Selecting Cells with Plasmid Vector b Many cells will not take up plasmid during transformation b Cells with plasmid can be identified because original plasmid contained gene for antibiotic resistance (ampicillin) b Use medium with ampicillin – if bacteria grow then plasmid must be present b But don’t know if plasmid had DNA insert

Selecting Cells with Plasmid that Carries DNA Insert b b Use beta-galactosidase system to do blue- white screening b b Host must lack enzyme and plasmid vector must carry gene for beta-gal b b Restriction site is within beta-gal gene b b Thus, if insert occurred within beta-gal gene, enzyme not produced b b X-gal = artificial substrate added to medium that turns blue if enzyme present, otherwise bacteria are normal white color

Cloning Considerations b b Choosing a vector Plasmids limited to small molecules Bacteriophages (phages) are viruses that infect bacteria Phages can carry DNA inserts up to 15,000 nucleotides long

Cloning Considerations Choosing a host Bacteria very good, but limitations Bacteria lack ability to modify proteins and limited size of insert Yeast (Saccharomyces cere isiae) excellent for many applications Occasionally necessary to clone genes into specific animal or plant hosts – more difficult but possible

Hosts & Vectors b Host systems: - Bacterium (E.coli) - Bacterium (E.coli) - Yeast (Saccharomyces cerevisea) - Yeast (Saccharomyces cerevisea) - Insect cells - Insect cells - Mammalian (Chinese Hamster Ovary cells) - Mammalian (Chinese Hamster Ovary cells) b Cloning vectors - derived from natural replicons - derived from natural replicons - Capable of replicating and isolation from host. - Capable of replicating and isolation from host. - Contain a selectable marker to distinguish host cells containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth conditions. - Contain a selectable marker to distinguish host cells containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth conditions.

Types of Vectors b Plasmid DNA E. coli vectors, extra-chromosomal and circular E. coli vectors, extra-chromosomal and circular b Bacteriophages Phage l – clone large DNA fragments and incorporate Phage l – clone large DNA fragments and incorporate into host genome into host genome Phage M13 – allows cloned DNA to be isolated in Phage M13 – allows cloned DNA to be isolated in single-stranded form single-stranded form b Cosmids hybrids of plasmid-bacteriophage l hybrids of plasmid-bacteriophage l b Artificial chromosomes - Cloning of very large genomic fragments - Cloning of very large genomic fragments - BACs (bacterial artificial chromosomes) - BACs (bacterial artificial chromosomes) - YACs (yeast artificial chromosomes - YACs (yeast artificial chromosomes

Types of Vectors TypesInserts Plasmid<10000 bp Lambda phage10-15 kbp Cosmids45 kbp BACs300kbp YACs1000kbp

Vectors used in different Hosts b Bacteria E. coli cloning and expression vectors E. coli cloning and expression vectors eg. pGEMT from Promega; pGEX from Invitrogen eg. pGEMT from Promega; pGEX from Invitrogen pQE from Qiagen pQE from Qiagen b Yeast yeast episomal plasmids for gene expression yeast episomal plasmids for gene expression eg. PICHIA expression vectors from Invitrogen eg. PICHIA expression vectors from Invitrogen b Plants Agrobacterium tumefaciens Ti plasmid introduce genes into plants Agrobacterium tumefaciens Ti plasmid introduce genes into plants b Eukaryotic cells Plasmid vectors used for gene expression and functional studies eg. Viruses – SV 40, baculovirus, retroviruses Plasmid vectors used for gene expression and functional studies eg. Viruses – SV 40, baculovirus, retroviruses

viruses that infect bacteria viruses that infect bacteria known dsDNA sequence of ~ 50 kb known dsDNA sequence of ~ 50 kb linear double-stranded molecule with single-stranded complementary ends linear double-stranded molecule with single-stranded complementary ends cohesive termini (cos region) cohesive termini (cos region)

can accept large pieces of foreign DNA can accept large pieces of foreign DNA tremendous improvement over the years tremendous improvement over the years can be reconstituted in vitro can be reconstituted in vitro Desirable properties of λ phage:

Bacteriophage  phage genome - linear 48.5 kb genome.  Each ends consists of cos (cohesive) sites – 12 bp cos ends Cos ends allows DNA circularization in the cell  Central region of genome are non- essential portions and can be replaced by foreign DNA (up to 23kb)

Bacteriophage Bacteriophage

b b Phage particles injects linear DNA into the cell b b DNA ligate to form circle b b Replicate to form many new phage particles which are released by cell lysis and cell death b b or DNA intergrate to host genome by site-specific recombination (lysogenic phase)

Lysis plaques of  phage on E. coli bacteria. bacteriophage plaques bacteria lawn Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

