Genetic engineering can be defined as transfer of DNA between hosts (o species) by in vitro enzymatic manipulations, This implies that the DNA to, be transferred will be duplicated in the new host. Since most DNA fragments are incapable of self-replication in E.coli or any other host cell, an additional segment of DNA, capable of autonomous replication, must be linked to the fragment to be cloned. This autonomously replicating | fragment is the molecular cloning vector and, by definition, plays a central role in recombinant DNA technology. Most cloning vectors were originally derived from naturally occurring extrachromosomal elements such as) bacteriophage and plasmids. Stanley Cohen and co-workers first reported the use of bacterial plasmids as | ; molecular cloning vectors. Since that initial report thousands of cloning | vectors have been constructed, and their versatility in terms of cloning sites, | host-range, and function appears to be limited only by the ingenuity and imagination of the inventor. Whereas insertion of foreign DNA fragments _ into pSC101 was limited to a single restriction enzyme cleavage site.and t0 | Evcoli as a host, most vectors currently in use carry multiple restriction- _ enzyme cleavage sites and have been modified for use with most common — laboratory microorganisms. Many vectors now include dual origins of © replication that enable th % on hosts as dissimilar as E. ae oe em to be‘shuttled between ‘hosts as -as E. Scanned wth CamScanner xGene Cloning Vectors e the nucleotide sequence of genes and segments of DNA. vecte the cloned gen>- 4, ggermine Toning vector is necessary tt carriers of sarily, @ tor, a DNA molecule Y a5 faced into a cell would be JFjated out by cell division and eventually lost. vce are 2 number of, features common to all cloning vectors as: 1, The vector must be able to replicate in the host cell. Thus, it must have: * 4 replication origin. Rep! jcation origin is a sequence element which is recognized bY the host cell's replication machinery and hence the a ‘rector can be amplified in the host cell. 2, Vector must contain a selectable marker for identification and isolation of subpopulation of facteria containing vector. ‘Actually, efficiency of transformation is very low and only or minority of cells in the population take Up ‘and retain the exogenous DNA. ‘Marker in vector helps in identifying transformed bacterial colony. an Ke 3, Most commonly used vectors contain a sh sequence with DNA many closely placed iction e I ‘sites. It is known as ail multicloning site_( CS) ‘or polylinkers. These MCS can also be a chemically synthesized in a véc and are very helpful during foreign mie | gene insertion. Any two sites within an MCS can be simultaneously ee cleaved with the help of range of restriction enzymes and that t aly | without excising the vector sequences. - ich as | This chapter deals with major types of cloning vectors. da esi VPlasmins Joning — g sites. A plasmid is a replicon (unit i : yn (unit of genetic material capable of independent ity and Toliation) that is s a ety Y i ax extachromasomal_siate (Fig 4 ee specific selection) they suvivaT of in which they reside, In fae are not certain environment, such as in aye however, Scanned wth CamScanner| Genetic Engineering and Its Applications | eis e) \ Plasmids | A Bacterial chromosome 19 chromosome: and plasmids. Fig. 4.1: Bacterium showin Replication of! ‘Plasmids ion of plasmids is of replication. el ( ‘The independent replicatic sequences acting as the origin DNA replicative enzymes ©} code for special enzymes necessary conditions, some plasm ‘They are called gpis replicate along, for their replication. into the bacteri sie of Plasmids ‘The size of the plasmid varies from less than Smaller, 1.0 kb Oe eae ‘are much fu 1 . Larges plasmids are less in a gene cloning - smaller ones are more in number. Table describing size of some plasmids. ‘ Size(Nuclestde ai 7 PERS a Scanned wth CamScanner39 Gene Cloning Vectors or Number smid found in a single bacterial cell is molecules of a pla i pa ae molec ranges from 1 to more than 50 per cell but this (ec, 1€siding in bacterial Ei : fic for a given plasmid number is speci Amplification of the Plasmid ‘The copy number of a plasmid can be increased manifold. This process is A. This property is exploited to plifcation