Amjad Khan Afridi

PHAGE PHAGE VECTORSVECTORS

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PHAGESPHAGES• Derivatives of phage have been developed as cloning vectors since Derivatives of phage have been developed as cloning vectors since

the early days of gene technology. the early days of gene technology. • The phage derivatives are considered to be the most suitable The phage derivatives are considered to be the most suitable

cloning vehicles for cloning genomic eukaryotic DNA because of the cloning vehicles for cloning genomic eukaryotic DNA because of the following advantages over the plasmids.following advantages over the plasmids.

Thousands of phage plaques can be obtained in a single petri dish.Thousands of phage plaques can be obtained in a single petri dish. Selection by Selection by DNA-DNA hybridization DNA-DNA hybridization is possible.is possible. In vitro packaging In vitro packaging into empty phage head is possible thus into empty phage head is possible thus

increasing phage infectivityincreasing phage infectivitySize selection of the packaged DNA is possible.Size selection of the packaged DNA is possible. Millions of independently cloned virus particle can be constituted Millions of independently cloned virus particle can be constituted

to form a gene library.to form a gene library.

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BACTERIOPHAGESBACTERIOPHAGES • Bacteriophage is a Bacteriophage is a genetically complex genetically complex but but very extensively studied virus of very extensively studied virus of E.coliE.coli. .

• The DNA of phage, is a The DNA of phage, is a linear duplex linear duplex molecule of 48502 bp molecule of 48502 bp (~49kb) in length.(~49kb) in length.

• The DNA isolated from virus particles is a The DNA isolated from virus particles is a double stranded linear molecule with short double stranded linear molecule with short complementary single stranded complementary single stranded projections of projections of 12 nucleotides at its 5’ 12 nucleotides at its 5’ ends. ends.

• These cohesive termini, also referred to as These cohesive termini, also referred to as coscos sites, sites, allow the DNA to be allow the DNA to be circularized circularized after infection of the host cell after infection of the host cell and also packaging of the DNA.and also packaging of the DNA.

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GENERAL STRUCTURE OF BACTERIOPHAGE DNAGENERAL STRUCTURE OF BACTERIOPHAGE DNA• The genetic map of phage comprises approximately 40 genes which The genetic map of phage comprises approximately 40 genes which

are organized in functional clusters. are organized in functional clusters. • Genes coding for head and tail are proteins (genes Genes coding for head and tail are proteins (genes A-JA-J) are on the ) are on the

left of the linear map. left of the linear map.

• The central region contains genes, such as The central region contains genes, such as int, xis, exo int, xis, exo etc., which etc., which are responsible forare responsible for lysogenisationlysogenisation i.e i.e the process leading to the the process leading to the integration of viral DNA and other recombination events. integration of viral DNA and other recombination events.

• Much of this central region is not essential for lytic growth.Much of this central region is not essential for lytic growth.• Genes to the right of the central region comprise Genes to the right of the central region comprise six regulatory six regulatory

genesgenes, two genes (, two genes (O O andand P P) which are essential ) which are essential for DNA replication for DNA replication during lytic growth and two more genes (during lytic growth and two more genes (SS and and RR) which are required ) which are required for the lysis of the cellular membranes.for the lysis of the cellular membranes.

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• In the phage DNA, larger central region is not essential for phage In the phage DNA, larger central region is not essential for phage growth andgrowth and

replication. replication. • This region of phage can be deleted or replaced without seriously This region of phage can be deleted or replaced without seriously

impairingimpairing

the phage growth cycle. the phage growth cycle. • Using this non-essential region of phage, several phage vector Using this non-essential region of phage, several phage vector

derivatives have been constructed for efficient gene cloning.derivatives have been constructed for efficient gene cloning.

