Lambda phage and cloning vectors

Sherko Naseri MS of Medical Virology Tehran University of Medical Science

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Introduction
Lambda phage is a type of virus that can infect E.coli. Size of gene : 48502bp Have linear double streand DNA with a single-stranded 5' extension of 12 bases at both ends although this linear DNA when entre to bacteria cell become circular! In lytic pathwey may be ss/dsDNA or RNA but in lysogenic pathwey just have DNA In genom structure have two complementary ends that call cohensive ends or COS Have two pathwey 1.lysogenic 2.lytic Lambda 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 type such as charons Lambda genom is devided to 2 part : 1 left hand 2.right hand Uniqe replication metod that this phage is use named DNA ROLLING CIRCLE Mechanism of DAN rolling circle : one strand DNA is cuted , then(leader strand)3 end become a site for nocleotid adding and in the end COS will identified with endonulease enzyme and take apart thats after that another strand is too replicated(leading straid 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

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Excisionase
binds integrase enables integrase to reverse integration process

antibiotics
Nephloxin kinolone

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fX174

T4

P22

fd

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 double-stranded

DNA phage linear genome with cohesive ends

circularizes upon entry into host

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N&CRO win

cI repressor win

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rate of production of cro and cI gene products determines if lysogeny or lytic cycle occurs
c&c are late primary gene

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Controlled lytic or lysogenic events can be achieved by using vectors such as Xgtll or XSV2 in which the temperaturesensitive cI repressor mutation (cI857) is introduced.

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

There are several classes of vectors in use:

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

2. Phage l: 5-20 kb fragments (its own genome is only 50 kb!) Commonly used in making genomic libraries. (very high efficiency of transfection( is the process of deliberately introducing nucleic acids into cells)) 3. Cosmids: 35-45 kb similar to plasmids (high efficiency for 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 of DNA into many copes. A DNA fragment must be inserted into a cloning vector. A cloning vector is a DNA molecule that has an origin of replication and is capable of replicating in a bacterial cell.
Most vectors are genetically engineered plasmids

or phages. There are also cosmid vectors, bacterial artificial chromosomes, and yeast artificial 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 host cell. 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.
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Plasmid Polylinkers and Marker Genes for Blue-White screening

A vector usually contains a sequence (polylinker) which

can recognize several restriction enzymes so that the vector can be used for cloning a variety of DNA samples. Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue. Example: pUC19 Resistant to ampicillin, has (ampr gene) Contains portion of the lac operon which codes for betagalactosidase. X-gal is a substrate of beta-galactosidase and turns blue in the presence of functional beta-galactosidase is added to the medium. Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and 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 is put in. The stuffer fragment keeps the vector at a correct size and carries marker genes that are removed when foreign DNA is inserted into the vector. Example: Charon 4A Lambda When Charon 4A Lambda is intact, beta-galactosidase reacts with Xgal 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-galactosidase is 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 plasmid and Lambda systems. They are predominantly plasmids with a bacterial oriV, an antibiotic selection marker and a cloning site, but they carry one, or more recently two cos sites derived from bacteriophage lambda. Cleaved cosmids are mixed with foreign DNA that has been cleaved with the same endonuclease. Recombinant cosmids are packaged into lambda caspids Recombinant cosmid is injected into the bacterial cell where the rcosmid arranges into a circle and replicates as a plasmid. It can be maintained and recovered just as plasmids.

Shown above is a 50,000 base-pair long DNA molecule bound with six EcoRI molecules, and imaged using the atomic force microscope. This image clearly indicates the six EcoRI "sites" and allows an accurate restriction enzyme map of the cosmid to be generated. http://homer.ornl.gov/cbps/afmimaging.htm

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Methods for Integration of DNA in Host Organisms

Transformation of prokaryote cells
Electroporation Optoporation(Laser-Assisted Permeation of Vertebrate Cell

Membranes) Chemical: Incubation in CaCl2 buffer and heat shock damage of bacterial cell walls and intrusion of foreign DNA

Transformation of yeasts cells

Electroporation Phages Chemical: Lithium acetate mediated transformation

plasma membrane and polyglycan shell become permeable for DNA

Transfection of mammalian cells

Electroporation Chemical: Calcium phosphate mediated transfection

uptake of calcium DNA complexes via endocytosis Liposomal transfection: Endocytosis of liposomes