of the plasmid \ alee the plasmid copy number.f When the bacterial culture is at its iMGonential phasc chloramphenicol is added ro the medium, This arrest exponeomal DNA replication (and cell division). The culture is then d for another 12 hrs for the “plasmid molecules to replicate. n_increases the ni r cell. Sometimes the PI d copy numbers may reach to several thousand. Types of Plasmids Plasmids can be categorized on the basis of number of copies per cell as, I, Relaxed plasmids, which are normally maintained at multiple copies per cell, and Il. Stringent plasmids, which have a limited number of copies per cell. Plasmids with larger copy mumber are more useful for i bs vi more for gene cloning Plasmids can also be classified as conjugative : and conjugative plasmids, depending on whether or Sea ca a Be of transfer gene called the tra genes. These tra genes promote bacterial conjugation. Generally conjugative plasmids are of high molecular we and are present as 1-3 copies per cell, whereas non-con last have low molecular weight and n copies per cell. isolation of P Cee Scanned wth CamScannerGenetic Engineering and Its Application, 1 40 sl and plasmid amplification: of bacter cer I cells to release their contents. ; terial 11 Breaking the bact {fl. Treatment of bacterial cell extracts to remove all components excep | the DNA. Plasmid DNA from the chromosomal DNA. IV. Separation of ae an containing required plasmid is grown usually on Lp Tre can ani medium).,LB medium contains trypton (10 gf), é jut tract (5 g/l) and sodium chloride (10 g/l) and pH of the medium jg Fisted to 7-5. Late long cultures are then transferred to LB medium | ‘ontaining appropriate antibiotic with small amount of chloromphenical for | 12-16 hours incubation. ‘ Isolation of DNA from such bacteria culture can be done by very gentle cell lysis, using lysozyme and then detergent, followed by clearing the lysate by centrifugation. Centrifugation sediments the high molecular weight DNA | redominantly chromosomal) and cell debris, leaving the small plasmid | Molecules and RNA in supematent. Undamaged plasmid is particularly | compact, since it is supercoiled as a consequence of having slightly too few | fims of double helix per unit length. Further purification of the plasmid can _ be achieved by caesium chloride density gradient ultracentrifugation of | the nucleic acid preparation in presences of ethidium bromide. Ethidium , bromide causes unwinding of DNA as it binds to it, simultaneously — producing a decrease in its buoyant density. Since supercoiled plasmid DNA can unwind to only a very limited extent, it will not bind the dye and q haye a higher density than other types of DNA (latter binds to dye). _ Because of this density difference, plasmid DNA can be separated front other DNA by isopycnic ultracentrifuge fm Plasmid Cloning ‘The following criteria are used to design an ideal cloning 2 id vector should be small apis possible to | . and should contribute as little Scanned wth CamScanner Gene clon more | which areas, | marke distin: 4, The P numb 5. Itshe A ple inact gene mole ‘These pr types of also kee cloning 1, The that mai CorApplications . Gone cloning Vectors ak the plasmid into two or more fragment more than one site will br which is avoided. confer readily selectable A asmid should have the ability 10 ae on the host cells that can be used to rents excep, $ > markers (phenotypic trait) “istinguish the transformed cells from the non-transformed ones. 4, The plasmids should be easily propagated in the desired host so that the ally on LR umber of recombinant DNA molecule can be amplified. on (10 g/l) 5, Itshould have @ large number of copies. > medium jg 4 'B medi plasmid vector should have an additional gen ic marker that can be . phenical $63 jnactivated by insertion of the foreign DNA, so that the inactivated for gene may help in distinguishing the cells harbouring recombinant : seecules on the basis of a readily altered phenotype. ] aan cell “These properties provide maximum flexibility in terms of cloning different Says by types of restriction fragments, In addition to these properties one should sieht NA aiso keep in mind following characters when designing future molecular Pl som cloning vectors. particularl; eS pe 1 oe vector should use, whenever possible, dominant