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TYPES OF PHAGE VECTORSTYPES OF PHAGE VECTORS• Wild type phage DNA itself cannot be used as a vector since it Wild type phage DNA itself cannot be used as a vector since it

contains too many restriction sites. contains too many restriction sites. • Further, these sites are often located within the essential regions Further, these sites are often located within the essential regions

for phage's growth and development. for phage's growth and development. • From these wild phages, derivatives with single target sites and From these wild phages, derivatives with single target sites and

two target sites have been synthesized. two target sites have been synthesized. • Phage vectors which Phage vectors which contain single site contain single site for the insertion of foreign for the insertion of foreign

DNA have been designated as DNA have been designated as Insertional vectorsInsertional vectors;;• vectors with vectors with two cleavage sitestwo cleavage sites, which allow foreign DNA to be , which allow foreign DNA to be

substituted for the DNA sequences between those sites, are known substituted for the DNA sequences between those sites, are known as as replacement vectorsreplacement vectors..

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INSERTIONAL VECTORSINSERTIONAL VECTORS• A large segment of the non-essential region A large segment of the non-essential region

has been deleted, and the two arms ligated has been deleted, and the two arms ligated together.together.

• An insertion vector possesses at least one An insertion vector possesses at least one unique restriction site into which new DNA can unique restriction site into which new DNA can be inserted.be inserted.

• Two popular insertion vectors are:Two popular insertion vectors are:• Egt10 : Egt10 : which can carry which can carry up to 8 kb up to 8 kb of new DNA, of new DNA,

inserted into a inserted into a unique unique EcoEcoRI RI site located in the site located in the ccI gene.I gene.

• Insertional inactivation of this gene means Insertional inactivation of this gene means that recombinants are distinguished as clear that recombinants are distinguished as clear rather than turbid plaques.rather than turbid plaques.

• EZAPIIEZAPII : insertion of up to 10 kb DNA into any : insertion of up to 10 kb DNA into any of 6 restriction sites within of 6 restriction sites within a polylinker a polylinker inactivates the inactivates the lacZlacZ′ gene carried by the vector.′ gene carried by the vector.

• Recombinants give clear rather than blue Recombinants give clear rather than blue plaques on X-gal agar.plaques on X-gal agar.

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E.COLI/E.COLI/ REPLACEMENT VECTORS REPLACEMENT VECTORS ExamplesExamples: : EMBL3 and EMBL3 and DASH. DASH. A representative scheme for A representative scheme for

cloning:cloning: 1. The 1. The vector DNAvector DNA is cleaved with is cleaved with

BamH1BamH1 and the long (19 kb) and and the long (19 kb) and short (9 kb) arms are purified; short (9 kb) arms are purified;

2. The 2. The target target fragments are prepared fragments are prepared by digestion, also with by digestion, also with BamBamH1 or a H1 or a compatible enzyme (compatible enzyme (SauSau3A); 3A);

3. The target fragments are treated 3. The target fragments are treated with with alkaline phosphatasealkaline phosphatase to to prevent them ligating to each prevent them ligating to each other;other;

4. The 4. The arms arms and the and the target target fragmentsfragments are ligated together at are ligated together at relatively high concentration to relatively high concentration to form long linear products.form long linear products.

B B

20kb

B

Can notParking

infectE.coli

Long armLong arm ShorShort t

armarmReplace.

48.5 kb

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PACKAGING AND INFECTIONPACKAGING AND INFECTIONThe The RecombinantsRecombinants that can that can notnot be packaged: be packaged:

1. Ligated 1. Ligated ends which do not contain an insert; ends which do not contain an insert;2. The insert is much smaller or larger than the 20 kb;2. The insert is much smaller or larger than the 20 kb;3. The recombinants with two left or right arms. 3. The recombinants with two left or right arms.

in vivo

B

Replication concata-mers

cleave individual genomesin vitroA mixture of phage coat proteins and the phage DNA-processing enzymes

Packaging:

Packaging

phage particles

Infection of E. coli109 recombinants per mg of vector DNA..

FORMATION OF PLAQUESFORMATION OF PLAQUES Plaques are the analogs of single bacterial colonies.Plaques are the analogs of single bacterial colonies. FormationFormation: :

The infected The infected E.coliE.coli cells from a packaging reaction are cells from a packaging reaction are spread on an agar plate, spread on an agar plate,

The plate has been pre-spread with uninfected cells, which The plate has been pre-spread with uninfected cells, which will grow to form a continuous lawn. will grow to form a continuous lawn.

