Gene Cloning & Cloning Vectors

KONGUNADU ARTS AND SCIENCE COLLEGE

(AUTONOMOUS)

Coimbatore – 641 029

Genetic Engineering

Dr. S. Viswanathan

U/TS/649

DEPARTMENT OF BIOCHEMISTRY (PG)

Gene Cloning - definition••

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Clone: from the Greek - klon, a twig

The production of exact copies (clones) of a particular gene orDNA sequence using genetic engineering techniques.

The DNA containing the target gene(s) is split into fragmentsusing restriction enzymes. These fragments are then insertedinto cloning vectors which transfer the recombinant DNA tosuitable host cells.

• Inside the host cell the recombinant DNA undergoesreplication; thus, a bacterial host will give rise to a colony ofcells containing the cloned target gene.

Significance•

•••

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A particular gene can be isolated and its nucleotide sequence determined

Control sequences of DNA can be identified & analyzed

Protein/enzyme/RNA function can be investigated

Mutations can be identified, e.g. gene defects related to specific diseases

Organisms can be ‘engineered’ for specific purposes

History• G. Mendel, in the middle of the 19thcentury coined the term gene

for the factor controlling the inheritance of biologicalcharacteristics of an organism.

• In 1903, W. Sutton proposed that genes reside on chromosomes.• In 1922 Gene mapping by T H Morgan and he analyzed the

relative positions of 2000 genes on 4 chromosomes of thefruitfly, Drosophila melanogaster.

• In 1944 Avery, Macleod and McCarty and in 1952 Hershey andChase explained the molecular nature of the gene to beDeoxyribonucleic acid.

• 1952 to 1966: The Structure of DNA, genetic code and theprocess of transcription and translation.

• 1971-1973: New technology like recombinant DNA technology orgenetic engineering or the process of Gene cloning developed.This technology helped in studying the regulation of genes,aberrations in the genes and then used genes for production ofdesired proteins.

• In 1985, Kary Mullis invented the Polymerase Chain reaction

The first cloning experiment done by Boyer and Cohen

Cloning Vector• It is the central

component of agene cloningprocess.

• A small piece of DNAinto which a foreignDNA fragment canbe inserted.

• The insertion of the fragment is carried out by treating the vector and the foreign DNA with a restriction enzyme that creates the same overhang, then ligating the fragments together.

Characteristics of a cloning vector

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Ori (Origin of replication) is a specific sequence of nucleotide from where replication starts

It should have selectable marker gene

It should have restriction sites: a synthetic multiple cloning site (MCS) can be inserted into the vector

Replicate inside the host cell to form multiple copies of the recombinant DNA molecule.

Less than 10kb in size.

Contd….

Origin of Replication: Allow

the vector as well as the

foreign DNA to amplify in

the host cell Selectable Marker:

Antibiotic resistance genes-

Allow the host to grow on

selective media; Can

selectively amplify this

specific vector in the host

cell Multiple Cloning Sites:

Allow insertion of foreign

DNA

Types of Cloning Vectors

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1.

2.

3.

4.

5.

6.

They allow the exogenous DNA to be inserted, stored, and manipulated mainly at DNA level.

Types

Plasmid vectors

Bacteriophage vectors

Cosmids

Phagemids

Fosmids

BACs & YACs

Plasmid Vector•

•

Plasmid vectors are double-stranded, extra-chromosomal DNA molecules, circular, self-replicating.

Advantages:

– Small, easy to handle

– Easy purification

– Straightforward selection strategies

– Useful for cloning small DNA fragments (< 10kbp)

• Disadvantages:

– Less useful for cloning large DNA fragments (> 10kbp)

1. Contains an origin of replication, allowing for replication

independent of host’s genome.

2. Contains Selective markers: Selection of cells containing a

plasmid

twin antibiotic resistance

blue-white screening

3. Contains a multiple cloning site (MCS)

4. Easy to be isolated from the host cell.

5. Plasmids range in size from 1.0kb to 250kb e.g. pUC8 is 2.1 kb

and TOL is 117 kb in size.