genetic markers lasmid can t can be expressed in a wide range of hosts.Cr (dominant genetic fuugation of marker) do not restrict the vector to a particular genetic Sea oa ‘and quantitative assys for all vector markers should also be ultaneously ry ed plasmid 2. Partitioning sequences such as par 01 5 ir cer should i he dye and into the vector to ensure cffcient segregation seg be ee ataadoe)s daughter cells. F oe rated from ‘alate a mechanism in for controlling vector copy number will” greatly increase the versatility of the vector. Plasmid vectors in which tie (inhibitor) Papeete plieet eee Cae Te ible’ promoter are available. With mice number can be lowered when RNAI i “— Scanned wth CamScanner42 Genetic Engineering and Its Application i Ge CLONING VECTORS BASED ON BACTERIAL PLASMIDS prs | i Bacteria are the common Source of plasmids. For the sole purpose of oe cloning experiments, new plasmids with specific features are constructed in | these vitro. Such artificial plasmids are superior and efficient. ‘The most populy | . genes plasmid vector thus obtained is pBR 322. ao This | Plasmid pBR322 ae pBR322 is an artificial plasmid, which is regarded as the 'work house! of | -BR- gene cloning laboratory. Its DNA is derived from there different but naturally occurring plasmids. The DNA sequence of entire plasmid has been mapped and published (Fig 4.2). 322 Ori: The occ 212 of 1 Col Fig. 4.2: The plasmid p8R922 amp" resistance to ampicillin and tetra ui tel" are genes for eee aes sais ater ian sn Cia). n endonucleases are indicat oie ee ee Scanned wth CamScannerene Cloning Vectors fe restriction sites for over 20 restriction endonucleases and it that give resistance against antibiotic ampicillin (Amp") and Hence cloning in pBR322 at any of the sites within 1] result in insertional inactivation of either of: these BR322 has contains genes tha tetracycline (Tet). J these two genes wil genes. Nomenclature: The plasmids are named on the basis of certain criteria. This can be explained with the help of pBR322. In this plasmid, Pp ‘denotes that it is a plasmid BR- indicates the laboratory in which the plasmid was originally constructed, BR stands for Bolivar and Rodriguez who constructed this plasmid. 322 Itis a number given to distinguish this plasmid from others ~ developed in the same laboratory. For example, there are plasmids PBR 325, pBR 327, pBR 328, pBR 345 etc. Origin of pBR 322 The plasmid pBR 322 was constructed by using three different naturally occurring plasmids. The ampicillin resistance gene was derived from RSF 2124, The tetracycline resistance gene was taken from pSC 101. The origin of replication was obtained from pMBI which is related to the plasmid ColE (Fig. 4.3). Scanned wth CamScanneron - oe Genetic engineering and Its APPIiatign, q natural plasmid, making jg on 44 yy smallet than ® creases the efficiency of ig muc’ eee and inc! 2 UDtaly 1. The plasmid stant to amas Or rmatiOO- Ween, foreign DN: ta fort 2, since this plasmid is na » eerted into this PIASTC” tibjotic resistance BI 3. It has two Tr sites, Insertion © ‘will inactivate Is harbouring as relaxed origin of replication (its multiplication ig yo ed to cell division). So in presence of chloromphenico i increase to 1000-3000. 5. ‘This plasmid is used as a raw material to construct several other usefy! plasmids or other cloning vectors. 6. Single restriction sites for various enzymes, is present around the plasmid, which can be used to open the circle at a specific point prior | to insertion of foreign DNA. | Though pBR 322 has been proved to be a usefull and effective vector, it also | has few disadvantages. Several derivatives with improved efficiencies have been constructed. Some disadvantages related with pBR 322 are as follows: 1. Instability: pBR 322 is lost in continuous culture (and i aa ili id - to a limited extent) in the absence of San cea may create oe for the large-scale fermentation of recombinat bacteria vestigatir i overcome ‘this meee cast opal es henee anne : Partition sequences have been. Rael ae “ | Copy number: Because of its relaxed mode of replication, pBR present in approxima i imacopy feanes canes eae copies per chromosome, ot too high for a particular : 1A upto 6 kb in length Cathe enes namely Amp" and Tet® ys anew gene at 2 site in any oye Sead the marker gene. This