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Lambda phage

andcloning vectors

Sherko NaseriMS of Medical Virology

Tehran University of Medical Science1

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IntroductionLambda phage is a type of virus that can infect E.coli.Size of gene : 48502bpHave linear double streand DNA with a single-stranded 5'extension of 12 bases at both

endsalthough this linear DNA when entre to bacteria cellbecome circular!In lytic pathwey may be ss/dsDNA or RNA but inlysogenic pathwey just have DNAIn genom structure have two complementary ends thatcall cohensive ends or COSHave two pathwey 1.lysogenic 2.lyticLambda is often lysogenic

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Wild type of this phage is not proper for gene

engineering and coloning , we can use of modyfied typesuch as charonsLambda genom is devided to 2 part :1 left hand 2.right hand

Uniqe replication metod that this phage is use namedDNA ROLLING CIRCLEMechanism of DAN rolling circle : one strand DNA iscuted , then(leader strand)3 end become a site fornocleotid adding and in the end COS will identified

with endonulease enzyme and take apart thatsafter that another strand is too replicated(leadingstraid became template)

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INDUCTION

triggered by drop in levels of lambda repressor caused by exposure to UV light and chemicals that cause

DNA damage

Excisionase binds integrase

enables integrase to reverse integration process

antibiotics

Nephloxin kinolone

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fX174

P22 fd

T4

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double-strandedDNA phage

linear genomewith cohesive

ends circularizes

upon entry intohost

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cI repressor win

N&CRO win

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rate of production

of cro and cI gene

products determines if

lysogeny or lytic cycle

occurs

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c&c arelate primary

gene

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Controlled lytic or lysogenic events canbe achieved by using vectors such asXgtll or XSV2 in which the temperature-sensitive cI repressormutation (cI857) is introduced.

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The DNA into which a foreign piece of DNA is cloned iscalled a VECTOR

There are several classes of vectors in use:

1. Plasmids:Accept up to ~10 kb foreign DNA

2. Phagel

: 5-20 kb fragments (its own genome is only 50kb!) Commonly used in making genomic libraries. (veryhigh efficiency oftransfection(is the process ofdeliberately introducing nucleic acids into cells))

3. Cosmids: 35-45 kb similar to plasmids (high efficiencyfor transformations)

4. YACs (Yeast Artificial Chromosomes): 300-2000 kb!(essential for cloning very large fragments)

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A vector is used to amplify a single molecule ofDNA into many copes. A DNA fragment must beinserted into a cloning vector. A cloning vector isa DNA molecule that has an origin of replication

and is capable ofreplicating in a bacterial cell.

Most vectors are genetically engineered plasmidsor phages. There are also cosmid vectors,bacterial artificial chromosomes, and yeastartificial chromosomes.

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Plasmid Cloning Vectors Plasmids are circular, double-stranded DNA

molecules that exist in bacteria and in the nuclei

of some eukaryotic cells.

They can replicate independently of the hostcell. The size of plasmids ranges from a few kb

to near 100 kb

Can hold up to 10 kb fragments

Plasmids have an origin of replication, antibiotic

resistance genes as markers, and several

unique restriction sites.

After culture growth, the clone fragment can be

recovered easily. The cells are lysed and the

DNA is isolated and purified.

A DNA fragment can be kept indefinitely if

mixed with glycerol in a

70 degrees C freezer.15sherko naseri

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Plasmid Polylinkers and Marker Genes forBlue-Whitescreening

Avector usually contains a sequence (polylinker) whichcan recognize several restriction enzymes so that thevector can be used for cloning a variety of DNA samples.

Colonies with recombinant plasmids are white, andcolonies with nonrecombinant plasmids are blue.

Example: pUC19 Resistant to ampicillin, has (amprgene) Contains portion of the lac operon which codes forbeta-

galactosidase.

X-gal is a substrate of beta-galactosidase and turns bluein the presence of functional beta-galactosidase is addedto the medium.

Insertion of foreign DNA into the polylinker disrupts thelac operon, beta-galactosidase becomes non-functionaland the colonies fail to turn blue, but appear white.

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Phage Cloning Vectors Fragments up to 23 kb can be may be accommodated by a phage

vector

Lambda is most common phage

60% of the genome is needed for lytic pathway.

Segments of the Lambda DNA is removed and a stuffer fragment isput in.

The stuffer fragment keeps the vector at a correct size and carries

marker genes that are removed when foreign DNA is inserted intothe vector.

Example: Charon 4A Lambda

When Charon 4A Lambda is intact, beta-galactosidase reacts with X-gal and the colonies turn blue.

When the DNA segment replaces the stuffer region, the lac gene is

missing, which codes for beta-galactosidase, no beta-galactosidaseis formed, and the colonies are white.