M13 phage vectors 1.Replication form (RF, dsDNA) of M13 phage can be purified and manipulated like a plasmid. 2.Phage particles (ssDNA): DNA can be isolated in a single-stranded form DNA sequencing Site-directed mutagenesis Cloning (RF, like plasmid)  transfection (recombinant DNA)  growth (plating on a cell lawn)  plaques formation (slow growth)

Small plasmid vectors (pBluescript) being developed to incorporate M13 functionality Contain both the plasmid and M13 origin of replication Normally propagate as true plasmids Can be induced to form single-stranded phage particles by infection of the host cell with a helper phage. Hybrid plasmid-M13 vectors

M13 phage  M13 phage contains a circular 6.7kb ssDNA  Replicate in E. coli cells as double-stranded circles (replicative form, RF), ~ 100 copies per cell  Cells are not lysed by M13, but grow slowly.  Recombinant M13 phage can produce either - dsDNA RF can be isolated & manipulated as plasmid - dsDNA RF can be isolated & manipulated as plasmid - ssDNA isolated from phage particles in growth medium - ssDNA isolated from phage particles in growth medium ( used for DNA sequencing and site-directed mutagenesis) ( used for DNA sequencing and site-directed mutagenesis)

M13 phage cloning vectors  M13 RF containing cloned fragment (eg. M13amp18 and 19) - Transfect into E.coli cells - Transfect into E.coli cells - plating in a lawn of cells produce plaques - plating in a lawn of cells produce plaques - Plaques consist of slow growth rather than lysis of infected cells - Plaques consist of slow growth rather than lysis of infected cells - Blue-white selection using MCS and lacZ - Blue-white selection using MCS and lacZ  Hybrid plasmid – M13 vectors (eg. pBlueScript) - eveloped to incorporate M13 functionality - eveloped to incorporate M13 functionality - contain plasmid & M13 origin of replication, minus the genes for full - contain plasmid & M13 origin of replication, minus the genes for full phage life cycle. phage life cycle. - propagate as true plasmid - propagate as true plasmid - can be induced to form single-stranded phage particles by - can be induced to form single-stranded phage particles by infection of the host cell with a helper phage, provides the gene products required for ss production and packaging infection of the host cell with a helper phage, provides the gene products required for ss production and packaging

Cosmid vectors 1.Utilizing the properties of the phage l cos sites in a plasmid vector. 2.A combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the l head. 3.The insert can be kb.

Digestion Ligation Formation of a cosmid clone

small circular dsDNA that autonomously replicates apart from the chromosome of the host cell small circular dsDNA that autonomously replicates apart from the chromosome of the host cell “molecular parasites” “molecular parasites” carry one or more genes some of which confer resistance tocertain antibiotics carry one or more genes some of which confer resistance tocertain antibiotics origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host origin of replication (ORI) --- a region of DNA that allows multiplication of the plasmid within the host plasmid replication: stringent or relaxed plasmid replication: stringent or relaxed

small size small size known DNA sequence known DNA sequence high copy number high copy number a selectable marker a selectable marker a second selectable gene a second selectable gene large number of unique restriction sites large number of unique restriction sites Desirable properties of plasmids:

modified plasmids containing cos sequences modified plasmids containing cos sequences carry an ORI & an antibiotic resistance marker carry an ORI & an antibiotic resistance marker can accommodate ~35 to 45 kb of foreign DNA can accommodate ~35 to 45 kb of foreign DNA can be propagated as plasmids can be propagated as plasmids can be introduced into host by standard procedures can be introduced into host by standard procedures chief technical problems occur when used for library construction chief technical problems occur when used for library construction

JUST A SUMMARY !!! Still remember transformation?

 Means of inserting foreign DNA into the vector Ligation of the DNA into the linearized vector two or more fragments of DNA (blunt/cohesive) two or more fragments of DNA (blunt/cohesive) buffer containing ATP buffer containing ATP T4 DNA ligase T4 DNA ligase Requirements for a ligation reaction:

 Method of placing the in vitro modified DNA into the host cell Transformation into the host cell bacterial cells take up naked DNA molecules bacterial cells take up naked DNA molecules cells are made “competent” cells are made “competent” cells treated with ice-cold CaCl 2 then heat-shocked cells treated with ice-cold CaCl 2 then heat-shocked efficiency of 10 7 to 10 8 transformed colonies/μg DNA efficiency of 10 7 to 10 8 transformed colonies/μg DNA maximum transformation frequency of maximum transformation frequency of 10 -3

Electroporation of the DNA into the host cell “electric field-mediated membrane permeabilization” “electric field-mediated membrane permeabilization” high strength electric field in the presence of DNA high strength electric field in the presence of DNA protocols differ for various species protocols differ for various species efficiencies of 10 9 per μg DNA (3 kb) & 10 6 (136 kb) efficiencies of 10 9 per μg DNA (3 kb) & 10 6 (136 kb)