helps in ty ad areal cel recombinant plasmids. Scanned wth CamScanner Gone cloning vy used for | several PB constructe< petection ‘Ap® and number of been con concentrat 5, Host rang as an E.co negative | determina organisms constructi later in t organism: Col El Plas Bacterial stra that inhibits ti 6 immunity tol immunity can Size Isolated fron ‘Unique sitesNore take 1 be vith C of not its ful the rior: 45 gene c1onina Vectors out impairing plasmid replication. i oses witht sed for cloning pur ith multiple cloning sites have been BR-derivative, vectors w! sea .d to enhance cloning versatility. : Convenient, reliable, and quantitative assays for pee sepemeye expression arc not available, Consequently, a AP a PBR 822 deatives with improved selectable markers have numberof fructed, These include resistance to. high tetracycline concentrations and resistance to neomycin kanamycin and thiostrepton. ost range limitation: Although pBR 322 was originally constructed "4s an E.coli vector, it can replicate in a few instances in other Gram- negative hosts like “Serratia marcescens. Since the AP* and Tc’ Teerminants on pBR 322 are known to function in a variety of organi: the host range of this plasmid has been broadened by the construction of bifunctional plasmids called shuttle vectors described later in this chapter), Shuttle vectors can replicate in two different organisms because they contain two origins for DNA replication. Ss constructe Col EI Plasmid DNA Bacterial strain containing Col E, plasmid produce Colicin El, a protein that inhibits the growth of other E.coli strains. Col El plasmid also ee - immunity to low level of colicin E1 for strains carrying the plasmid. Colicin immunity can be tested by challenging stains with Colicin E1: Size 44 x 109 Da or 6,600 bp Isolated from E.coli strain 600 : Unique sites With the gene for Colicin producti ion - Ay RI, Sac, Scal,Smal, ae Col E 1 Amp Plasmid DNA This strain carries col El Amp Scanned wth CamScannerEngineering and Its Applications a Genetic ee cn 7.0 M D2 oF 6,000 bp yy j strain HB101 Isolated-from Ecole Unique sites vu | ‘Amplcilin resistance Pat, Taneel resistance am Hl, Bco RV, Neu I, Sal l, Sph | y rc henicol resistance Eco RI é Sane sites ‘Ava, Bal I, Belt, Cla, Hind I, Nde te t pMB9 Plasmid DNA Size 3.5 M Da; Approx $300 bp. 7 Isolated from _ Evcoli strain RRI. Unique sites’ L._ Within the gene _for tetracycline resistance BA MH I, Hind II, Sal Confers tetra cycline resistance and colicin immunity. pTZ Plasmids A versatile cloning vector containing F and pBR322 origins of replication u allowing replication of the episomal DNA in ds or ss form. For st 5 replication, helper phage M13 KO7 is required. pTZ also feature the Cc n multiple cloning sites from pUC 18 or pUC 19 to facilitate cloning. The 8- galactosidase gene provides easy identification of recombinant colonies by color detection when employing X-Gal (5-bromo, 4-chloro- 3-indoyl -8D- galactopyranosidase) in the growth medium, Size ar 2880 bp Unique sites (wien Begalactont ictosidase ‘ili rea aac LSal, ampicillin The T? promoter, located within i d for in vivo or in vitro synthesis ee ae Bene, serves as a tersplatt_ DNA. The in vitro synthesized RNA may be used as Scanned wth CamScanner= i 47 ‘ one cionind Vectors __Viel DNA Discard inessential a oa reemena pee cloned Scanned wth CamScanner48 Genetic Engineering and Its Applications PUB110 Plasmid DNA, Size Isok 3.0 x 106 Daltons Approx. 4500 bp. ‘solated from Bacillus subtilis strain BD170 Comments This Staphylococcus aureus plasmid replicates in Baéillus subtilis. The plasmid confers Fs resistance to kanamycin. Unique sites a) Within gene for Bgl II kanamycin resistance b)- Other unique sites Bam HI, Eco RI, Xba I. Dee ee ae “BACTERIOPHAGE VECTORS FOR E.COL/ The cloning of single genes is usually best carried out using plasmids, since the insert will rarely be larger than about 2 kb. But, for cloning of larger pieces of DNA (e.g. during gene library construction), these plasmids are not suitable as larger inserts increase the plasmid size, making the transformation inefficient. Large molecules can be injected in host bacterial cell by viral particles (bacteriophages). Commonly used bacteriophages are M13, fl, fd and 2 phage. 