Cloning vectors

Plasmids, phage-vectors, phagemids, cosmids, animals and plant vectors

Properties of cloning vectors

Vector is DNA, which can replicate independently and is inherited in extrachromosomal manner.

Fragment of DNA is inserted in cloning vector, which can replicate itself in a cell. Vectors should

• Reasonably small and manageable DNA• Easily Move from cell to cell • Easily generate and purify large amount of DNA

Additional features of widely used vectors • Detection mechanism for the presence of vector• Easy selection of cell with vector• Easy to insert DNA into the vector • Detection mechanism for the insert in vector• Plasmid, viruses and chromosomes are used as

vector

• Vectors are used for cloning gene, gene manipulation and to transform DNA into cell.

• Generally vector means plasmid and phage DNA

• Cosmid, phage, phagemids, yeast artificial chromosome, bacterial artificial chromosome and transposons are some other vectors

• Artificial vectors are developed suitable for definitive function and host- replicate and transferred

• Vectors are host specific, some vectors are developed which can function in different cells

• Most vectors are developed for E.coli cells, this bacteria is the experimental organism and used as work house for cloning and preparation of DNA.

• DNA is generally first worked in E.coli before it is introduced in other cell.

• Cloning vectors are developed for E.coli, Yeast, other fungi, plants and animals

Desired features of vectors• Origin of Replication: necessary to replicate

independently in host cells. Must contain at least one ori. The origin of replication is specific to certain cell.

• Cloning sites: The vectors should contain cloning sites, the unique recognition sequence for RE where foreign DNA can be cloned. Artificially constructed recognition sequences for a number of restriction enzymes- The Multiple Cloning site(MCS) serves to facilitate the insertion of a foreign DNA in artificially constructed cloning plasmid.

• Selectable marker gene: To distinguish transformed cells from non transformed cells eg. antibiotic resistance gene.

• Control elements: some contain control elements (promoter) for the expression of inserted genes in host cell.

• Size: smaller size is preferred-less than 10 kb • High copy number: Plasmids are Low copy (1

to 4/25 per cell) High copy (10 to100 or more copies per cell) Copy number depends on origin of replication.

• Polyhistidine sequence: Some with 6 His coding CACCACCACCACCACCAC for His tagging of expressed protein at N or C terminal. Help to purify the protein by nickel column

Plasmid as Cloning vectors• Plasmids are the main cloning vector. They are

the extrachromosomal DNA of bacteria. Plasmid is double stranded, circular closed DNA with origin of replication. Non-integrative plasmid andepisomes

• Plasmid with tra and mob genes are transferred by conjugation- conjugative vectors. Non-conjugativ vectors lack such genes. Plasmids used in laboratory are with deleted tra and mobgene to prevent self-mobilization

• Size of plasmid: less than 1 kb to over 250 kb. Small plasmids are preferred as small plasmids are easy to handle as large often degrade when handling. Size of plasmid used in cloning is reduced so that large gene can be cloned in it.

• Stringent and relaxed plasmid: Stringent are with Rop gene, which multiply with chromosome so in low number. Relaxed are with mutated Ropgene so multiply independently and is in high copy number.

• Incompatible plasmid: Plasmids with same replication system can not coexist in a cell. E.coli can harbour up to 7 compatible plasmids.

• F-plasmid: Plasmid with information to its transfer to other cell

• R-plasmid: Plasmid with antibiotic resistance gene

• Cryptic plasmids are without functional gene• A bacterial cell may contain 0 to many types of

plasmid

SizePlasmidkb MDa

pUC8 2.1 1.8 E.coliColE1 6.4 4.2 E.coliRP4 54 36 PseudomonasF 95 63 E.coliTOL 117 78 PseudomonaspTiAch5 213 142 Agrobacterium

Organism

Plasmid transfer by conjugation

Replication of non-integrative plasmid and episome

Plasmid classificationClassified according to the main characteristic coded by the plasmid genes

• F-plasmid: Carry tra gene- so conjugal transfer of plasmid possible

• R-plasmid: confer resistance to antibiotics, mercury etc. Gene amp or bla make ampiciline resistance

• Col plasmid: code for colicin that kill other bacteria.• Degradative plasmid: with gene to mtabolize unusual

molecules as toluene. Eg TOL of Pseudomonas putida• Virulence plasmid: confer pathogenicity on the host

bacterium. Eg. Ti plasmid of Agrobacterium

Plasmids also found in organisms other than bacteria.2µm circle found in many strains of the Saccharomycescerevisiae. Plasmids are not found in other eukaryotes.

