Many cells will not take up plasmid during transformation Cells with plasmid can be identified because original plasmid contained gene for antibiotic

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

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

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

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

7 Host systems: - Bacterium (E.coli) - Yeast (Saccharomyces cerevisea) - Insect cells - Mammalian (Chinese Hamster Ovary cells) Cloning vectors - derived from natural replicons - 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.

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

9 Types Inserts Plasmid <10000 bp Lambda phage kbp Cosmids 45 kbp BACs 300kbp YACs 1000kbp

10 Bacteria E. coli cloning and expression vectors eg. pgemt from Promega; pgex from Invitrogen pqe from Qiagen Yeast yeast episomal plasmids for gene expression eg. PICHIA expression vectors from Invitrogen Plants Agrobacterium tumefaciens Ti plasmid introduce genes into plants Eukaryotic cells Plasmid vectors used for gene expression and functional studies eg. Viruses SV 40, baculovirus, retroviruses

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

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

14 Bacteriophage l l 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)

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

17 plaques bacteria lawn l bacteriophage Plaques: the clear areas within the lawn where lysis and re-infection have prevented the cells from growing.

18 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)

19 Hybrid plasmid-m13 vectors 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.

20 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 - ssdna isolated from phage particles in growth medium ( used for DNA sequencing and site-directed mutagenesis)

21 - eveloped to incorporate M13 functionality - contain plasmid & M13 origin of replication, minus the genes for full phage life cycle. - propagate as true plasmid - 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 M13 RF containing cloned fragment (eg. M13amp18 and 19) - Transfect into E.coli cells - plating in a lawn of cells produce plaques - Plaques consist of slow growth rather than lysis of infected cells - Blue-white selection using MCS and lacz Hybrid plasmid M13 vectors (eg. pbluescript)

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23 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.

24 Formation of a cosmid clone Digestion Ligation

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

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

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

30 Still remember transformation?

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

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

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

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