3 Phage Aas a vector A commonly used vector is that of the 4 phage, which is 49 kb in length. For the cee of long DNA fragments, up to about 20 kb much of the inessential A DNA is removed and replaced by the insert. The recombinant _DNA is then packaged within viral particles in vitro, and these are allowed 4o infect bacterial cells which have been plated out on agar Fig. 4.4). Since the DNA is injected into the cells, a hi iciency of varsfomati can be abianed, Once inside the ells the Yeeombinat viel ‘s replicated. All the genes needed for normal lytic growth are stil of cel ysis and infection of sumounding cele ees oe ‘ysed cells on a background, or fa ii cells, Cloned DNA can b Fae un wo ba es Cloned DNA on ‘There are two Scanned wth CamScanner Gen in! Beoieer 5 > a49 Gene Cloning Vectors cement vectors t vectors contain a restriction site for phage propagation | host, Remaining part of lambda genome is removed ‘A. Ligation is performed at & ratio of arms to f very long concatemers, In which vector Lambda repla La replacemen! in suitable bacterial js replaced by foreign DN: target that favours the formation 0! ‘ and target molecules are interspersed. ; eplacement vectors (e.g. EM BL 4) the internal region that is ae oe the target contains a gene that renders the phage inviable in an appropriate E.coli host. Vector molecule containing target DNA and those without target DNA can be selected by infecting host. Recombinant phage in which the internal region is replaced by target DNA is viable and are mostly used for cloning eukaryotic DNA fragments. t _ Lambda insertion vectors When cloning into an insertion vector, the phage DNA is cleaved with a restriction enzyme that cuts it only once, and the target is inserted into this site. No phage DNA is removed therefore, much lower size of DNA can be inserted. In commonly used"vector 4 gt 10 the E col RII cloning site is in a gene which is deleterious to phage replication in certain host strains. This allows selection against non-recombinant phage. Filamentous Phages as Cloning Vector It included Ff class of filamentous phages, including strains fl, fd and M13, which infect E.coli cells displaying E.coli. These Ff virions are long and thin and contain a closed loop of single stranded DNA. Because the | Scanned wth CamScannerg and Its Applications Genetic Engineer" Fig, 4.5: Simplified diagram of the life cycle of bacteriophage M13. ‘The genetic information of the virus is stored in single-stranded DNA packaged within a long threadlike or filamentous virion. The virus enter the cell through an F pilus. The coat Proteins are removed from the DNA, and it replicates by the rolling circle ‘mechanism. Progeny single strands of DNA are packaged in new Coats, andthe progeny virions are extruded through the cell envelope without killing the host cell. The teplicating DNA molecules are shown as relaxed Circles; in reality, they are Supercoiled throughout the duplication process. Scanned wth CamScanner51 gone cloning Vectors J-chloroform extractions. The same DNA strand of the-virus sd it att lement is the ne packay .d; it is called the "* strand (its comp i a 0 Te eae "4" strand has the same "sense" as the mRNA; its strand). cleotide triplets correspond to the mRNA codons, but with T in place of cm U. Major simple phenol : 2 pa en f the M13 life cycle are illustrated in Fig. 4.5, Packaging’ o le eet phage DNA in progeny phage provides a neat biclogi¢al svnfcation of single-stranded DNA. Importantly, this will be true for a , cloned in the'viral chromosome just as for the phage genes foreign gene ¢ ‘ themselves. This property is exploited for its use as vector. Genetic Organisation of Wild Type BActeriophage M13 ‘The DNA of wild type M13 bacteriophage is single stranded and circular. It is 6407 bases long it has 10 genes that are closely packed. All these genes are essential for the replication of the phages. There is a segment of 507 nucleotide intergenic sequence (IS) which contains origin of replication (OR). This intergenic region can be manipulated for cloning without disrupting the origin of replication. Hence the wild M13 phage has limited use in gene cloning experiments. The size of the phage particle is decided by the size of a pa DNA. Upto six times the normal length of M13 DNA carl be Construction of M13 Based Vectors eh be manipulated for cloning. As this regi ion sites loning. As