Bacteriophage(viruses infecting bacteria)

Structure of phage

The lytic infection cycleof bacteriophage

Lysogeniccylcle of lamda

Infection by some animal viruses

Major functions: Synthesis of genome and protein, and assembly

Infection of M13- new phage particles are continuously assembled and released without killing the host cell

Only λ and M13 are used as cloning vectors

M13 is with single stranded circular DNA (+) synthesis of – strand produce replicative form. Later + strand accumulate and phage assemble.

Gene organization in The λ DNA molecule• Head and tail phage, head with DNA• λ-DNA is 49 kb in size- studied well by both gene

mapping and DNA sequencing.• Genes for one function are clustered together

The λ genetic map showing position of genes and functions

Linear and circular form of phage DNA

• In phage head it is linear with 12 nucleotide long complementary single strand at two ends. The two end can come together and become circular- the λcohesive ends are called cos sites.

• Cos site is necessary for DNA packaging• Cos sites allow it to circularize in bacteria

–necessary to be integrated in bacterial genome.

• Phage DNA replicationis by rolling circlemechanism- producecatenanae of many λ-genome in seriseconnected by cos sites

• Cos site is therecognision sequencefor an endonuclease of phage (gene A) thatcleaves at the cos siteproducing single strand sticky end help in λ-DNA packing in phagehead.

• The cleavage and packing is cos sitespecific

M13 filamentous phage• Filamentous phage, smaller

genome of 6407 nucleotides-circular and single stranded

• Capsid made of 3 different proteins (15 in case of λ)

• Infection occurs throughpilus.Simpler infection cycle and does not need genes for insertion.

• Double stranded DNA synthesisedin host cell (RF). The DNA is notinserted in bacterial genome. Dividethere produce about 100 copies and also transfered to daughter cells

• Phage particles released withoutkilling host cell.

• About 1000 new phages areproduced in each generation of host

M13 as a cloning vector• Small genome size <10 kb• Double stranded replicative form works as

plasmid.• Easily prepared from culture and can be used for

transfection• Single stranded DNA can be produced- used for

sequencing and in vitro mutagenesis

Viruses as cloning vector• Viruses as cloning vectors for higher organisms-

as plasmids are found only in bacteria and yeast• Mammalian viruses simian virus (SV40), adeno

virus and retrovirus• Insect virus -baculovirus

Cloning vectors for E. coli

• E.coli cell as workhouse and experimental organism since last 50 years.

• Microbiology, genetics and biochemistry of E.coli is most known among bacteria.

• For genetic engineering new sophisticated cloning vectors are developed for various purposes.

Vectors based on coli plasmids• Large no of desirable vectors are available also

commercially- easy to purify, high transformation efficiency, markers for selection-transformantand recombinant, able to clone large fragments up to 8 kb

• pBR322 is one of the first vectors developed.• Plasmid cloning vectors are preferred for routine

works• Nomenclature: p is for plasmid, BR is the

laboratory where developed (name of scientists Bolivar and Rodriguez, who developed), the number distinguish with other plasmids (from plasmid like pBR325, pBR 327 etc.)

pBR322Properties:• Size: 4363 bp• Double antibiotic resistance-

amp and tet• Marker genes: β-lactamase

for amp. Resistance and tetracycline (detoxify tetracycline by a set of genes).

• Unique restriction site in each marker- insertion here inactivate resistance

• CoE1 ori makes it low copy number 15 mol/cell - but can be increase to 1000-3000 by plasmid amplification using protein synthesis inhibitor like chloramphenicol.

Fig. 6.1

A map of pBR322 sowing position of antibiotic resistantgenes, ori and someimportant restrictionsites

pBR322 is a designed vector

• Developed in 70s (first publication 1977)

• Constructed to have the desirable properties

• Consists of components of three natural plasmids

• AmpR from plasmid R1, tetR from R6-5, the replication of origin from pMB1 (related to ColE1)

• Different other plasmids are developed from pBR322 fulfilling specific functions.

pBR327 is derived from pBR322• Deletion of 1089 bp from pBR322, double

antibiotic resistance remained• High copy number 30-45/cell in normal cell• Deletion destroys the conjugative ability of 322.• Non-conjugative, so can not direct own transfer• No possibility of recombinant pBR327 escaping

to colonize bacteria• Preferred if the cloned gene is potentially

harmful-to avoid accident.• These two plasmid are not used today as many

other desirable plasmids are developed.

pUC 8 (Mesing et al, University of California 1983)

• Descended from pBR322 with its ori and ampR

gene.• ampR gene changed and no more contain unique

restriction sites.• Lac operon with truncated LacZ’ gene (β-

galactosidase gene)-Blue white screening possible

• Restriction sites clustered into a short segment in Lac Z’ gene-Multiple cloning site (MCS).