Lambda phage is proper cloning vector but not expression!!

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Cosmid Cloning Vectors Fragments from 30 to 46 kb can be accommodated

by a cosmid vector.

Cosmids combine essential elements of a plasmidand Lambda systems.

They are predominantly plasmids with a bacterialoriV, an antibiotic selection markerand acloning site,but they carry one, or more recently two cossites derived from bacteriophage lambda.

Cleaved cosmids are mixed with foreign DNA that hasbeen cleaved with the same endonuclease.

Recombinant cosmids are packaged into lambdacaspids

Recombinant cosmid is injected into the bacterial cellwhere the rcosmid arranges into a circle andreplicates as a plasmid. It can be maintained andrecovered just as plasmids.

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Shown above is a 50,000 base-pair long DNAmolecule bound with six EcoRI molecules,and imaged using the atomic forcemicroscope. This image clearly indicates thesix EcoRI "sites" and allows an accuraterestriction enzyme map of the cosmid to begenerated.

http://homer.ornl.gov/cbps/afmimaging.htm
http://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://homer.ornl.gov/cbps/afmimaging.htmhttp://homer.ornl.gov/cbps/afmimaging.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htmhttp://dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/www.brunel.ac.uk/depts/bl/project/genome/moltec/library/phagevek.htm

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Methods for Integration of DNA in Host Organisms

Transformation of prokaryote cells Electroporation

Optoporation(Laser-Assisted Permeation of Vertebrate CellMembranes)

Chemical: Incubation in CaCl2 buffer and heat shockdamage of bacterial cell walls and intrusion of foreign DNA

Transformation of yeasts cells Electroporation Phages

Chemical: Lithium acetate mediated transformationplasma membrane and polyglycan shell become permeablefor DNA

Transfection of mammalian cells Electroporation

Chemical: Calcium phosphate mediated transfectionuptake of calcium DNA complexes via endocytosis

Liposomal transfection: Endocytosis of liposomesElectroporation device with

electroporation cuvettes

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f l D A f

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Vectors for larger DNA fragmentsl vectors

- Can insert fragments of DNAup to 25 kb.

- Can introduce into cells at avery high efficiency

BAC vectors (bacterial artificial chromosomes)

- Contain sequences from the E. coli F plasmid present at one copy per cell.- Can clone up to 200-300 kb per BAC clone.

YAC vectors (Yeast artificial chromosomes)- Contains sequences required to replicate and maintain chromosome in

budding yeast (like l, end up as a linear molecules)

- a yeast origin of replication, a centromere, and a telomere at each end.

- Can clone >2,000 kb (2 Mb).

- p1 phage(lambda like/125kb)

- PACs(pi-derived artificial chromosomes)(combination of p1&BAC/300kb)

- YIp5(yeast integrative plasmid)(shuttle>E.coli-sacaromyses)E.coli origine ofreplication,URA3(a fragment of s.c)

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Bacterial Artificial Chromosomes(BACs) and

Yeast Artificial Chromosomes(YACs)

BACs can hold up to 300 kbs.

The F factorof E.coli is capable ofhandling large segments of DNA.

Recombinant BACs are introduced intoE.coli by electroportation ( a brief high-voltage current). Once in the cell, therBAC replicates like an F factor.

Example: pBAC108L

Has a set of regulatory genes, OriS,and repEwhich control F-factorreplication, and parA and parB whichlimit the number of copies to one or

two. A chloramphenicol resistance gene,

and a cloning segment.

In human genom progect widely used

YACs can hold up to 500 kbs.

YACs are designed to replicate asplasmids in bacteria when no foreign DNAis present. Once a fragment is inserted,YACs are transferred to cells, they thenreplicate as eukaryotic chromosomes.

YACs contain: a yeast centromere, twoyeast telomeres, a bacterial origin ofreplication, and bacterial selectablemarkers.

YAC plasmidYeast chromosome

DNA is inserted to a unique restriction site,and cleaves the plasmid with another

restriction endonuclease that removes afragment of DNA and causes the YAC tobecome linear. Once in the cell, the rYACreplicates as a chromosome, alsoreplicating the foreign DNA.