this region has only two restriction Hoeven v8 1), wild type phage i phage is not an efficient vector, : ton sae lowever, can be modified 0 erodes acti anal Sica sites. A few In M13 phage DNA, the intergenic sequence is the only region which can M13 pI and M13 mp2 Scanned wth CamScanner =—— ~e Engineering and Its Appiicay, ‘Ong Genetic 3 i ene This can be tested x-gal agar where the plaque gives ue a enzyme. colour Perches 2 t cloning vector derives 13 pha The M13 mp2 is the a aa ee DNA eral ts e insertion inactivates lacZ’ (insert | the unique Eco RI a i ends can be _ ins I. ee he ages fail to produce blue pla S ' vee foreig inactivation). The recombinant gal agar, instead, they produce clear plaques naner DNA M13 MP7 will p saat ee comp! MIS mp7 is a derivative of M13 mp2. When a polylinker is inserted inty be isc the Eco RI site of lacZ’ gene; the M13 mp2 becomes M13 mp7. Thy The \ polylinker is designed in such a way that it does not inacti gene. vate the lacr (whe an It as the Eco RI sticky ends and has restriction sites for Bam HI Sal I an (lowe: Pst L Thus M13 mp7 is a more complex vector with four possible carlie from | insertional sites, Foreign DNA with corresponding sticky ends can be inserted into these sites. The recombinant M13 mp7 phage cannot produce . blue ploaques an X- te “ibis! inecavaeened sing ee gla plates du fo insertional inactivation of lez pee When M13 mp7 is cut with Eco RI, a _ When tw , Bam HI, S i pue ee or tat of it is excised. Foreign DNA wit he eee a : . inserted to product i eae = © inserted @ recombinant Insertion nee Pein Seles the lacZ’ gene a ee e and Pl will a plaques an X-gal agar by the recpnaine aoe ‘by the formation ofles Reon ; recombinant ’ The in ‘MI3-Plasmid Hybrid Vectors ies Hybrid “nin Scanned wth CamScanner53 j Gene Cloning Vectors que 118 AND pUC 119 ‘ ‘he phage-plasmid "hybrid" veetor pUC 118 and pUC 119 are a palr of ially identical except that the regions into which vectors tat > Sinserted are present in opposite orientations (turned end- farien velative to the rest of the genes of the vector. Thus, if a foreign forend) Wetted into a specific restriction site in both vectors, one vector DNs. tae one strand of the gene, and the other vector will package the “omplemeatary strand of the gene. Therefore, both strands’ of the gene can be isolated, sequenced, subjected to site-specific mutagenesis, and $0 on. The vectors were designated pUC for plasmid, University of California (where ‘the first pUC vectors in the series were constructed by J. Messing and J, Vieira), and 118, 119 to. distinguish them from earlier members (lower numbers) of the series. Vectors pUC 118 and pUC 119 differ from earlier vectors in the pUC series by the addition of the origin of replication from phage M13. This permits pUC 118 and pUC 119 to replicate eitlier (1) as a double-stranded plasmid in the absence of “helper” phage or (2) as a single-stranded DNA that is packaged in M13 phage coats and extrude. from the cell in the presence of “helper” phage. In the absence of “helper” phage, replication is controlled by the plasmid origin of replication. Th Vectors lite pBR322, contain an origin of replication initially pre... s pli ColEL, In the presence of “helper” oe replication of pUC 11 Pl is directed by the M13 igi icatic ado Bee Phage origin of replication that has The important ing vector Sign features of the pUC 118 and pUC 119 cloning vector are the” 1. small, supercoiled, covalent circular DNA. sug ly closed - DNA-t and manipulated in vitro; al Scanned wth CamScannersonst Engineering and Its APP “ oie fe ee ee a 6 he i nee poten fusion Produc’ ty par mae peti HN fe ay a plasmid ae ‘of double-stranded p! NAS ingy ” DNA procs er pha sence of “hel x e M13 origin of carat DNA and pcan phages presence of "helper ‘ons are present in pUC 118 and puc 10, 9. the pobelonty etre if pUC 118 packages one strand Of 8 clon gene, pC 119 will package the complementary strand, ; i in different, but ji f the pUC vector series contain dif , but rela DE clatase sites. The polycloning regions of pic iis aH pUC 119 contain’ 10 clustered restriction enzyme cleavage sie; ‘Some of these sites are substrates for two or more different restrict enzymes. ‘ ‘The utility of the pUC vectors is greatly enhanced by a simple color tes that allows one to distinguish