• Lac promoter before MCS so it is an expression vector. Lac operon is inducible- active when Isopropyl thiogalactopyranoside (IPTG), an analog of allolactose is provided.

• Due to a chance mutation in ori result plasmid with a copy number of 500-700 even before amplification.

• Identification of recombinant by plating on amp plus X-gal plate (with 5 bromo, 4-chloro-3 indolyl-β-galactoside)

• Different pUC vectors with various restriction sites on MCS are developed.

• Cloned DNA in pUC are easily transferred to other vectors.

The pUC plasmids: pUC8, pUC18 and shuttling a DNA fragment from pUC8 to M13mp8

Blue white screening• using β-galactosidase and

X-gal that change colour when insert presnt.

• β-galactosidase split X-gal and produce blue colour

• The 5‘ end of lacZ gene (~ 3 kb) encode α-fragment β-galactosidase (N-terminal 146 AA).

• In special host cell with LacZ missing the front protion the plasmid complement and produce active enzyme-αcomplementation

pGEM3Z for in vitro transcription• Similar to pUC vector with ampR and lac Z’ genes

and MCS• Additional two recognition sequences for

attachment of RNA polymerase present.• The T7 and SP6 promoter of phage are in two

sides of MCS, so transcription of introduced gene is possible.

• The T7 and SP6 promoter is not recognised by E.coli RNA polymerase.

• T7 and SP6 is recognised by T7 and SP6 phage RNA polymerases.

• Phage promoters are active promoter- the lyticcycle completes in 20 min- can produce 1-2 µg RNA/min.

• In vitro transcription possible

pGEM3Z: the vectorand in vitro RNA synthesis. R: MCS with restriction sitesfor EcoRI, SacI, KpnI, AvaI, SmaI, BamHI, XbaI, SalI, AccI, HincII, PstI, SphI and HindIII

Cloning vector based on M13 bacteriophage

• Plasmid needs suitable ori for multiplication in host cell using enzymes of host cell.

• Phage replication is complicated and needs several phage genes- for coat protein and phage specific enzymes. Deletion of these genes will impair the phage replication.

• Phage modification is difficult and phage cloning vectors are slightly different form original molecule.

The M13 genome

M136.4 kb with 10 closely packed genes essential for replication. 507 intergenic sequence (IS) where DNA can be inserted.Limited scope to modify M13M13 produce single strand DNA is one of the attraction to modify it as cloning vector

How M13 cloning vectors are developed?• M13mp1: Lac Z’ (Lac

I’OPZ’) introduced into IS which produced blue plaques if X-Gal is added in plate

• M13mp2: Single nucleotide change of the GGATTC found near the start of LacZ’gene is changed to EcoR I recognition site GAATTC, the change do not bring significant change in β-galactosidase enzyme-DNA inserted by EcoRIcleavage mack LacZ‘gene inactive andproduce clear plaque.

Construction of cloning vectors from wild type M13 genome

• M13mp7: Artificial synthesized polylinker (MCS) is introduced in EcoRI restriction site of LacZ’ gene

• The MCS do not disturb the reading frame of gene and produce altered but functional β-galactosidase

Fig 6.7

Construction of M13mp7- withMCS

Cloning with M13mp7

Cloning by EcoRI, BamHI restriction in M13mp7 will result: New DNA is inserted or the polylinker is reinserted or the vector selfligate- in first case clear plaque and later two case blue plaquesare produced

DNA cloned in M13mp7 is recovered easily- useful in Gene bank

Fig 6.9

More complex M13 vectors

• M13mp8: with complex polylinkertwo enzyme digestion and foreign DNA insertion possible.

• M13mp9 polylinkerin reverse orientation-advantage in DNA sequencing

• Other M13 vector pairs are available with different MCS.

Hybrid Plasmid-M13 vectors

• M13 vectors can clone only small fragments of DNA- only ~1500 bp

• Hybrid vectors phagemids like pEMBL8 made by PUC 8 and 1300 bp fragment of M13. Can produce single strand DNA in E. coli cell with the help of helper phage

• The phagemid is with LacZ’ and polilinker so blue white screening is possible.

• Up to 10 kb fragment cloned can be changed into single strand.

Phagemids are with• ColE I ori and Ampr –propagation and selection

as plasmid• Phage ori enables ssDNA production with helper

plasmid.• MCS in LacZ’ so blue white selection• pBluescript is one common phagemid designed

by combining features of filamentous phage and plasmid so Propagate cloned DNA as like plasmid but when infected with helper phage, the mode of replication changes and replicate like phage and ssDNA or dsDNA

pBluescript shows following characters.• Size only 2.985 kb with Bacterial plasmid (eg.