A plasmid vector for cloning

pBR322• It was one of the first vectors to be developed in 1977.• The ‘p’ indicates that it is plasmid, ‘BR’ indicates Bolivar and

Rodriguez• ‘322’ distinguishes it from the other plasmids produced in the

same laboratory e.g. pBR325, pBR327, pBR328.• It is 4363bp in size i.e. less than 10kb• It carries two sets of antibiotic resistance genes i.e. either

ampicillin or tetracycline can be used as a selectable marker.• Each of the marker genes carries unique restriction sites and

insertion of DNA into these sites inactivates the specific markersite. e.g. insertion of new DNA with Pst1, Puv1, Ppa1 or Sca1inactivates the ampRgene.

• It has a high copy number. They are about 15 molecules presentin transformed cells but it can be increased to 1000 to 3000 byplasmid amplification in the presence of protein synthesisinhibitor i.e. chloramphenicol.

• The vector comprises DNA derived from three differentnaturally occurring plasmids: the ampRgene is from R1 plasmid,tetRfrom R6-5 plasmid and the ori gene from pMB1 plasmid.

Features of pBR322

yes

yes

B X B

B

B

Ampr

ori

Ampicillin resistant? yes

Tetracycline resistant? No

Ampr

Tcr

ori

pBR322

Ampr

X

Tcr

ori

Screening by insertional inactivation of a resistance gene

Replica plating: transfer of the colonies from one

plate to another using absorbent pad or Velvet

transfer of colonies

+ampicillin + ampicillin+ tetracycline

these colonies have bacteria with

recombinant plasmid

Screening bacteria by replica plating

pBR327•••

•

It was produced by removing a 1089bp segment from pBR322.

The ampRand tetRgenes are intact.

It has high copy number than pBR322 i.e. 30-45 molecules per E.

coli cell. Thus, more the copies of the cloned genes more will bethe effect of the cloned gene on the host cell detectable.

The deletion destroys the conjugative ability of the vectorwhich is important for biological containment.

FROM pBR322 to pUC

• pBR322 requires double screening

• pBR322 has limited number of restriction site

For these reasonspUC (on the left)was engineered

pUC8- Lac selection plasmid

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It is 2750bp in size and is one of the most popular E. coli cloningvectors.

Derived from pBR322 in which only the ori and the ampRgenesremain.

The nucleotide sequence of ampRgene has been changed so thatit no longer contains the unique restriction sites.

The restriction sites are clustered into the lac Z’ gene.

It has a high copy number of 500-700 molecules per cell evenbefore amplification.

The identification of the recombinant cells can be achieved by asingle step process i.e. by plating onto agar medium containingampicillin and X-gal.

Conti-• The clustering of the restriction sites allows a DNA fragment

with two different sticky ends to be cloned without resortingto additional manipulations.

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•

pUC18/pUC19 is a vector having different combinations ofrestriction sites and provide greater flexibility for the DNA tobe cloned.

The restriction site clusters in these vectors are the same as theclusters in M13mp series of vectors. Thus, the DNA cloned inpUC vectors can be transferred directly to its M13mpcounterpart enabling the cloned gene to be obtained as a singlestranded DNA.

Plasmid vectors

The pUC18 cloning vector

Next Major Advance in Plasmid(ology)

The inclusion of polylinkersinto plasmid vectors

Polylinker is a tandem array ofrestriction endonuclease sitesin a very short expanse ofDNA

For example, pUC18’spolylinker

Sites for 13 RE’s Region spans

equivalent of 20the

aminoacids or 60 nucleotides

The Polylinker Advantage

Unique sites (usually)

Insert excision facilitated

Restriction endonuclease mapping and Subcloning made easier

Directional cloning

Blue-White screening for pUC18

• Colonies with recombinant plasmidsare white, and colonies withnonrecombinant plasmids are blue.

• Resistant to ampicillin, has (amprgene)• Contains portion of the lac operon

which codes for beta-galactosidase.• X-gal is a substrate

galactosidase and turnsof beta-

blue in thepresence of functional beta-galactosidase is added to the medium.

• Insertion of foreign DNA into the

beta-galactosidase becomespolylinker disrupts the lac operon,

non-functional and the colonies fail to turn blue, but appear white.