cell harboring plasmids with foreign DNA inserts from those horboring plasmids with no insert. The basis of this color ication- ductic i replication thus, pro On of 5; ff this DNA in phage ocat ie indicator test is the functional inactivation of the 5' segment of the lacz . gene present in the vector by the insertion of foreign DNA into the polycloning. Tegion, * Z . The pEMBL8 Cloning Vehicle ‘ {he PEMBLS cloning vehicle is ing a 13004 fragment of the constructed by transferring a 131 MIB genome bas the ga MH Phage into the pUCE pa This Scanned wth CamScanner G2 Recomb’ ene aon vectors inant pEMBL8 genome can be packaged into the capsid of ~ MiB phage- 7 nage particles containing the recombinant pEMBL8 genome are rl as the wild type M13 phages in their ability to infect E.coli falls snd then in the expression of the foreign gene. Recombinant pEMBL8 genome can be easily screened using * inedium containing X-gal. M13 replication protein ° Cluster of restriction ~ sites With the help of M13 protein pEMBL & replicates into single ‘stranded DNA Scanned wth CamScannereS i i id Its Ay engineering ar PPlicay, Genetic ny 2 Gene CI Ge z ; is ene . n live a dual life. Their op’ cosmins apna niles es replication Plage ce osmids are hybrid ticate aS Pegente of marker gene. Cleavage: Be! col 4] part enable’ "election due t2. the re mbda part (COS SEQUENCES) allows 4." into MI also helps iP Trarker gene. THEM he transduced to a recipient by recomb! located me in a age COM AME Og" genes for viral proteins there, — 10 infection a in the host. Host cell lysis is also absent, 7," ie viral particles are not form ager DNA 10 increase the size of. Packaging a vector x aa “ih ae ed, This requirement is the basis for the sip ee used for cloning. A ‘Two cosmids are described below: Cosmid pHC79 Itis specially suitable for the cloning of large DNA fragments (upto 40 it) in connection with an in vitro packaging system. It is 6.5 kb in size and contains part of the pBR 322 sequence and a fragment of A-phage DNA containing the cos region. The cos region is required for packaging in ® phage heads. The pBR 322 segment has the genes for Ampicillin ani Tetracycline resistance. S Cosmid pJB8 This cosmid is $4 kb in size, It has ampicillin resistant genes of plasmil PBR 322 and the cos site derived iden from i + ae considered in using a cosmi ict peace oat, DUM Two facts 1f_ LA ™ SBpeeeee L. The enzymes that only theca ses none ee DNA Molecule into the viral coat! 2. The in vitro Packag : c ie . J fanges between 37.59 Gos eee any fragment of D! Provided that the DNA caer DNA) ms Scanned wth CamScannera Gene Cloning Vectors ed up 008 | openel that concatemers soncatemers of Teco into & phage heads. reeombinant COS Amp R Bam Ht A wih ton! —Cal — ‘COs: 57 ‘mids to form recombinant molecules. Ligation occurs is suc! of recombinant cosmids are formed. These mbinant cosmid molecules are then packaged in vil When these phage particles infect bacterial. cells the mids will be transferred into the bactetial cells. cos * __AmpR —S Restrict, Sp eee an Linear pJBS Scanned wth CamScannerEngineering and Its App, 2 canes ton, Cloni Gene ombinant cosmid DNA nds and replicates ag q Pe 58 i the linear TO°' serial cell Ot the 008 © inside the es pase pairing at ME circular CELLS ‘on ANIMAL yinus VECTORS Fl sr the animal coll through certain iimias the natural ability of these vine 4 Foreign DNA is introt tT xplo Perea virases. An experin ‘Some of the animal viruses used as y, avy cls 20 ile ey ovine Papilloma Virus (PY), vat : * are: one lovirus, Retroviruses ‘and Adenoviruses. Vins, Advantages of Virus Vectors 1, Animal viruses ean introduce the genes directly by infecting the jy, cells. 2. They contain powerful promoters which are very much needed jy, a gene expression. ee 3. They have the capacity to replicate their genomes into large number, shown This will help to have high level of gene expression. 