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Cloning Vectors

plasmids viruses bacteriophage

lambda (l) filamentous

(ssDNA) combination

large phage that infects E. coli phage attaches to bacteria andinjects DNA

complex genetics and life

cycle with two phases: lytic

lysogeny

Phage l

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Cl i V

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l as a Cloning Vector infectious l can be assembled in vitro foreign DNA can be incorporated into

the l genome non-essential genes removed phage assembly can occur with 40-52 kb of DNA

(wild-type l 50kb)

13 kb stuffer fragment (lysogenygenes) discarded

accommodates 11-20 kb foreign DNA

Insertion Vector Replacement Vector

accommodates up to 7-10 kbforeign DNA (depending onvector)

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Genetic studies of specialized transducingbacteriophages showed, however,that the centralone-third of the genome, i.e., the region betweenthe J and N genes, is not essential for lyticgrowth.The presence of a nonessential middle fragmentof the phage genome was also revealed duringconstruction of viable deletion mutants

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Lambda phage DNA isaolation

neednt to cell culture and cell concentrateCan use of infected bacteria with phageA bige problem!! There need tovast amount of phage forextract DNAExpected number of phage is 10*10 per mlThis amount can get about 500ngr phage DNAfor providig above, we need to bacterial cultureLambda phage commonly is a lysogenic phageFor change phage strategy can use ofshockcI gene is responsible for keeping lysogenic cycle

With induced mutation in cI temperature sensitivegene(cIts)This shock can do with increase of temp from 30C to 42CIn this temp (cIts) is inactive AND lytic pathway is run

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Purification of Lambda Phage DNA

Sometimes just one level ofDeproteination is enough BUT in lambdaphage we need a another mediate step to

catch Purify DNA

This mediate step is ULTRA CENTRIFUGEWITH CSCL GRADIENT

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After centrifuge and cachedlambda phage concentart , in nextlevel we should eliminate thecapsids.1.adding K proteinase or pronase

for diget capside protein2.adding phenol orphenol/cloroform compound(1:1)The latest step is centrifuge

Deproteination

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DNA DETECTIONDetection and identification ofproduct done with Electrophoresis

use of labeledprobe(Bloting)(autoradiography)ETIDIOMBROMIDE is used fordetection of DNA in CSCL gradient

OR gel electrophoresis

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DNA Modification Enzymes

Neucleasei. Endonucleaseii. Exonuclease Ligase(T4),Linkers,Adaptors(Adherent ) Polymerase Alkaline phosphatase Polynucleotide kinase(T4) Terminal deoxynucleotidyl transferase Restriction Enzyme Topoisomerase(5P>5Ohwith ALP for

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Recombinant phage plaque selection

lacZ gene induced inactivation : -galactosidase is inactivate and rPlaque become

white and Normal plaque become bluecI gene induced inactivation : plaque

apearance is changed,it means rPlaque becomeclear and nrPlaque become turbidSpi phenotype : lambda phage normally cantinfect E.colis that already infeced with p2 phageLambda genom size : 37-52bp can locate in

capsid and the part less than 37 kb cant packedalso with this vectors just rphages can replicating

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1 Prepare foreign DNA

2 Prepare vector DNA3 Mix vector, foreign DNA and

ligase4 In vitropackaging5 Infect host E. coli6 Screen plaques

7 Plaque purification8 Subclone fragment into

plasmid

Library Construction in lVector DNA purchase pre-cut and

dephosphorylated

EcoRI BamHI

AATTCGAACCCCTTCG GATCCNNNNNN----

GCTTGGGGAAGCCTAG GNNNNNN----

Genomic DNA Options preliminary Southern blots

optimal enzyme (s) size fractionation adaptors

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1 Prepare foreign DNA

2 Prepare vector DNA3 Mix vector, foreign DNA and

ligase4 In vitropackaging5 Infect host E. coli6 Screen plaques

7 Plaque purification8 Subclone fragment into

plasmid

Library Construction in l

COSLLLLLLLLGAATTCFFFFFFFGAATTCRRRRRRRRR

||||||||| ||||||||| ||||||||||

LLLLLLLLCTTAA GFFFFFFFCTTAA GRRRRRRRRRCOS

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Plaque assay technique

for quantification ofbacterial viruses.

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Plaque Lift


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Library Construction

1 Prepare foreign DNA2 Prepare vector DNA3 Ligation reaction4 In vitropackaging5 Infect host E. coli(plate on

lawn)6 Screen plaques

7 Plaque purification8 Subclone fragment intoplasmid

punch out plaque(s)with Pasteur pipette

elute phage particlesfrom agar

re-plate and re-screenas needed

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Plaque hybridization

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(CLONE)

All phage plaques takentogether represent genomiclibrary...

Infect a bacterial cell with arecombinant phage

(m.o.i. = 1) mix with uninfectedbacteria and plate

Creating a genomic libraryusing phage vectors...


li l t

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replica plateplaques orcolonies onto

filter denature DNAon filter

incubate with

radiolabeledprobe

detect cells thacontain correct

gene byautoradiography

grow upappropriateclonesherko naseri

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Library Construction

1 Prepare foreign DNA2 Prepare vector DNA3 Ligation reaction4 In vitropackaging5 Infect host E. coli(plate on

lawn)6 Screen plaques7 Plaque purification8 Subclone fragment into

plasmid

amplify cloned phage purify phage DNA excise insert ligate into plasmid

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Mammals

Birds

Reptiles

Amphibians

Fish

Insects

Plants

Protists

Bacteria

105 107 108 109 1010 1011106

base pairs per haploid genome

Vector Insert kbplasmid 0-5*

l insertion 0-10l replacement 11-20cosmid 35-45YAC 100-2000*transformation efficiency with size(10-20 kb is generally upper limit)

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Screening a Phage LibraryThe phage library can be screened for the presence of adesired gene by using either specific immunological ornucleic acid probes.