Another Major Advance: Blue-White Screening

Alpha complementation

LacZ Beta galactosidase (Homotetramer)• 1021aa 3 , 1 kbp• Bacteria carry mutant allele

(LacZΔM15) lacking N-terminal domain inactive protein

peptide

• Alpha peptide carried by vector plasmid

• MCS inserted into LacZ alpha

With insert = white colonies Without insert = blue

colonies

• Exploits X-Gal (5-bromo-4-cloro-3-indonyl-Betagalactoside), a chromogenic substrate analog to galactose

What are advantages of pUC over pBR322?

• Single step screening

• MCS increases the number of potential cloning strategies available

X-gal (also abbreviated BCIG for bromo-chloro-indolyl-galactopyranoside) is an organic compound consisting of galactoside linked to indole.

X-gal is cleaved by β-galactosidase yielding galactose and 5-bromo-4-chloro-3-hydroxyindole.

The latter is then oxidized into 5,5'-dibromo-4,4'-dichloro-indigo, aninsoluble blue product.

Thus, if X-gal and an inducer of β-galactosidase (usually IPTG) is containedwithin an agar medium on a culture plate, colonies which have afunctional lacZ gene can easily be distinguished.

pGEM3Z••

It is very similar to pUC vector and is of same size (2750bp).

It carries the ampR and Lac Z gene. The cluster of restriction sites is present in the Lac Z gene.

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It has two promoter sequences i.e. T7 promoter (RNApolymerase of T7 bacteriophage) and SP6 (RNA polymerase ofSP6 phage) promoter sequences that lie on the either side ofthe cluster of restriction sites.

These promoters act as the sites for the attachment of RNApolymerase. Thus if a recombinant pGEM3Z is mixed with RNApolymerase in a test tube, transcription occurs and RNA copiesof the cloned fragment are synthesized.

Bacteriophage• These are the viruses that specifically infect bacteria and during

infection inject the phage DNA into the host cell where itundergoes replication.

• The phages are simple in structure and consist of DNA moleculehaving several genes for phage replication which is surrounded

by a capsid made up of proteins.

Types of Phages

• On the basis of structureHead and Tail phages: e.g. λ phage Filamentous phages: e.g. M13

•On the basis of phage infection cycle Lytic Phage:The infection cycle is completed very quickly and the release of

new phage particles is associated with the lysis of the host cell

Lysogenic phage:

The phage DNA gets integrated into the bacterial DNA known asPROPHAGE and after many cell divisions released by the lysis ofthe host cell e.g. λ phage.

Some phages do not form prophages and the new phageparticles are continuously assembled and released from thehost cell without the lysis of the host cell e.g. M13 phage.

λ Phage• It is 49kb in size and is used as a

cloning vector because: The genes related in terms of

function are clustered together in thegenome and allows the genes to beswitched on and off as a group ratherthan individually.

The linear double stranded DNAmolecule has a stretch of 12nucleotides at its either ends whichact as sticky ends or cohesive ends(cos sites)

They can base pair to form a circularDNA molecule which is important forinsertion into the bacterial genome.

Another role of cos sites is in theformation of large number of λ DNAmolecules by rolling circle mechanismof replication (Catenane).

λ Phage Cloning Vector

• “Head and Tail” phage, very well-studied• Large, linear genome of ~49.0 kb

• Two lifestyle modes– Lytic: replicative mode– Lysogenic: latent mode

• It has a large size genome (49kb) and only 3kb new DNA can beinserted because if the size of the molecule is more than 52kbthen it can not be packaged into the head of the phage.

• The phage has more than one recognition sequence for almostall the restriction endonucleases. So the use of any restrictionenzyme will break the phage DNA into number of smallfragments.

• Despite these disadvantages, λ phages are used to clone largeDNA (5kb to 25kb) molecules.

Lytic and Lysogenic cycle

Recombination and Lysogeny

Cos site: At the ends short (12bp) ss- complementary region “cohesive or sticky” ends--- circulation after infection

Left Arm: Structural genes for head and tail Central Region: genes for lysogenic growth and

recombination/insertion of genome into baterial genome Right Arm: genes involved in DNA replication and lytic growthOnly 30 kb is required for lytic growth. Thus, one could clone 19 kb of “foreign” DNA. Packaging efficiency 78%-105% of the lambda genome.

