniently employed. The types of replication functions determine the copy number and the stability of the plasmids in bacterial cells.

14. BACTERIAL SELECTION MARKER Antibiotic resistance genes in common cloning vectors, such as genes that can confer resistance to: Kanamycin Carbenicillin Gentamicin Spectinomycin Chloramphenicol Tetracycline all employed in plant transformation vectors. In the process of plant transformation, A. tumefaciens should be removed from plant cells by antibiotics after infection.

15. PLASMID MOBILIZATION FUNCTIONS The plasmids with OriT or the bom may be mobilized from E. coli to A. tumefaciens aided by a conjugal helper plasmid, such as pRK2013. This function is not necessary when vectors are introduced into A. tumefaciens by electroporation or freeze-thaw methods, but it is a good idea to have a wider option because the conjugal transfer is a very efficient process.

16. PROMOTERS Selectable markers need to be expressed in calli , in cells from those plants that are being regenerated , or germinating embryos to facilitate plant transformation. Therefore, promoters for constitutive expression are preferred . Promoters used mainly for dicotyledonous plants include the 35S promoter from cauliflower mosaic virus and promoters derived from Ti plasmids , such as nopaline synthase ( Nos ), octopine synthase ( Ocs ), mannopine synthase (Mas), gene 1, gene 2, and gene 7

17. 3' Signal DNA fragments of a few hundred bases derived fromthe 3' ends of the CaMV 35S transcript and Agrobacterium Nos and otherT -DNA genes are carried by many of the binary and super-binary vectors

18. TYPE OF BINARY VECTOR 1. pGA series vectors . 2. pCG series vectors 3. pCIT series   4. pGPTV   5. pBECK2000 series 6. Binary-BAC ( BiBAC ) vector 7. pGreen series

19. 1) pGA series vectors , which contain: An ori derived from RK2 for replication in E. coli and Agrobacterium . A tetracycline resistance gene. The cis -acting factor required for conjugal transfer. The right (RB) and left (LB) T-DNA borders. A neomycin phosphotransferase ( nptII ) gene, which confers resistance to kanamycin and G418 in transformed plants . A polylinker site ( multicloning site).

20. pGPTV ( glucuronidase plant transformation vector) series , which have: Different plant selectable marker genes close to the left T-DNA border. This design overcomes problems inherent with the preferential right to left border transfer of T-DNA and improves the chances of having the gene of interest transferred to the plant cell in cells expressing the selectable marker gene.

21. Binary-BAC ( BiBAC ) vector Based on a bacterial artificial chromosome (BAC) vector and is suitable for Agrobacterium -mediated transformation of high-molecular-weight DNA. Comprises low-copy number origins of replication for both E . coli and Agrobacterium to ensure replication of the plasmid as a single-copy in both bacteria; and A helper plasmid carrying additional copies of vir -genes in order to clone very large T-DNAs (up to 150 kb) into the plant genome.

22. pGreen series , small plasmids of around 3.2 Kb containing: A broad host range replication origin ( ori pSa ) and a ColE1 origin derived from pUC , A pSa replicase gene ( rep A) that provides replication functions in trans and is located in a compatible plasmid ( pSoup ) in Agrobacterium , and Multiple cloning sites based on the pBlueScript vector, which allow any arrangement of selectable marker and reporter genes.

23. TRANSFARMATION PROCUDER In general, the transformation procedure is as follows: The recombinant small replicon is transferred via bacterial conjugation or direct transfer to  A. tumefaciens  harboring a helper Ti plasmid. The plant cells are co-cultivated with the  Agrobacterium , to allow transfer of recombinant T-DNA into the plant genome, and Transformed plant cells are selected under appropriate conditions .

24. ADVANTASE Compared with co-integrated vectors, binary vectors present some advantages: No recombination process takes place between the molecules involved. Instead of a very large, recombinant, disarmed Ti plasmid, small vectors are used, which increases transfer efficiency from E. coli to Agrobacterium.

25. DISVANTASE A possible disadvantage may ensue from the fact that the stability of wide host range replicons in  E. coli  and  Agrobacterium  varies considerably. Depending on the orientation, plasmids with two different origins of replication may be unstale in  E. coli  where both origins are active.

26. REFFERENCE An Introduction to Genetic Analysis, Griffiths et al., 7th ed.  ISBN 0-7167-3771-X Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907–1392 Lan -Ying Lee and Stanton B. Gelvin (2007) Molecular Cell Biology, Lodish et al., 6th ed.  ISBN 1-4292-0314-5 Principles of Genetics, Snustad & Simmons, 4th ed.  ISBN 0-471-69939-X Sherman, Fred(2007). "9 Yeast Vectors "  http://dbb.urmc.rochester.edu/labs/Sherman_f/yeast/9.html . Slater, Adrian; Scott, Nigel; Fowler, Mark (2008). Plant Biotechnology the genetic manipulation of plants. Oxford University Press Inc., New York .

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