After incubation, phage-infected cells result in clear areas, After incubation, phage-infected cells result in clear areas, that are plaques, where cycles of lysis and re-infection that are plaques, where cycles of lysis and re-infection have prevented the cells from growing. 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 lawnPlaques

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RF

BACTERIOPHAGE M13BACTERIOPHAGE M13Genome features:Genome features: Size is small (6.7 kb); Single- Size is small (6.7 kb); Single-

stranded; Circular genome; DNA; Positive-sense. stranded; Circular genome; DNA; Positive-sense.

g3pg6p

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.

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E.COLIE.COLI/M13 PHAGE /M13 PHAGE VECTORSVECTORS

StructureStructure: : The The phage particlesphage particles containcontain a 6.7 kb circular a 6.7 kb circular ssDNAssDNA. . After infection of a sensitive After infection of a sensitive E. coliE. coli host, the complementary strand is host, the complementary strand is synthesized, like a synthesized, like a plasmidplasmid, and , and the DNA replicated as a the DNA replicated as a dsDNAdsDNA, , the replicative form (RF). the replicative form (RF).

FeaturesFeatures: : The host cells can The host cells can continue to grow slowly.continue to grow slowly.

• ssDNAssDNA: The single-stranded forms : The single-stranded forms are continuously packaged and are continuously packaged and released from the cells as new released from the cells as new phage particles. ssDNA has a phage particles. ssDNA has a number of applications, including number of applications, including DNA sequencing and DNA sequencing and site- site-directed mutagenesis. directed mutagenesis.

• dsDNAdsDNA: The RF (dsDNA) can be : The RF (dsDNA) can be purified purified in vitroin vitro and manipulated and manipulated exactly like a plasmid. exactly like a plasmid.

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CLONING IN M13CLONING IN M13PurposePurpose: When the : When the single-stranded DNA single-stranded DNA of a fragment is required, a M of a fragment is required, a M

13 vector can be used as a common cloning tool. 13 vector can be used as a common cloning tool. PreparationPreparation of ssDNA: of ssDNA: 1. 1. CloningCloning: standard plasmid cloning method can be used to : standard plasmid cloning method can be used to

incorporate recombinant DNA into M13 vectors;incorporate recombinant DNA into M13 vectors;2. 2. TransformationTransformation: the M13 then infects sensitive : the M13 then infects sensitive E. coliE. coli cells; cells;3. 3. PlatingPlating: the host cells grow to form the plaques; : the host cells grow to form the plaques; 4. 4. IsolationIsolation: the ssDNA may then be isolated from phage particles in : the ssDNA may then be isolated from phage particles in

the growth medium of the plate. the growth medium of the plate. ScreeningScreening: Blue-white screening using MCSs and lacZ' has been : Blue-white screening using MCSs and lacZ' has been

engineered into M13 vectors. engineered into M13 vectors. ExamplesExamples: The : The M13mpl8M13mpl8 and and M13mp19M13mp19, which are a pair of vectors in , which are a pair of vectors in

which the MCS are in opposite orientations relative to the M13 origin which the MCS are in opposite orientations relative to the M13 origin of replication. of replication.

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HYBRID PLASMID-M13 VECTORSHYBRID PLASMID-M13 VECTORS

DefinitionDefinition: A number of small plasmid vectors, for example pBlue-script, : A number of small plasmid vectors, for example pBlue-script, have been developed to incorporate M13 functionality. have been developed to incorporate M13 functionality.

StructureStructure: They contain both plasmid and M13 origins of replication, but : They contain both plasmid and M13 origins of replication, but do not possess the genes required for the full phage life cycle. do not possess the genes required for the full phage life cycle.

Working waysWorking ways: : 1. 1. Plasmid wayPlasmid way: they normally propagate as true plasmids, and have the : they normally propagate as true plasmids, and have the

advantages of rapid growth and easy manipulation of plasmid vectors;advantages of rapid growth and easy manipulation of plasmid vectors;2. 2. Phage wayPhage way: they can be induced to produce single-stranded phage : they can be induced to produce single-stranded phage

particles by co-infection with a fully functional particles by co-infection with a fully functional helper phagehelper phage, which , which provides the gene products required for single-strand production and provides the gene products required for single-strand production and packaging. packaging.

