DNA CLONING DNA CLONING Dr.Sarookhani Dr.Sarookhani / / - - PDF document

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• DNA CLONING

DNA CLONING

Dr.Sarookhani Dr.Sarookhani

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• Dr.Sarookhani

Dr.Sarookhani

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• Dr.Sarookhani

Dr.Sarookhani

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• Dr.Sarookhani

Dr.Sarookhani

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• WAYS OF GENERATING

WAYS OF GENERATING DNA DNA FRAGMENTS

FRAGMENTS

1.

• 1. Non

Non-

• specific

specific generation generation of

• f truly

truly random random fragments fragments (by (by mechanical mechanical shearing shearing or

• r digestion

digestion with with non non-

• specific

specific nucleases) nucleases)

2.

• 2. Through

Through reverse reverse transcription transcription of

• f mRNA

mRNA into into DNA DNA

3.

• 3. Highly

Highly specific specific amplification amplification of

• f a

a chosen chosen piece piece of

• f

DNA DNA by by PCR PCR

4.

• 4. The

The use use of

• f synthetic

synthetic DNA DNA

5.

• 5. Restriction

Restriction endonucleases endonucleases digestion digestion

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• ENZYMES IN CLONING

ENZYMES IN CLONING

Cutting and joining DNA Cutting and joining DNA

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• RESTRICTION ENDONUCLEASES

RESTRICTION ENDONUCLEASES

• Enzymes

Enzymes

• Recognizes

Recognizes a a short, short, symmetrical symmetrical DNA DNA sequence sequence

• Hydrolyzes/cuts

Hydrolyzes/cuts the the DNA DNA backbone backbone in in each each strand strand

– – Specific

Specific site site within within that that sequence sequence

– – Foreign

Foreign DNA DNA is is degraded degraded into into short short fragments fragments

– – Finger

Finger printing printing

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• RESTRICTION ENDONUCLEASES

RESTRICTION ENDONUCLEASES

2.

• 2. Part

Part of

• f the

the restriction restriction-

• modification

modification defense defense mechanism mechanism against against foreign foreign DNA DNA

3.

• 3. Basic

Basic tools tools of

• f gene

gene cloning cloning

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• RESTRICTION ENDONUCLEASES

RESTRICTION ENDONUCLEASES

3 types types Type I Type I Type II Type II Type III Type III

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• TYPE II REs

TYPE II REs

• commonly

commonly used used in in cloning cloning

• recognize

recognize and and cut cut within within (or (or immediately immediately adjacent adjacent to) to) specific specific target target sequences sequences

– – generate

generate specific specific fragments fragments

• a

a small small number number

– – cut

cut the the DNA DNA at at a a defined defined distance distance (usually (usually only

• nly a

a few few bases) bases) away away from from the the recognition recognition site site

– – limited

limited applications applications

• requirement

requirement: : Mg Mg2

2+ +

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• CHARACTERIZATION AND IDENTIFICATION

CHARACTERIZATION AND IDENTIFICATION

1.

• 1. The name of the organism from which

The name of the organism from which they are obtained they are obtained

2.

• 2. Write in

Write in italics italics

• The first letter of the genus

The first letter of the genus

• The first two letters of the species

The first two letters of the species name name

3.

• 3. A suffix indicating the specific enzyme

A suffix indicating the specific enzyme from that species from that species

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• CHARACTERIZATION AND

CHARACTERIZATION AND IDENTIFICATION IDENTIFICATION

• Example:

Example:

– – Pst

PstI from I from Providencia stuartii Providencia stuartii

– – Hae

HaeI, I, Hae HaeII and II and Hae HaeIII, three different enzymes, III, three different enzymes, with different specificities from with different specificities from Haemophilus Haemophilus aegyptius aegyptius

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• THE PRODUCT OF REs DIGESTION

THE PRODUCT OF REs DIGESTION

1.