Engineered version of bacteriophage (infects E. coli). Central region of the chromosome (linear) is cut with a

restriction enzyme and digested DNA is inserted.DNA is packaged in phage heads to form virus particles. Phages with both ends of the chormosome and a 37-52 kb

insert replicate by infecting E. coli. Phages replicate using E. coli and the lytic cycle. Produces large quantities of 37-52 kb cloned DNA. Like plasmid vectors, large number of restriction sites available;

phage cloning vectors are useful for larger DNA fragmentsthan pUC19 plasmid vectors.

Phage cloning vectors:

But bacterial transformation with recombiant lambda phages is very ineffective

In Vitro Packaging

DNA can be packaged into phage particle in vitro

cos sequences

The packaged

phage particles

are infectious

How to transfer

recombinant lambda

into cells?

The infection process is aboutthousand times more efficienct

than transformation with

plasmid vectors.

106 tansformed colonies permicrogram of plasmid vector

109 plaques per microgram ofrecombinant Lambda vector

Strategies

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For the construction of the cloning vector, the non-essentialregion (between positions 20 and 35) in the genome of thephage was removed which decreased the size of the moleculeby 15kb. Thus, upto 18kb of new DNA can be inserted

By in vitro mutagenesis and by natural selection one or tworestriction sites are removed e.g. EcoR1.

Insertion Vectors• In these vectors the non-essential region is deleted and the two

arms ligated together. The vector possesses one unique

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restriction site.

λgt10: It can clone up to 8kb of new DNA inserted in the EcoR1 site located in the c1 gene.

λZAP11: In this vector up to 10kb of DNA can be inserted in toany of the 6 restriction sites within the polylinker. This inactivates the lac Z gene of the vector

Replacement Vectors•

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This vector has two recognition sites for the restriction endonuclease used for cloning.

These sites replace the segment of DNA (Stuffer fragment) from the vector genome by the DNA to be cloned.

These vectors can carry large pieces of DNA than insertion vectors.

λEMBL4: This vector can be used to insert up to 20kb of DNA.

Replacement vectors

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Left arm:– head & tail proteins

Right arm:– DNA synthesis

– regulation

– host lysis

• central

&

Deleted region:– integration

excision

– regulation

M13 Phage• It is 6407 nucleotides in length, circular and consists of a single

stranded DNA molecule and is used as a cloning vector because I t is less than 10kb insize.

plasmid.

mutagenesis.

The double stranded replicative form of the genome acts like a

Genes cloned can be obtained in the form of single stranded DNA which is helpful in gene sequencing and in vitro

I t is easily prepared from the culture of infected E. colicells.

Single-stranded, circular genome, 6.4 kb Infect only F+ bacteria, using pilus F- coded Can clone pieces of DNA up to 6X the M13 genome size (36 kb) --

but the larger the DNA, the less stable the clone is…..

• Drawback: foreign DNA can be unstable (slows down host cell growth, so deletions confer a selective advantage)

M13 Phage Cloning Vector• The M13 genome is 6.4kb in length• Consists of ten closely packed genes for replication of the

phage.• There is a single 507 nucleotide intergenic sequence (IS) into

which new DNA can be inserted• This region includes the ori gene

Useful for– Sequencing– Site-directed mutagenesis (later)– Any other technique that requires single stranded DNA

M13 structure

Used in

‘phage display’ techniques

M 1 3 l i f ecycle: a n overvi ew

ss

ss

dsIsolate for cloning

M13 doesn’t lyse cells, but it does slow them down

M13 infections form plaques, but they are “turbid”

“lawn” ofE. coli

M13 Vectors•

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M13mp1-The lac Z gene was introduced in the IS and it does nothave any unique restriction site in the gene.

M13mp2- This vector was constructed by including the EcoR1site in the lac Z gene. This was done by a single nucleotidechange in GGATTC near the start of the lac Z gene to GAATTC by

•

in vitro mutagenesis. The β-galactosidase enzyme remainsfunctional in the vector.

M13mp7- It was formed by inserting a polylinker into the EcoR1site of lac Z gene of M13mp2. A polylinker is a short nucleotidesequence that consists of number of restriction sites (EcoR1,BamH1, Sal1 and Pst1) and has a EcoR1 sticky ends. Thispolylinker does not disrupt the function of Lac Z gene.