Electroporation device with electroporation cuvettes

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Vectors for larger DNA fragments

l vectors
- Can insert fragments of DNA up to 25 kb. - Can introduce into cells at a very 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 of replication,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 factor of E.coli is capable of handling large segments of DNA. Recombinant BACs are introduced into E.coli by electroportation ( a brief highvoltage current). Once in the cell, the rBAC replicates like an F factor. Example: pBAC108L Has a set of regulatory genes, OriS, and repE which control F-factor replication, and parA and parB which limit 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 as plasmids in bacteria when no foreign DNA is present. Once a fragment is inserted, YACs are transferred to cells, they then replicate as eukaryotic chromosomes. YACs contain: a yeast centromere, two yeast telomeres, a bacterial origin of replication, and bacterial selectable markers. YAC plasmidYeast chromosome DNA is inserted to a unique restriction site, and cleaves the plasmid with another restriction endonuclease that removes a fragment of DNA and causes the YAC to become linear. Once in the cell, the rYAC replicates as a chromosome, also replicating the foreign DNA.

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Phage l
Cloning Vectors plasmids viruses bacteriophage lambda (l) filamentous (ssDNA) combination

large phage that infects E. coli phage attaches to bacteria and injects DNA complex genetics and life cycle with two phases: lytic lysogeny

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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)

Insertion Vector

Replacement Vector

accommodates up to 7-10 kb foreign DNA (depending on vector)

13 kb stuffer fragment (lysogeny genes) discarded accommodates 11-20 kb foreign DNA

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Genetic studies of specialized transducing bacteriophages showed, however,that the central one-third of the genome, i.e., the region between the J and N genes, is not essential for lytic growth. The presence of a nonessential middle fragment of the phage genome was also revealed during construction of viable deletion mutants

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Lambda phage DNA isaolation

neednt to cell culture and cell concentrate Can use of infected bacteria with phage A bige problem!! There need to vast amount of phage for extract DNA Expected number of phage is 10*10 per ml This amount can get about 500ngr phage DNA for providig above, we need to bacterial culture Lambda phage commonly is a lysogenic phage For change phage strategy can use of shock cI gene is responsible for keeping lysogenic cycle With induced mutation in cI temperature sensitive gene(cIts) This shock can do with increase of temp from 30C to 42C In this temp (cIts) is inactive AND lytic pathway is run
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Phage Accumulation from culture media

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Purification of Lambda Phage DNA

Sometimes just one level of Deproteination is enough BUT in lambda phage we need a another mediate step to catch Purify DNA This mediate step is ULTRA CENTRIFUGE WITH CSCL GRADIENT

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Deproteination
After centrifuge and cached lambda phage concentart , in next level we should eliminate the capsids. 1.adding K proteinase or pronase for diget capside protein 2.adding phenol or phenol/cloroform compound(1:1) The latest step is centrifuge
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DNA DETECTION
Detection and identification of product done with Electrophoresis use of labeled probe(Bloting)(autoradiography) ETIDIOMBROMIDE is used for detection of DNA in CSCL gradient OR gel electrophoresis

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DNA Modification Enzymes

Neuclease i. Endonuclease ii. Exonuclease Ligase(T4),Linkers,Adaptors(Adherent ) Polymerase Alkaline phosphatase Polynucleotide kinase(T4) Terminal deoxynucleotidyl transferase Restriction Enzyme Topoisomerase(5P>5Ohwith ALP for adherance)
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Recombinant phage plaque selection

lacZ gene induced inactivation : galactosidase is inactivate and rPlaque become white and Normal plaque become blue cI gene induced inactivation : plaque apearance is changed,it means rPlaque become clear and nrPlaque become turbid Spi phenotype : lambda phage normally cant infect E.colis that already infeced with p2 phage Lambda genom size : 37-52bp can locate in capsid and the part less than 37 kb cant packed also with this vectors just rphages can replicating

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Library Construction in l
1 Prepare foreign DNA 2 Prepare vector DNA 3 Mix vector, foreign DNA and ligase 4 In vitro packaging 5 Infect host E. coli 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

Vector DNA
purchase pre-cut and dephosphorylated

Genomic DNA Options

preliminary Southern blots optimal enzyme (s) size fractionation adaptors

EcoRI BamHI AATTCGAACCCCTTCG GCTTGGGGAAGCCTAG

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GATCCNNNNNN---GNNNNNN---34

Library Construction in l
1 Prepare foreign DNA 2 Prepare vector DNA 3 Mix vector, foreign DNA and ligase 4 In vitro packaging 5 Infect host E. coli 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

COSLLLLLLLLG AATTCFFFFFFFG AATTCRRRRRRRRR ||||||||| ||||||||| |||||||||| LLLLLLLLCTTAA GFFFFFFFCTTAA GRRRRRRRRRCOS

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Library Construction
1 2 3 4 5 Prepare foreign DNA Prepare vector DNA Ligation reaction In vitro packaging Infect host E. coli (plate on lawn) 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

Plaque: clear zone on bacterial lawn cause by lytic phage

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Plaque assay technique for quantification of bacterial viruses.