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COSMIDSCOSMIDS• Cosmids are Cosmids are hybrids between hybrids between a a phagephage DNA molecule DNA molecule

and a and a bacterial plasmidbacterial plasmid, and their design centers on , and their design centers on the fact that the enzymes that package the the fact that the enzymes that package the DNA DNA molecule into the phage protein coat need only the molecule into the phage protein coat need only the cos sites in order to function.cos sites in order to function.

• The in vitro packaging reaction works not only with The in vitro packaging reaction works not only with genomes, but also with any molecule that genomes, but also with any molecule that carries carries coscos sites sites separated by 37–52 kb of DNA.separated by 37–52 kb of DNA.

• A cosmid is basically a plasmid that carries a A cosmid is basically a plasmid that carries a coscos site . site .• It also needs a It also needs a selectable markerselectable marker, such as the , such as the

ampicillinampicillin resistance gene, and a plasmid resistance gene, and a plasmid origin of origin of replicationreplication, as cosmids lack all the , as cosmids lack all the genes and so do genes and so do not produce plaques.not produce plaques.

• Instead colonies are formed on selective media, just Instead colonies are formed on selective media, just as with a plasmid vector.as with a plasmid vector.

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• The following table provides a list cosmid vectors The following table provides a list cosmid vectors and their structural features.and their structural features.

• Cosmid Size(kb) Cosmid Size(kb) Cleavage sites Size of Cleavage sites Size of insertion (kb)insertion (kb)

• MUA3 MUA3 4.76 4.76 EcoRI/PstI/PvuII/PvuI EcoRI/PstI/PvuII/PvuI 40 – 4840 – 48• pJB8 pJB8 5.40 5.40 BamHI BamHI 32 – 4532 – 45• Homer I 5.40Homer I 5.40 EcoRI/ClaI EcoRI/ClaI 30 – 4730 – 47• Homer II 6.38 Homer II 6.38 SstI SstI 32 – 4432 – 44• pJC79 pJC79 6.40 6.40 EcoRI/ClaI/BamH IEcoRI/ClaI/BamH I 32 – 32 –

4444

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BACTERIAL ARTIFICIAL BACTERIAL ARTIFICIAL CHROMOSOMES (BAC)CHROMOSOMES (BAC)

BACs are based on bacterial mini-F BACs are based on bacterial mini-F plasmids, which are small pieces of plasmids, which are small pieces of episomal bacterial DNA that give the episomal bacterial DNA that give the bacteria the ability to initiate bacteria the ability to initiate conjugation with adjacent bacteria. conjugation with adjacent bacteria. They have a cloning limit of 75-300 They have a cloning limit of 75-300 kb.kb.

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YEAST ARTIFICIAL CHROMOSOMES (YAC)YEAST ARTIFICIAL CHROMOSOMES (YAC)

YACs are artificial chromosomes that replicate in yeast cells. They consist YACs are artificial chromosomes that replicate in yeast cells. They consist of of TelomeresTelomeres, which are ends of chromosomes involved in the replication , which are ends of chromosomes involved in the replication and stability of linear DNA.and stability of linear DNA.

Origin of replication Origin of replication sequences necessary for the replication in yeast cells.sequences necessary for the replication in yeast cells. A A yeast centromereyeast centromere, which is a specialized chromosomal region where , which is a specialized chromosomal region where

spindle fibers attach during mitosis.spindle fibers attach during mitosis. A selectable marker for identification in yeast cells.A selectable marker for identification in yeast cells.Ampicillin resistance Ampicillin resistance gene for selective amplification.gene for selective amplification. Recognition sites for restriction enzymes.Recognition sites for restriction enzymes.