ColE1ori)-high copy• Selectable marker (antibiotic resistance for amp)• A filamentous phage origin of replication (for

ssDNA production with the help of helper plasmid)

• Multiple cloning site in Lac Z’ gene after Lac Z promoter with possibility of blue/white screening

• Expression of insert DNA produce protein as β-galactosidase fusion protein.

• MCS bordered by T3 and T7 promoters reading in opposite direction

• Synthesis of sens and antisens strand possible in host cell with M13 helper plasmid as F1+ or F1- ori present.

pBS

Cloning vector based on λ-bacteriophage

• Size of DNA fragment that in λ-DNA insertioncan is limited to 3 kb as more than 52 kb cannot packed in its headand λ-DNA itself is 49 kb.

• Large genome so has more than onerecognition sequencesfor almost all enzymes-make insertionproblematic.

• The fragments can notrejoin and form viable λ-genome.

• However, variety of λ-cloning vectos aredeveloped to clonelarge piece of DNA from5 to 25 kb fragmennt

λ-genome

• 49 kb large• Large segment in central region can be removed without

affecting its infection ability• Non essential region between 20 to 35 decrease size by

15 kb and 18 kb DNA can be added.• The non-essential region is for integration and excision

of λ-prophage from E.coli chromosome.• The λ thus is non lysogenic and go only lytic infection

cycle- it is desirable as plaques are formed without induction

Fig. 2.9/6.13

How RS removed?• λ-genome consists of

multiple cleavage sitefor restrictionenzymes. How is itremoved?

• In vitro mutagenesiscan remove such sitesas by changingGAATTC to GGATTC, the recognition site forE.coli. But hard to remove as many such sites are present.

• Natural selection was used to find λ that lack the unwanted RS forRE.

Fig 6.14Natural selection method to find phage lacking EcoRI sites

Insertion vector

Large nonessential part is deleted and religated so that one unique restriction site is resulted, where foreign DNA can be inserted- size of insertion depends on size deleted.

• λ-gt10: EcoRI RS at cI gene where up to 8 kb fragment canbe inserted. Inactivation of this gene result clear plaqueinstead of turbid plaque.

• λZAPII: insertion of up to 10 kb into any RS within MCS inactivates LacZ‘ gene carried by vector. Recombinantproduce clear plaques rather than blue with x-gal.

λ insertion vectors. P polylinker (MCS)

Replacement vector• With two RS for cloning that flank the DNA fragment

replaced (stuffer fragment) by DNA to be cloned.• The stuffer consists of extra RS for the RE so that

religation of the fragments is not possible.• Large DNA fragment can be cloned and non

recombinant will not be packed in phage head as the size will be too small.

• λ-EMBL4: Can carry up to 20 kb fragment by replacingsegment flanked by RE. Rcombinant selection by size orutilizing Spi phenotype (phage P2 and Spi+ and Spi-).

• λGEM11 or λGEM12: Capacity to clone 23 kb(maximum as all nonessential DNA removed). Stufferflanked by polylinker with 7 RS. One RE is SfiI, whichreconizes rare sequence GGCCNNNNNGGCC which ismostly not found in cloned sequence- used to cut out thecloned DNA. Size and Spi phenotype is used forselection

Replacement vectors

Spiphenotype: Stufferfragment with red and gamgene which inhibit wild type λ-phage multiplication in lysogenic E.coli prophage

Cloning strategy• Like in plasmid: λ-

moleculesrestricted, foreignDNA ligated thenthe product is usedto transfect E. colithat needs circularvector.

• Better resultsproducing large number of recombinant is byusing linear vector– by in vitropackaging, all worked in test tube

Cosmid• Sophisticated type of λ-based vector- a hybrid of

phage and plasmid DNA.• Only cos site is necessary to pack DNA in phage

particles. Any molecule of DNA separating twocos sites by 37-52 kb is packed in phageparticle.

• Cosmids are Plasmid vectors with bacteriophageλ-cos site

• The cos-site is about 200 bp DNA sequence : required for in vitro packing and to circularize DNA in bacteria. Cleavage at cos site during packing results 12 bp long complementary single stand at two end. Linear cosmid with cossites at both end

• For successful packaging cosmid needs two cos-DNA sequences separated by short DNA sequence of 37 to 52 kb fragment.

• In a 5 kb cosmid cloning vector 35 to 45 kb insert can be cloned.