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Plaque Lift

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Library Construction
1 2 3 4 5 Prepare foreign DNA Prepare vector DNA Ligation reaction In vitro packaging Infect host E. coli (plate on lawn) 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

punch out plaque(s) with Pasteur pipette elute phage particles from agar re-plate and re-screen as needed
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Plaque hybridization

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Creating a genomic library using phage vectors...

Infect a bacterial cell with a recombinant phage (m.o.i. = 1) mix with uninfected bacteria and plate (CLONE)

All phage plaques taken together represent genomic library...

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 replica

plate plaques or colonies onto filter denature DNA on filter incubate with radiolabeled probe

detect cells that contain correct gene by autoradiography grow up appropriate clone

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Library Construction
1 2 3 4 5 Prepare foreign DNA Prepare vector DNA Ligation reaction In vitro packaging Infect host E. coli (plate on lawn) 6 Screen plaques 7 Plaque purification 8 Subclone fragment into plasmid

amplify cloned phage purify phage DNA excise insert ligate into plasmid
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Bacteria Protists Plants

Vector
plasmid l insertion l replacement cosmid YAC

Insert (kb)
Fish 0-5* 0-10 Amphibians 11-20 Reptiles 35-45 100-2000 Birds

Insects

*transformation efficiency with size

(10-20 kb is generally upper limit)

Mammals

105

106

107

108

109

1010

1011

base pairs per haploid genome

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Screening a Phage Library

The phage library can be screened for the presence of a desired gene by using either specific immunological or nucleic acid probes.

Immunological screening. Immunological

selection can be used for the vectors in which the desired gene is expressed as 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 for detection of gene product is not available

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Storage of Lambda Stocks

Most of the lambda strains are stable for

several 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 lambda are kept in 0.7% (vol/vol) dimethyl sulfoxide at-70C for long-term storage. Klinman and Cohen have developed a method for storage of a phage library at-70C by 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 the lambda cos ends (15). They are 4 to 6 kb in size and are specifically designed for cloning of large DNA fragments (40 to 50 kb). They have (i) a drug resistance marker, (ii) a plasmid origin of replication, (iii) a fragment carrying the ligated cohesive ends (cos) of phage lambda,and (iv) one or more unique restriction sites for cloning.

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Yeast Artificial Chromosomes (YACS)

replicates as linear chromosome 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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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 III AGCT 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 30 kb 20 kb 10 kb 5 kb
-cut out & use DNA from this region

Make recombinant DNA in appropriate vector

clone into vector

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

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Making cDNA
the reverse transcriptase is not highly processive, so you end up with some incompletelysynthesized first-strand DNA normally RNAseH is used in this step the 3 end of any particular ssDNA may not form such a hairpin one normally uses a mixture of random primers in this step this step is not necessary if you use random primers
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end result: cDNA where not all molecules are full-length

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An example: Get the gene for albinism

1. 2. 3. Mechanism based: we know defect is in an enzyme (tyrosinase) Purify enzyme, inject rabbit, pull serum (use as is or purify back IgG) From mRNA+ cells known to synthesize tyrosinase, make a cDNA expression based library (lambda)

4.
5. 6. 7.

cDNA+ clones identified by an AB screen with above probe

cDNA clone grown sequenced and shown to be 1590 bp long (only Exons) cDNA now used to back hybridize/screen a human genomic DNA library Genomic clone isolated, sequenced

8.
9.

Exon/intron arrangements worked out

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

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

**Not feasible as yet.

Coding

5 reg. elements

3 stuff

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

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Refrence
Bacteriophage Lambda as a Cloning Vectort VIJAY M. CHAUTHAIWALE,lt A. THERWATH,2 AND VASANTI V. DESHPANDEl* Methods for identification of recombinants of phage l (-galactosidase/immunodetection/nucleic acid hybridization/recombinant DNA molecules) BRIGITTE SANZEY, ODILE MERCEREAU, THERESE TERNYNCK, AND PHILIPPE KOURILSKY
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BIOMST BIOTECHNOLOGICAL TASKS FOR MICROSYSTEM TECHNOLOGY

Recombinant DNA Technology in Todays Medicine.

Shelley M. Martineau Jessica A. Matthews Catherine C. Miller Carol D. Riley Institute of TIP Productions, Inc

Gnter Roth, Felix von Stetten

Optoporation

Laser-Assisted Permeation of Vertebrate Cell Membranes A thesis submitted in partial fulfillment of the requirement for the degree of Bachelor of Science in Physics from The College of William and Mary in Virginia,
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