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THE PROCEDURE FOR THE PROCEDURE FOR MAKING YAC VECTORS IS MAKING YAC VECTORS IS

AS FOLLOWSAS FOLLOWS1. The target DNA is partially digested by a 1. The target DNA is partially digested by a restriction endonuclease, and the YAC restriction endonuclease, and the YAC vector is cleaved by restriction enzymes.vector is cleaved by restriction enzymes.

2. The cleaved vector segments are ligated 2. The cleaved vector segments are ligated with a digested DNA fragment to form an with a digested DNA fragment to form an artificial chromosome.artificial chromosome.

3. Yeast cells are transformed to make a 3. Yeast cells are transformed to make a large number of copies.large number of copies.

They are the largest of the cloning vectors, They are the largest of the cloning vectors, with a with a cloning limit of 100-1000 kbcloning limit of 100-1000 kb, , however they have very low efficiency.however they have very low efficiency..

YEAST/YAC VECTORSYEAST/YAC VECTORSCEN4CEN4 is the centromere of is the centromere of

chromosome 4 of chromosome 4 of YeastYeast. The . The centromere will segregate the centromere will segregate the daughter chromosomes. daughter chromosomes.

ARSARS is autonomously replicating is autonomously replicating sequence, its function is as a sequence, its function is as a yeast origin of replication. yeast origin of replication.

TRP1 TRP1 and and URA3URA3 are yeast are yeast selectable markers, one for each selectable markers, one for each end, to ensure the right end, to ensure the right reconstituted YACs survive in the reconstituted YACs survive in the yeast cells. yeast cells.

TELTEL is the telomeric DNA sequence, is the telomeric DNA sequence, which is extended by the which is extended by the telomerase enzyme inside the telomerase enzyme inside the yeast cell.yeast cell.

SUP4SUP4 is a gene, which is is a gene, which is insertionally inactivated, for a insertionally inactivated, for a red-white color test, like blue-red-white color test, like blue-white screening in white screening in E. coliE. 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

SnaBI

BamHI

.

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SHUTTLE VECTORSSHUTTLE VECTORSDefinitionDefinition: They are the vectors that : They are the vectors that

can shuttle between more than one can shuttle between more than one host, for example, one is host, for example, one is E. coliE. coli and and the other is yeast.the other is yeast.

Structure and functionStructure and function: Most of the : Most of the vectors for use in eukaryotic cells are vectors for use in eukaryotic cells are constructed as shuttle vectors. constructed as shuttle vectors.

• InIn E. coliE. coli: : • This means that they can survive This means that they can survive

and have the genes (ori and ampand have the genes (ori and ampr r ) ) required for replication and required for replication and selection in selection in E. coliE. coli. .

• In the In the desired eukaryotic cellsdesired eukaryotic cells: : • They can also survive in the They can also survive in the

desired host cellsdesired host cells, and let the , and let the target insert sequences take target insert sequences take effects.effects.

E.coli

Yeast

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YEAST EPISOMAL PLASMIDSYEAST EPISOMAL PLASMIDSStructureStructure of YEps of YEps

a a oriori: for replication in : for replication in E.coliE.colia a ampamprr: for selection in : for selection in E. coliE. colia a 22 origin: for replication in origin: for replication in

yestyestLEU2LEU2: is homologous gene : is homologous gene

and a selectable marker in and a selectable marker in yeast, involved in leucine yeast, involved in leucine synthesis. synthesis.

X geneX gene: a shuttle sequence.: a shuttle sequence.

ori

ampr

2 originLEU2X gene

Function of YEps• It replicates as plasmids• It integrates into a yeast

chromosome by homologous recombination.

YEps

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EXPRESSION VECTORSEXPRESSION VECTORS• In DNA cloning experiments all the genes cloned are In DNA cloning experiments all the genes cloned are not expressed not expressed fully because fully because

of of weak promoters weak promoters in vector DNA. in vector DNA. • This can be dramatically improved by placing such genes This can be dramatically improved by placing such genes downstream of strong downstream of strong

promoters. promoters. • An additional problem in maximizing expression of cloned genes in E. coli which 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 is frequently encountered with genes from a heterologous source is that the gene gene carries no translation start signal carries no translation start signal which can be efficiently recognized by which can be efficiently recognized by the E. coli translation system.the E. coli translation system.

