Selecting Cells with Plasmid Vector

Many cells will not take up plasmid during Many cells will not take up plasmid during transformationtransformation

Cells with plasmid can be identified because Cells with plasmid can be identified because original original plasmid contained gene for antibiotic plasmid contained gene for antibiotic resistanceresistance (ampicillin) (ampicillin)

Use Use medium with ampicillin medium with ampicillin – if bacteria grow – if bacteria grow then plasmid must be presentthen plasmid must be present

But don’t know if plasmid had DNA insertBut don’t know if plasmid had DNA insert

Selecting Cells with Plasmid that Selecting Cells with Plasmid that Carries DNA InsertCarries DNA Insert

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

Cloning ConsiderationsCloning Considerations

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 ConsiderationsCloning 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 & VectorsHosts & Vectors

Host systems: Host systems: - Bacterium (- Bacterium (E.coliE.coli)) - Yeast (- Yeast (Saccharomyces cereviseaSaccharomyces cerevisea)) - Insect cells- Insect cells - Mammalian (Chinese Hamster Ovary cells)- Mammalian (Chinese Hamster Ovary cells)

Cloning vectors 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 - Contain a selectable marker to distinguish host cells

containing the vector from amongst those that do not (eg. containing the vector from amongst those that do not (eg. antibiotic resistancy or survival under certain growth antibiotic resistancy or survival under certain growth conditions.conditions.

Types of VectorsTypes of Vectors Plasmid DNAPlasmid DNA E. coliE. coli vectors, extra-chromosomal and circular vectors, extra-chromosomal and circular Bacteriophages Bacteriophages Phage l – clone large DNA fragments and incorporate Phage l – clone large DNA fragments and incorporate into host genomeinto host genome Phage M13 – allows cloned DNA to be isolated in Phage M13 – allows cloned DNA to be isolated in single-stranded formsingle-stranded form CosmidsCosmids hybrids of plasmid-bacteriophage lhybrids of plasmid-bacteriophage l Artificial chromosomesArtificial 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 VectorsTypes of Vectors

Vectors used in different HostsVectors used in different Hosts Bacteria Bacteria E. coli E. coli cloning and expression vectorscloning and expression vectors eg. pGEMT from Promega; pGEX from Invitrogeneg. pGEMT from Promega; pGEX from Invitrogen pQE from QiagenpQE from Qiagen

YeastYeast yeast episomal plasmids for gene expression yeast episomal plasmids for gene expression eg. PICHIA expression vectors from Invitrogeneg. PICHIA expression vectors from Invitrogen

PlantsPlants Agrobacterium tumefaciensAgrobacterium tumefaciens Ti plasmid introduce genes into Ti plasmid introduce genes into

plantsplants

Eukaryotic cellsEukaryotic cells Plasmid vectors used for gene expression and functional Plasmid vectors used for gene expression and functional

studies eg. Viruses – SV 40, baculovirus, retrovirusesstudies eg. Viruses – SV 40, baculovirus, retroviruses

http://dwb.unl.edu/Teacher/NSF/C08/C08Links/mbclserver.rutgers.edu/~sofer/lambdaMap.gif

• viruses that infect bacteriaviruses that infect bacteria

• known dsDNA sequence of ~ 50 kbknown dsDNA sequence of ~ 50 kb

• linear double-stranded molecule with linear double-stranded molecule with

single-stranded complementary endssingle-stranded complementary ends

• cohesive termini (cos region)cohesive termini (cos region)

• can accept large pieces of foreign DNAcan accept large pieces of foreign DNA

• tremendous improvement over the tremendous improvement over the

yearsyears

• can be reconstituted can be reconstituted in vitroin vitro

Desirable properties of Desirable properties of λλ

phage: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

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)

Lysis plaques of Lysis plaques of phage onphage on E. coliE. coli bacteria. 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 vectors1. 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)

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

M13 phage contains a circular 6.7kb ssDNAM13 phage contains a circular 6.7kb ssDNA

Replicate in Replicate in E. coli E. coli cells as double-stranded circles cells as double-stranded circles (replicative form, RF), ~ 100 copies per cell(replicative form, RF), ~ 100 copies per cell

Cells are not lysed by M13, but grow slowly.Cells are not lysed by M13, but grow slowly.