• A Cosmid consists of Cos-siteOrigins of replication selectable marker (antibiotic resistance)Multiple cloning site (MCS)

• Cosmids do not produce plaque. • Cosmid and λ are used to clone DNA fragments

for gene linrary

Cosmid and cloning of long DNA fragment

The λ-phage can infect E. coli cells but can not produce plaques. Infected cells selected by antibiotic resistance. All colonies ae recombinant, nonrecombinant is not packed

Vector insert sizePlasmid 8 kbM13 3 kbλEMBL4 20 kbCosmid 45 kb

Large DNA fragments are cloned in other modified vectors• High capacity vector based on P1 bacteriophage can

clone 110 kb DNA in its capsid.• Cosmid type vector based on bacteriophage P1 is used

to clone 75 to 100 kb DNA• Bacterial artificial chromosome (BAC): Based on F-

plasmid. Can clone 100 to 300 kb fragment. • Vector with combined feature of P1 and BAC called P1

derived artificial chromosome (PAC) can clone up to 300kb fragment.

Vectors are developed for several other bacteria like Streptomyces, Bacillus and Pseudomonas

Specific to host or broad host range plasmid, that are able to replicate in variety of bacterial hosts.

Some also derived from bacteriophage specific to the host.

BAC and PAC

• PAC vectors are derived from P1 a temperate phage with genome 100 kb.

• BAC consists of following featuresSequence needed for autonomous replication , copy number like oriS, repE, parA etc. derived from F-factorChloramphenicol resistance as marker gene. Cloning sites (HindIII and BamHI) and other RS.Phage λcosN site and P1 loxP site. Cos for linearize and lox is recognised by Cre recombinase for cre.mediated recombinationMCS within lacZ‘ gene flanked by T7 and sp6 promoter sequences

Advantages of BAC and PAC:• The DNA is circular molecule and replicate in

bacteria.• Easy to prepare like plasmid.• DNA in BAC and PAC is easily analysed as in

cosmid.• Bacteria are grown in agar plate and screening

is done by colony lift hybridization.

Disadvantage: • The DNA cloned is still not sufficient for gene

bank of most eukaryotic organism

Cloning vectors for Eukaryotes

• Wide varieties of cloning vectors are developed for E.coli- used in molecular experiments

• Cloning vectors needed to other organism- if expression system is to be studied eukaryotes, to change the property of organism

• Cloning vectors are also developed for yeast and fungi, higher plants, animals

Vectors for yeast and other fungi

• Yeast is another important organism for biotechnology: brewing and as host organism for production of biopharmaceuticals from cloned gene.

• Discovery of plasmid 2µm in S. cerevisiae, one of the limited number of plasmid found in eukaryotes created more interest to yeast.

• Its 6 kb size is ideal for cloning vector, copy number in yeast cell 70 to 200

• Plasmid with ori (ARS); 599 bp FLP inverted repeat sequence called "flip" site and encodes a site-directed recombinase called FLP, the "flip" protein promotes recombination between these repeats-convert A form of plasmid to B form, where the gene order is rearranged by intramolecular recombination;

• REP2, REP1, and D are the three genes which involved in replication of plasmid, encode proteins required for regulation of FLP expression

The 2 µm

Selectable marker: • Some cloning vector

carry gene resistance for inhibitor like methotrexate and copper.

• LEU2 codes for β-isopropyl-malatedehydrogenase, an enzyme for Leucinesynthesis

• Auxotrophic mutantyeast with non-functional LEU2, cangrow in minimal mediumonly in presence of plasmid with LEU2 gene

• Selection of transformed cells are thus possible

Cloning in yeast using the LEU2 gene as a selectable marker

Yeast episomal plasmids (YEps)• Derived from 2µm plasmid-

may contain entire 2µm or only its origin like YEp13. YEp13: Hybrid of 2µm plasmid and pBR322. 2µm ori, LEU2 gene and entire pBr322. It is a shuttle vector can be replicated and selected for both E.coli and yeast.

• Episomal plasmid: the LEU2 gene and its mutant present in yeast chromosome . Due to the homologous recombination between two genes may result insertion of plasmid in one chromosome. Remain integrated or excised.

• High transformation rate 10,000 to 100,000/µg DNA

• Copy number: 20-50 7.3 and 7.5

Episomal plasmid, YEp13

• The new DNA is cloned in tetR gene and cloned in E.coli. The E.coli with recombinant plasmid will loss the tetracyclinresistance character-selection is possible. Then transformed to yeast

• In yeast cell it is selected by using LEU2 gene.

• Remain primarily as plasmid in yeast cell so recovering the recombinant DNA from transformed yeast is possible

• Vectors which integrate in chromosome -purification may be impossible-an disadvantage.