M13 mp18: engineered for alpha complementation

Bacteriophage vectorsAdvantages:– Useful for cloning large DNA fragments (10 - 23 kb)– Inherent size selection for large inserts

Disadvantages:– Less easy to handle

Uses of Bacteriophages:

Lambda phage cloning vectors:• For gene cloning of large DNA fragments (eukaryotic genes)• Excellent selection capability (stuffer stuff)

for library• Clone lots of precisely-sized DNA fragments construction

M13 based cloning vectors:• Single-stranded DNA• Sequencing• Site-directed mutagenesis

P h a g e m i d s / P h a s m i d s : p l a s m i d / F 1 M 1 3hybr i d s A phagemid or phasmid is a plasmid that contains an f1

origin of replication from an f1 phage. It can be used as a type of cloning vector in combination with

filamentous phage M13. A phagemid can be replicated as a plasmid, and also be packaged as single

stranded DNA in viral particles. Phagemids contain an origin of replication (ori) for double stranded

replication, as well as an f1 ori to enable single stranded replication and packaging into phage particles.

transformation and electroporation.

Many commonly used plasmids contain an f1 ori and are thus phagemids. Similarly to a plasmid, a phagemid can be used to clone DNA fragments

and be introduced into a bacterial host by a range of techniques, such as

However, infection of a bacterial host containing a phagemid with a'helper' phage, for example VCSM13 or M13K07, provides the necessaryviral components to enable single stranded DNA replication andpackaging of the phagemid DNA into phage particles.

into the cytoplasm of the host cell.

The 'helper' phage infects the bacterial host by first attaching to the hostcell's pilus and then, after attachment, transporting the phage genome

Inside the cell, the phage genome triggers production of single strandedphagemid DNA in the cytoplasm. This phagemid DNA is then packagedinto phage particles.

P h a g e m i d s / P h a s m i d s : p l a s m i d / M 1 3

The phage particles containihngyb r i d s ssDNA are released from the bacterial host cell into the extracellular environment.

Filamentous bacterial

phages growth

retardbut,

contrasting with thelambda phage and the T7 phage, are not generally lytic.

Helper phages are usuallyengineered to package lessefficiently (via a defective phageorigin of replication)\ than thephagemid so that the resultantphage particles containpredominantly phagemid DNA.

F1 Filamentous phage infectionrequires the presence of a pilusso only bacterial hostscontaining the F-plasmid or itsderivatives can be used togenerate phage particles.

A cosmid is a type of hybrid plasmid that contains a Lambda phagecos sequence.

the lambda phage.

normal bacteriophage packaging size.

Cosmids (cos sites + plasmid = cosmids) DNA sequences are originally from

They are often used as a cloning vector in genetic engineering. Cosmids can be used to build genomic libraries. They were first described by Collins and Hohn in 1978. Cosmids can contain 37 to 52 (normally 45) kb of DNA, limits based on the

They can replicate as plasmids if they have a suitable origin of replication: for example SV40 ori in mammalian cells, ColE1 ori for double-stranded DNAreplication or f1 ori for single-stranded DNA replication in prokaryotes.

be unable to grow.[3]

They frequently also contain a gene for selection such as antibiotic resistance, so that the transformed cells can be identified by plating on a medium containing the antibiotic. Those cells which did not take up the cosmid would

Unlike plasmids, they can also be packaged in phage capsids, which allowsthe foreign genes to be transferred into or between cells by transduction.

Plasmids become unstable after a certain amount of DNA has been insertedinto them, because their increased size is more conducive to recombination.To circumvent this, phage transduction is used instead. This is made possibleby the cohesive ends, also known as cos sites. In this way, they are similar tousing the lambda phage as a vector, except all the lambda genes have beendeleted with the exception of the cos sequence.

Cosmids

Features of both plasmid and lambda phage cloning vectors.

Circular.Do not occur naturallyOrigin (ori) sequence for E. coli. Selectable marker, e.g. ampR.Restriction sites (for cloning). Contain Phage (lambda) cos sites

which permits packaging into phage heads and thereforeintroduction to E. coli cells.