Recombinant M13 phage can produce either 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 vectorsM13 phage cloning vectors

M13 RF containing cloned fragment (eg. M13amp18 and 19)M13 RF containing cloned fragment (eg. M13amp18 and 19)

- Transfect into - Transfect into E.coli E.coli cellscells - 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)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 infection of the host cell with a helper phage, provides the gene products

required for ss production and packagingrequired 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 37-52 kb.

Digestion

Ligation

Formation of a cosmid clone

• small circular dsDNA that autonomously replicates small circular dsDNA that autonomously replicates

apart apart from the chromosome of the host cellfrom the chromosome of the host cell

• ““molecular parasites”molecular parasites”

• carry one or more genes some of which confer carry one or more genes some of which confer

resistance toresistance tocertain antibioticscertain antibiotics

• origin of replication (ORI) --- a region of DNA that origin of replication (ORI) --- a region of DNA that

allows multiplication of the plasmid within the allows multiplication of the plasmid within the

hosthost

• plasmid replication: stringent or relaxedplasmid replication: stringent or relaxed

small sizesmall size

known DNA sequenceknown DNA sequence

high copy numberhigh copy number

a selectable markera selectable marker

a second selectable genea second selectable gene

large number of unique restriction siteslarge number of unique restriction sites

Desirable properties of plasmids:Desirable properties of plasmids:

http://www-micro.msb.le.ac.uk/109/GeneticEngineering1.gif

• modified plasmids containing modified plasmids containing coscos sequences sequences

• carry an ORI & an antibiotic resistance markercarry an ORI & an antibiotic resistance marker

• can accommodate ~35 to 45 kb of foreign DNAcan accommodate ~35 to 45 kb of foreign DNA

• can be propagated as plasmidscan be propagated as plasmids

• can be introduced into host by standard can be introduced into host by standard

proceduresprocedures

• chief technical problems occur when used for chief technical problems occur when used for

library constructionlibrary construction

JUST A SUMMARY !!!JUST A SUMMARY !!!

Still remember Still remember transformation?transformation?

Means of inserting foreign DNA into the vectorMeans of inserting foreign DNA into the vector

LigationLigation of the DNA into the linearized vector 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 ATPbuffer containing ATP

• T4 DNA ligaseT4 DNA ligase

Requirements for a ligation reaction:Requirements for a ligation reaction:http://www.vivo.colostate.edu/hbooks/genetics/biotech/enzymes/

ligation.gif

Method of placing the Method of placing the in vitroin vitro modified DNA modified DNA

into the host cellinto the host cell

TransformationTransformation into the host cell into the host cell

• bacterial cells take up naked DNA moleculesbacterial cells take up naked DNA molecules

• cells are made “competent”cells are made “competent”

• cells treated with ice-cold CaClcells treated with ice-cold CaCl22 then heat-shocked then heat-shocked

• efficiency of 10efficiency of 1077 to 10 to 1088 transformed colonies/ transformed colonies/μμg DNAg DNA

• maximum transformation frequency of 10maximum transformation frequency of 10-3-3

ElectroporationElectroporation of the DNA into the host cell of the DNA into the host cell

• “ “electric field-mediated electric field-mediated

membrane membrane permeabilization” permeabilization”

• high strength electric field in the high strength electric field in the

presence of DNApresence of DNA

• protocols differ for various speciesprotocols differ for various species

• efficiencies of 10efficiencies of 1099 per per μμg DNA (3 g DNA (3

kb) kb) & 10& 1066 (136 kb) (136 kb)

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