Cloning strategy Cloning with an E.coli-yeast shuttle vector

Recombination betweenplasmid and chromosomalLEU2 gene

• pBR322 with inserted URA3 gene, which code for orotidine-5’ phosphate decarboxylasenecessary for pyrimidine synthesis- used as selectable marker. It survive in yeast cell only when integrated in yeast chromosome. Transformation rate less than1000 transformant/µg mostly 1-10 as integration is rare. Just 1 copy per cell. Used when stable transformation is required.

Yeast intregativeplasmid (YIps):

• It is a replicative plasmid with yeast chromosomal ori and TRP1 gene involve in tryptophan biosynthesis. Show high transformation rate 10 00 to 10,000 transformant/1µg Copy number:5-100. Unstable, used if protein should obtain from cloned gene.

• Various modification may make stable YEps and YRps.

Yeast replicativeplasmid (YRps):

• Key components Chromosome identifiedCentromere: for distribution correctly during cell divisionTelomeres: For correct replication of end and protect from exonucleaseOri: along the chromosome where DNA replication initiates.

• Possibility of formation of YAC by bringing the important components together, such vector allow to clone large DNA fragment of several hundred kb or more than 1 Mb DNA as present in yeast chromosome.

Yeast Artificial chromosome (YAC)( a linear vector)

Structure of YAC• Several YAC developed but all similar to pYAC3• pYAC3 consists of pBR322 with yeast genes like

TRP1 and URA3 (selectable marker of YIp5 and YRp7 respectively).

• The TRP1 and ori is extended to centromereregion CEN4

• Two Telomere sequences, which are not complete telomere sequence. The complete telomere synthesis takes place inside nucleus.

• One another selectable marker gene SUP4 with restriction site for SnaBI where new DNA is inserted

A YAC and cloning strategy to clone large pieces of DNA

URA3: Orotidine-5'-phosphate decarboxylase :

Cloning strategies• Three restriction sites two for BamHI and one for SnaBI is

used for cloning. SnaBI recognise TAGCTA sequence and result blunt end necessary to clone chromosomal DNA.

• Vector restricted with BamHI and SnaBI resulting three fragments. Fragment flanked by BamHI is discarded. The two other arms are flanked by TEL and SnaBI cleaved site.

• The DNA cloned is legated between two blunt ends of two fragments.

• The artificial chromosome thus produced is with centromereand telomere is transformed to yeast protoplast of S. cerovisiae.

• The yeast strain used for transformation is double auxotrophic mutant trp- and ura3-. Succesful transformation change it to trp+ and ura3+

• Only transformat can grow on minimal medium• Presence of the insert DNA in the vector can be tested by

insertional inactivation of SUP4 resulting white colonies. SUP4 result red colonies.

Application for YAC vectors• YAC originally used to study structure and behaviour of

chromosome- YAC are found stable during cell propagation of yeast. Thus on its possible use as vector to clone long DNA fragment was thought.

• Some genes are more than 100 kb (cystic fibrosis gene is 250 kb)

• YAC can be propagated also in mammalian cell- thus functional analysis of gene in its original host is possible

• In YAC routinely 600 kb fragment is cloned, but some special type can handle up to 1400kb. But such mega YAC have insert stability problem.

• To clone long DNA fragment for use in large scale DNA sequencing.

• To establish eukaryotic gene bank• The screening of YAC is not easy as colony lifting as in

bacteria is not possible and it should be done one by one• One YAC clone is fragmented and cloned in another

clone where the screening is easy.

Number of clonesaSpecies Genome size 17 kbb 35 kbc 300 kbd 1400kbe

E. Coli 4.6 × 106 820 410 50 11

Saccaromycescerevisiae

1.8 × 107 3225 1500 187 41

Doroaophiamelanogaster

1.2 × 108 21500 10,000 1240 266

Rice 5.7 × 108 100,000 49,000 6079 1304Human 3.2 × 109 564.000 274,000 33995 7269

Frog 2.3 × 1010 4,053000 1969000 244292 52423

Number of clones needed for genomic libraries of organisms

a calculated for a probability of 95% that any particular gene will be present in the libraryb Fragment suitable for a replacement vector such as λEMBL4c. Fragment suitable for cosmid d. Fragment suitable for YAC (about)eFragment suitable for special YAC (about)

• Episomal plasmid like 2µm are able to replicate also in some other yeast than S.cerevisiae, but the range of host is narrow.

• Integrative plasmid like YIPs produce stable recombinant

• Effective intrigative vectors are now available for Pichia pastoris, Kluveromyces lactis, filamentous fungi like Aspergilus nidulans and Neurosporacrasa.