4. . Certain animal viruses (retroviruses) naturally integrate their DNA inp the host chromosome. This helps in the propagation of the vial SV40 as ¢ genome throughout the cell line. anes . difficulties Simian Virus 40 (SV40) Mp: : Sen Cloning 4 2 ‘ S ammatian vine ancl is done by using vectors derived from cera a Ps iruses, It is Gen oman Virus 40 (SV40) belonging» aa rs Tech ebetical and the coat proteins are aang F waa aonmetry, Each capsomere is a 47000 kDa polypeptide, Ths itcular DNA molecule (5.2 xb). It as? ate grouped as early genes and late Teplication of the DNA. The late Viral particle ? Scanned wth CamScannercinig host 59 Bam HI Hind in Fig, 4.8: The SV40 genome showing the sites for the early and the late genes. This sites of several restriction endonucleases are also shown. (SV40 as a Vector ‘The wild type of SV40, cannot be used as a gene cloning vector. The difficulties posed by SV40 life cycli include: 1 - Integration SV40 DNA is accompanied by i ~ An SV40 vector has been Presence of relatively small genone (5.24 kb) and no part of it is dispénsible to allows insertion of target DNA, If native genes of the " 8V40 are removed, certain viral proteins cannot be synthesised. This situation can be overcome by co-infection with helper virus which Contains the missing genes of the recombinant SV40 DNA. unpredictable and Sequence rearrangements in the viral DNA, host DNA or both. constructed, known as SVGT- “ement between Hind IM and Bam HI v 5 the ‘abbit B-globin gene (RaSG) in SVGT-S vector with the host cells, The which has Scanned wth CamScannerd Its A : Engineering an Pi @ Genetic Ct, Hind UI apbit B-globin gene Bam HI © i : $V40 DNA containing the late genes is ra Aen Bam HI digestion to give the SVGT.5 vector. This gap is filled with RaBG gene. SV40 genome with the insertio.. an SV40 -plasmi cn Of ai is dering Past vectors, 4 kb in length, This COS Cells and SV40 08 cells are lines developed by Y. Guzman, which contain SV40 DNA i with UV-inactivated replication origin, The modified DNA cannot s teplicated but is able to synthesize T antigen. The latter is utilize to eplicate P vector DNA. ‘Thus several copies of vector are made, each one of which a expresses the inserted gene, : ir d Construction of SVAO-Plasmid Vectors : c c ; Scanned wth CamScanner‘tions INA nnot icate hich gone cloning Waclols. Fig. 4.10: Gene map of pSV1GTS vector it is constructed by inserting a 4 kb segment of the plasmid pBR322 into SVGTS. This segment codes for lactamase and contains the origin of replication of pBR322. This vector can be used as a shuttle vector. VECTORS FOR PLANT CELLS Several vector systems are used for the introduction of foreign genes into plant cells. Among them, the Ti-plasmid of Agrobacterium tumefaciens and 4 few plant viruses such as Caulimoviruses and Geminiviruses are the most important ones. The role of Ti-plasmid of Agrobacterium tumefaciens is discussed in detail in chapter 7. Cauliflower Mosaic Virus (CaMV), one of the Caulimoviruses is discussed in this section. Caulimoviruses Scanned wth CamScannerie Enginoerting 2nd IS APplcaya,. et r Gen ae gene C10" CaMV Chic ' : nome er 11). Gan Tae tl ‘ig. 4.11)- A placement. i as six 8 Biot the virus. Gene 11 is respongist phe oF . restric’ cll to cell SPC sion factor. Gene TI encode, » strand DS ane aphid tamer ces the coat Protein. Gene complete sein, Gene IV, PON Gene VE produces the inci reverse transct reduces the enzyme fay protein. 4 Fig. 4.11: Diagram of the circular double stranded DNA of CaMV showing the three gaps: G1, G2 and G3 (after D. Grierson and S.N. Covey, 1988. Plant Molecular Biology, Blackie and Son Ltd., Glasgow). Multiplication Cycle of CaMV Scanned wth CamScannerS | - 63 Gene Cloning Vectors i minus RNA also functions as 2 template for the synthesis of the : a0 thesis. Thus.a new i faa DNA will be the template for eee syn Y complete viral DNA is synthesised (Fig: 4.14). 1 Emiers the Nucleus ‘ofthe host ‘Overiaps : Vira NA 2 Plus stand, y Gaps sealed Mini or + 7 388 gerome Minus stand ie Reverse Tans e Fig. 4.12: Replication cycle of CaMV viral DNA inside the host. ‘The circular DNAs are finally assembled into viral particles, which’ can move from cell within the host plant. During the life cycle of CaMY, its DNA does not integrate with the host DNA. This virus also does not cause cell lysis. CaMV as a Gene Vector The naked CaMV DNA is capable of infecting host cells. Infections initiated by inoculating the host plant with DN. °CAMV DNA has a sings Sal I site. The Sal I linearised DNA is infectious in vitro in the absence of religation. Recircularization occurs in the plant cell, The viral, pa assembled in the host cells have @ systemic virus. Plant cells Scanned wth CamScanner