Immunological screening. Immunologicalselection can beused for the vectors in which the desired gene is expressedas a protein (antigen)

Nucleic acid probes.Screening with nucleic acid probes is convenient both when the

cloned gene is not expressed and when an easy method fordetection of gene product is not available

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Storage of Lambda Stocks

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Storage of Lambda Stocks

Most of the lambda strains are stable forseveral years

when stored at 4C in SM buffer(NaCl,MgSO4,Tris pH 7.5, Gelatin) containing 0.3%

freshly distilled chloroform

The master stocks of bacteriophage lambdaare kept in 0.7% (vol/vol) dimethyl sulfoxideat-70C for long-term storage.

Klinman and Cohen have developed amethod for storage of a phage library at-70Cby using top agar containing 30% glycerol.

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Cosmids

cosmid vectors are plasmids with cos sequences cos sequence permits in vitro packaging infection produces colonies

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Cosmids (e.g., pWE15 and pWE16[118]) are plasmids containing thelambda cos ends (15). They are 4 to 6kb in size and are specifically designedfor cloning of large DNA fragments (40to 50 kb). They have (i) a drugresistance marker, (ii) a plasmid originof replication, (iii) a fragment carryingthe ligated cohesive ends (cos) of

phage lambda,and (iv) one or moreunique restriction sites for cloning.

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Yeast Artificial Chromosomes (YACS)

replicates as linearchromosome in yeast

can incorporate 100 kb

- >2 Mb of foreign DNA vector contains:

bacterial ori and ampr yeast centromere and ARS ciliate telomere

yeast selectable marker

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C t ti G i Lib

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Constructing a Genomic Library- extract genomic DNA

- cut with a restriction enzyme (want only partial cutting*)

- mix with an excess of plasmid cut with the same enzyme- ligate

- transfer (transform) into bacteria.

Pick a 4-cutter enzyme ie. Hae IIIAGCT

Partial Digestion

* why would you only want partial cutting of the DNA?


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Select out pieces of ~20 kbases long, by electrophoresis

40 kb

-cut out &use DNAfrom thisregion

30 kb

20 kb

10 kb

5 kb

Make recombinant DNA inappropriate vector

cloneintovector

- you can obtain a collection of clones of different sequences that includethe entire genome of the organism

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An example: Get the gene for albinism

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An example: Get the gene for albinism1. Mechanism based: we know defect is in an enzyme (tyrosinase)

2. Purify enzyme, inject rabbit, pull serum (use as is or purify back IgG)

3. From mRNA+ cells known to synthesize tyrosinase, make a cDNA expressionbased library (lambda)

4. cDNA+ clones identified by an AB screen with above probe

5. cDNA clone grown sequenced and shown to be 1590 bp long (only Exons)

6. cDNA now used to back hybridize/screen a human genomic DNA library

7. Genomic clone isolated, sequenced

8. Exon/intron arrangements worked out

9. Structure/function studies of tyrosinase carried out (Mutagenesis of gene)

10. **FUTURE: carry out gene therapy expts to correct defect in somaticcells.

**Not feasible as yet.

5 reg. elementsCoding

3 stuff

Delivery vehicle: replication proficient, integrative?,harmless to host, tissue specificitymany others?sherko naseri

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Bacteriophage Lambda as a Cloning VectortVIJAY M. CHAUTHAIWALE,lt A. THERWATH,2 AND VASANTI V. DESHPANDEl*

Methods for identification of recombinants of phage l(-galactosidase/immunodetection/nucleic acid hybridization/recombinant DNAmolecules)BRIGITTE SANZEY, ODILE MERCEREAU, THERESE TERNYNCK, AND PHILIPPEKOURILSKY

Refrence

Recombinant DNATechnology in Todays Medicine.

Shelley M. Martineau

Jessica A. Matthews

Catherine C. Miller

Carol D. Riley

Institute of TIP Productions, Inc

BIOMST BIOTECHNOLOGICAL TASKSFOR

MICROSYSTEM TECHNOLOGY

Gnter Roth, Felix von Stetten

OptoporationLaser-Assisted Permeation of Vertebrate Cell MembranesA thesis submitted in partial fulfillment of the requirementfor the degree of Bachelor of Science in Physics fromThe College of William and Mary in Virginia,

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