Vectors for fungi

Vectors for higher plants

Mainly three types of cloning vectors are in use for plants:

• Vectors based on naturally occurring plasmid of Agrobacterium

• Direct gene transfer using various types of plasmid vectors

• Vectors based on plant viruses

Agrobacterium tumefacience(a natural genetic engineer)

• Plasmid in plants are not known, but bacterial Ti plasmid is of great importance in Genetic transformation of plant

What is Agrobacterium tumefacience?• A soil bacterium causing crown gall disease in many

dicotyledons.• Gall will develop if the bacteria invade plant through

wound• Crown develops after cancerous proliferation of the

infected tissue.• The cancerous development is associated with the Ti-

plasmid present in the bacterium• After infection part of the Ti-plasmid called T-DNA is

integrated in plant genome and produce unusual compounds used as nutrient by bacteria-natural genetic engineer. T-DNA is necessary for tumour development.

Agrobacteriuminfection and crown gall disease

Ti-plasmid

T-DNA, transferred to plant cell is with eight genes that express in plant cell-responsible for tumour development and synthesis of unusual compounds-amino acid derivatives called opine.

Greater than 200 kb

The Ti plasmid and its integration into plant chromosomal DNA after infection

•Independent replication unit- ori

•It consists of virulence region (vir), origin of replication (ori), conjugative transfer region, opine synthesis, opine catabolism, and tumor inducing region (onc).

The T-DNAA portion of Ti plasmid transformed to plant cell and integrated into the host genome. This region is responsible for tumorous response. It consists of

• Right and left border of 25 bp repeat sequence, involved in infection

• Any DNA placed between RB and LB will be transferred to plant cell

• Nopalin T-DNA is about 23 kb; octopine T-DNA consists of about 13 kb left and 8 kb right piece.

• TL is oncogenic; produce auxin and cytokinin (aux and ipt), responsible for crown gall

• TR consists of gene for opine synthesis (ocs and nos), Gene determining tumor size (tml)

• Disarming of T-DNA.

Use of Ti-plasmid to introduce new genes into a plant cell

• Ti-plasmid can be a transporters of gene to plant cell. • Difficulties

Large size-difficult to handleUnique restriction site for cloning is almost impossible.

• A number of disarmed Ti cloning vectors are developed• New strategies were developed to overcome the

difficulties

The binary vector strategy

• For infection,T-DNA can be in other plasmid than Ti-plasmid• T-DNA in small plasmid and other Ti genes in other plasmid is

also equally effective in T-DNA transfer- some strains of A. tumifaciens have naturally binary system.

• Small plasmid with T-DNA is with unique restriction sites so can be manipulated

• pBIN19 is a binary vector within LB and RB a copy of the lacZ’gene with MCS, Kan resistance gene that function after the integration of the vector sequence into the plant genome

• Cloning and manipulation done in pBIN19 in E.coli, the recombinant plasmid is then transferred to A.t. Transformed plant selected in medium containing Kan.

Fig 7.11

The co-integrationstrategy

• A small plasmid like pBR322 with small portion of T-DNA is used to clone the foreign DNA

• A.t. consists of large Ti-plasmid.

• Due to the homology between the two plasmid- if present in the same A.t. cell natural recombination can integrate the small plasmid in the T-region of Ti-plasmid.

• Infection of the plant insert the new gene into the plant chromosome The cointegration strategy

The Ri plasmid• Plasmid of Agrobacterium rhizogenes-

cause hairy root disease. Possible use in high expression cloned DNA.Limitation of cloning with Agrobacteriumplasmids

• Easy to clone in dicot but difficult in monocot.

• Direct gene transfer by bombardment with microprojectiles is utilized in genetic modification of monocots.

Plant viruses as cloning vectors• Virus infecting plants are also possible vectors

for cloning plant DNA• Transformation by virus infection is easily

achieved and virus spread through out the plant by natural infection process.

• One draw back is most of the plant viruses are RNA viruses.

• The two classes of DNA viruses caulimovirusesand geminiviruses are considered as potential. Neither is ideally suited for gene cloning. Narrow host range

Caulimoviruses:• Like in λ-phage constarin in packaging DNA,

even after delation of non-essential section of virus DNA insertion capacity is limited.

• Helper virus strategy is effective: CaMV lackingessential genes can carry large insert but cannot infect.Together with normal CaMV the gene is packed in viral coat and spread in plant.

• Plant viruses are of extremely narrow host rangeas CaMV for some brassicas.

Geminivirus• Maize and wheat are the natural host , so

potential vector for monocot.• Geminiviruses undergo rearrangment and

deletion of genome during infection cycle, whichmay delete the added gene

Cloning vectors for animalsWhy cloning vector for animal is needed?• For the synthesis of recombinant proein from

genes that are not expressed correctly in E.coli and yeast.