Packaging only occurs with 37-52 kbfragments - selection for large fragments

Useful for 37-52 kb. Packaged DNA is inserted into cells

and then replicates as a very largeplasmid

Cosmids

Cloning in a cosmid

Desired

ligation

Products-

these are

packaged

Cloning in a cosmid

Instead of transformation,

desired ligation products are

packaged and then transfected

into cells

Selection for colonies, not

screening of plaques (not

infectious)

1. Based on the E. colibacterial F-plasmid.

2. Can insert 40 kbfragment of DNA.

3. Low copy number in the host (e.g., 1 fosmid).

4. Fosmids offer higherstability than comparablehigh copy numbercosmids.

5. Contain other featuressimilar toplasmids/cosmids such asorigin sequence andpolylinker.

Fosmid:

Vectors that enable artificial chromosomes to be created and cloned into E. coli.

Features:

Useful for cloning up to 200-300 kb, but can be handled like regular bacterial plasmid vectors.

Useful for sequencing large stretches of chromosomal DNA;frequently used in genome sequencing projects.

F factor.

Like other vectors, BACs contain: Origin (ori) sequence derived from an E. coli plasmid called the

Multiple cloning sites (restriction sites). Selectable markers (antibiotic resistance).

Bacterial Artificial Chromosomes (BACs):

BACs: Bacterial Artificial Chromosomes

Based on the F factor of E. coli: 100 kb plasmid, propagates throughconjugation low copy number (1-2 copies percell) 2 genes (parA and parB): accurate partitioning during cell

division

BACs: just have par genes, replication ori, cloning sites, selectable marker

Can propagate very large pieces of DNA: up to 300 kb

Relatively easy to manipulate: move into cells by transformation (electroporation)

General BAC vector

replication

selection

Cloning, etc

7 kb

Vectors that enable artificial chromosomes to becreated and cloned into yeast.

•

• Based on the chromosome of Yeast

Features:••••

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•••

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CEN1, centromere sequencesegregationTEL, telomere sequencesextremity protection ARS1, autonomous replicating sequencereplicationSelectable marker (amino acid dependence, etc.) oneach arm.Ampori, origin of replication for propagation in an E. colihost.Restriction sites (for DNA ligation).Acquiring 150kbp it acquires chromosome like features SUP4 gene, a suppressor tRNA gene which overcomes the effect of the ade-2 ochre mutation and restores wild-type activity, resulting in colorless colonies.The host cells are also designed to have recessive trp1and ura3 alleles which can be complemented by thecorresponding TRP1 and URA3 alleles in the vector,providing a selection system for identifying cellscontaining the YAC vector.Useful for cloning very large DNA fragments up to 500kb; useful for very large DNA fragments.

YACs: Yeast Artificial Chromosomes

DESADVANTAGES OF YAC

Very fragile and prone to breakage,

Unstable, with their foreign DNA inserts often being deleted

Loss of the entire YAC during mitotic growth

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• Difficult to separate the YAC from the other host chromosomes

• The yield of DNA is not high

• Chimaerism

http://cwx.prenhall.com/bookbind/pubboo

ks/horton3/medialib/media_portfolio/23.ht

ml

1. Capable of replicating in two or more types of hosts.

2. Replicate autonomously, or integrate into the host genome and replicate when the host replicates.

3. Commonly used for transporting genes from one organism to another (i.e.,transforming animal and plant cells).

Example:

*Insert firefly

luciferase gene

into plasmid and

transform

Agrobacterium.

*Grow Agrobacterium

in large quantities

and infect tobacco

plant.

Shuttle vectors:

What determines the choice of vector

• insert size

vector size

restriction sites

copy number

cloning efficiency

ability to screen for inserts

Not all vectors permit

the identification of the desired

clones by simple selection or

color based strategies.

In the majority of cases we

need alternative approaches!!!!

References

1. Glover D.M. and Hames B.D. (1995). P Cloning 1 and 2. IRL Press.

2. Primrose et al., (2001). Principles of gene manipulation. 6th edition, Blackwell

Scientific Publishers.

3. T.A. Brown. (2010). Gene cloning and DNA analysis: an introduction. 6 th edition,

Brown, T.A. (Terence A.), Wiley Blackwell.