• This problem may arise for heterologous genes cloned into any host. Thus, even 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 though the gene can be transcribed from a promoter within the vector, the resulting resulting mRNA is poorly translated and little or no protein product will be mRNA is poorly translated and little or no protein product will be synthesized.synthesized.

• In such cases alternative strategies available are In such cases alternative strategies available are fusing the gene to amino fusing the gene to amino terminal region of vector gene that is efficiently translated in the host terminal region of vector gene that is efficiently translated in the host or or coupling the gene to a DNA fragment carrying both strong promoter and a coupling the gene to a DNA fragment carrying both strong promoter and a ribosomal binding site. ribosomal binding site.

• Vectors with this additional feature are called Vectors with this additional feature are called expression vectorsexpression vectors...

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T7expressional

vector

E.COLIE.COLI/T7 EXPRESSION /T7 EXPRESSION VECTORSVECTORS

• Definition ofDefinition of expression vectors expression vectors: : Cloned geneCloned geneexpression vectorexpression vector hosthostfusion fusion proteinprotein..

• StructureStructure• T7 promoterT7 promoter: a strong promoter; : a strong promoter; • RBSRBS: ribosome: ribosome binding site;binding site;• ATGATG: translation initiation : translation initiation

condon condon • MCSMCS: Multiple cloning sites : Multiple cloning sites • TTTT: transcription terminator.: transcription terminator.• ampamprr,,.. ori, ori,

• His-tagHis-tag: Some expression vectors : Some expression vectors are designed to have six histidine are designed to have six histidine codons that encode a hexahistidine codons that encode a hexahistidine tag at the N terminus of the tag at the N terminus of the expressed protein, which allows expressed protein, which allows one-step purification on an affinity one-step purification on an affinity column containing Nicolumn containing Ni2+2+..

T7

RBS MCS

TTATG

INSECT CELL/BACULOVIRUSINSECT CELL/BACULOVIRUSDefinitionDefinition: Baculovirus is an : Baculovirus is an insect virusinsect virus

which can be used for the overexpression of which can be used for the overexpression of animal proteinsanimal proteins in in insect cell cultureinsect cell culture. .

MechanismMechanism: : • Viral promoterViral promoter: This viral gene has an : This viral gene has an

extremelyextremely active promoter. active promoter. • Insect cell cultureInsect cell culture: The same promoter can be : The same promoter can be

used to drive the over-expression of a foreign used to drive the over-expression of a foreign gene engineered into the baculovirus gene engineered into the baculovirus genome. genome.

FunctionFunction: This method is being used : This method is being used increasingly for large-scale culture of proteins increasingly for large-scale culture of proteins of of animal originanimal origin, since the insect cells can , since the insect cells can produce many of the post-translational produce many of the post-translational modifications of animal proteins, which a modifications of animal proteins, which a bacterial expression system bacterial expression system cannotcannot. . Baculovirus-infected SF21 cells .

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MAMMALIAN CELL/VIRAL VECTORSMAMMALIAN CELL/VIRAL VECTORS• SV40SV40: This virus can infect : This virus can infect a number of mammalian a number of mammalian species. The SV40 genome species. The SV40 genome is only 5.2 kb in size. is only 5.2 kb in size. • Since it has packaging Since it has packaging constraints similar to constraints similar to phage phage , so it can be not , so it can be not used for transferring used for transferring large large fragments. fragments.

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MAMMALIAN CELL/VIRAL MAMMALIAN CELL/VIRAL VECTORSVECTORS

• RetrovirusesRetroviruses: They have a ssRNA : They have a ssRNA genome, which is copied into dsDNA genome, which is copied into dsDNA after infection. The DNA is then after infection. The DNA is then stably integrated into the host stably integrated into the host genome by a transposition genome by a transposition mechanism. mechanism.

• They have some strong promoters, They have some strong promoters, and they have been considered as and they have been considered as vectors for vectors for gene therapygene therapy, since the , since the foreign DNA will be incorporated into foreign DNA will be incorporated into the the host genomehost genome in a in a stable mannerstable manner..