• For gene therapy, in which a disease is treated by introducing cloned gene into the patient.

Attention has been focused on cloning vector for clinical uses in mammals.Novel type of vector are found as insect vector

Cloning vectors for insects• Drosophila melanogaster is still one model

organism for biologists. Morgan used first it as model organism.

• Discovery of homeotic selector genes of Drosophila that control the overall body plan of the fly-it is a model for developmental process.

• Vectors for cloning genes in Drosophila are available.

• No plasmid is known in Drosophila and though viruses infect the insect a different rout is followed for cloning vector.

• A transposon called the P-element is used in cloning.

P-element, a transposon• Transposon are small DNA (usually 10 kb) that can move from

one position to other in a chromosome- it is a common element in all organisms.

• P-elements is one transposon of Drosophila- it is 2.9kb in length with three genes flanked by short inverted repeat sequences of 31 bp at two ends

• The genes code for transposase and the inverted sequences works as recognition sequence for transposase.

• Trasposase cut the transposon and insert randomly P-element can also jump within a chromosome or between chromosomes-also between plasmid carrying P-element and fly chromosome. Shows the possibility of using cloning vector

• Non-autonomous P elements can also move within the genome if there are autonomous elements to produce transposase.

• The vector is a plasmid with two P elements. One element with insertion site where new DNA inserted and transposase gene is disrupted (inactive element). The element is moved if transposae present

• The second p-element with intact trasposase gene but without flanking inverted sequence- ‘wing clipped’ so it is not moved

P element vector of Drosophila. a. structure of P-elememt b. Transposition of P-element from plasmid to chromosome c. The P element cloning vector with two P elements. The left element with cloning site R causing disruption of transposase gene. The right P-element with intact transposase gene but itself can not transpose as it is wings clipped.

Transformation process• Gene of interest is inserted into the vector.• The plasmid inserted into fly embryo by

microinjection.• The transposase synthesised by wing clipped

element transpose enginered P-element into one of the fuit fly chromosomes.

• If this happens within germline nucleus then the adult developed from the embryo carry copies of the cloned gene.

• First P element cloning was in 1980s and has made great contribution in Drosophila genetics.

Cloning vectors based on insect viruses• Baculoviruses have played an important role in gene

cloning with other insects• The virus is mainly used in production of recombinant

protein.• Insect cells can provide high yields of protein.• The expression system of baculoviruses infecting insects

but not vertebrate is used• The poyhydrin gene of virus accumulate as nuclear

inclusion bodies makes up to 50% of total cell protein.• Similar production possible if normal gene is replaced by a

foreign one.• Autographa californica multinucleocapsid

nucleopolyhedrovirus (AcMNPV) infect also some mammalian cell lines.The virus genome do not replicate but maitained stably in mammalian cell for enough time for expression.

• Mammalian cell makes the protein stable by glycosylation• Bombyx mori nucleopolyhedrovirus (BmNPV) is used to

produce recombinant protein in silkworm

Cloning in mammals

Carried out for following reasons• To achieve gene knockout: generally

carried out in mice to determine function of an unidentified gene.

• For production of recombinant protein in mammalian cell culture- biotech pharming

• Gene therapy: cells engineered to treat diseases

Viruses as cloning vectors

• Simian virus 40 (SV40) first cloning experiment carried in 1979. The virus show host specific lytic and lysogeniccylce. The genome is 5.2 kb with early genes expressed earlier coding for protein for viral DNA replication and late genes coding for virus capsidprotein. It has also packaging constraints, used as replacement vector replacing late genes but early genes can be also replaced.

• Adenoviruses (AV): 1n 1979 further viruses were used to clone. AV can clone up to 8 kb fragment. AV have larger genome so are difficult to handle.

• Papillomaviruses: have relatively high capacity for DNA insert and obtain stable transformed cell.

• Adeno-associated virus (AAV): Found mostly in same infected cell with AV, use protein for replication synthesised by AV. In absence of helper virus integrate in host DNA, within human chromosome 19. It is important if out come of cloning should be rigorously checked like in gene therapy

• Retroviruses: Produce random but stable integration so has higher potential in gene therapy.

• Bovine papillomavirus (BPV): Viruses also kill the host cell so using viruses needs special trick and must done care fully. BPV causing warts on cattle. It produces about 100 copy in side cell without killing cell and BPV are transferred to daughter cell.

• Shuttle vector: With BPV and pBR322 sequences is capable to replicate in mouse and E.coli are developed and used to produce recombinant proteins in mouse cell lines

• Microinjection: Gene cloning without a vector: safe procedure.

References