• 1. Products

Products with with protruding protruding ends ends known known as as cohesive cohesive or

• r ‘

‘sticky sticky’ ’ ends ends

• Fragments

Fragments with with unpaired unpaired single single-

• stranded

stranded sequences sequences either either at at the the 5 5’ ’ or

• r 3

3’ ’ ends ends

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• 5’-GAATTC-3’

3’-CTTAAG-5’ 5’-G-3’ 3’-CTTAA-5’ 5’-AATTC-3’ 3’-G-5’ + EcoRI 5’ overhang forms cohesive ends 5’-CTGCAG-3’ 3’-GACGTC-5’ 5’-CTGCA-3’ 3’-G-5’ 5’-G-3’ 3’-ACGTC-5’ + (a) (b) PstI 3’ overhang forms cohesive ends

THE PRODUCT OF REs DIGESTION THE PRODUCT OF REs DIGESTION

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• THE PRODUCT OF REs DIGESTION

THE PRODUCT OF REs DIGESTION

2 2. . Products with blunt ends Products with blunt ends

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• Example

Example

• Advantage

Advantage: : they they can can be be joined joined to to any any other

• ther

blunt blunt-

• ended

ended fragment fragment

• Disadvantage

Disadvantage: : less less efficiently efficiently ligated ligated

5’-CCCGGG-3’ 3’-GGGCCC-5’ 5’-CCC-3’ 3’-GGG-5’ 5’-GGG-3’ 3’-CCC-5’ + SmaI

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• Enzymes

Enzymes Recognition site Recognition site Number of Number of bases bases Ends generated Ends generated Source Source Eco EcoRI RI G/AATTC G/AATTC 6 6 5 5’ ’ sticky sticky Escherichia coli Escherichia coli RY RY13 13 Bam BamHI HI G/GATCC G/GATCC 6 6 5 5’ ’ sticky sticky Bacillus Bacillus amyloliquefaciens amyloliquefaciensH H Bgl BglII II A/GATCT A/GATCT 6 6 5 5’ ’ sticky sticky Bacillus globigii Bacillus globigii Pst PstI I CTGCA/G CTGCA/G 6 6 3 3’ ’ sticky sticky Providencia stuartii Providencia stuartii Xma XmaI I C/CCGGG C/CCGGG 6 6 5 5’ ’ sticky sticky Xanthomonas Xanthomonas malvacearum malvacearum Sma SmaI I CCC/GGG CCC/GGG 6 6 blunt blunt Serratia marcescens Serratia marcescens Sau Sau3 3A A /GATC /GATC 4 4 5 5’ ’ sticky sticky Staphylococcus aureus Staphylococcus aureus 3 3A A Alu AluI I AG/CT AG/CT 4 4 blunt blunt Arthrobacter luteus Arthrobacter luteus Not NotI I GC/GGCCGC GC/GGCCGC 8 8 5 5’ ’ sticky sticky Nocardia otitidis Nocardia otitidis-

• caviarum

caviarum Pac PacI I TTAAT/TAA TTAAT/TAA 8 8 3 3’ ’ sticky sticky Pseudomonas Pseudomonas alcaligenes alcaligenes

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• VECTOR

INSERT CIRCULAR MONOMERS LINEAR DIMERS (OR HIGHER MULTIMERS CIRCULAR DIMERS RECOMBINANT PLASMID SOME POTENTIAL PRODUCTS OF LIGATION

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• OTHER ENZYMES IN CLONING

OTHER ENZYMES IN CLONING

• Nucleases

Nucleases

• Ligases

Ligases

• Phosphatase

Phosphatase and and Kinases Kinases

• DNA

DNA synthesizing synthesizing enzymes enzymes

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• NUCLEASES

NUCLEASES

Cutting/degrading DNA Cutting/degrading DNA ENDONUCLEASES ENDONUCLEASES EXONUCLEASES EXONUCLEASES MULTIFUNCTIONAL NUCLEASES MULTIFUNCTIONAL NUCLEASES

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• LIGATION

LIGATION

• The

The next next stage stage in in gene gene cloning cloning

– – joining

joining the the DNA DNA fragment fragment to to a a vector vector molecule molecule

• DNA

DNA ligase ligase

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• OH

p OH p p OH OH p LIGATION vector recombinant molecule

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• PHOSPHATASES AND KINASES

PHOSPHATASES AND KINASES

• Removing or adding respectively phosphate groups

Removing or adding respectively phosphate groups

• Examples:

Examples:

– – alkaline phosphatase: removes the

alkaline phosphatase: removes the 5 5’ ’ terminal terminal phosphate from a DNA molecule leaving an OH phosphate from a DNA molecule leaving an OH group group

– – polynucleotide kinase: adds a phosphate group

polynucleotide kinase: adds a phosphate group to a free to a free 5 5’ ’-

• terminus

terminus

• reverses the effect of alkaline phosphatase

reverses the effect of alkaline phosphatase

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• nick

nick fragment to be inserted ligase alkaline phosphatase phosphate groups removed from vector ligase dimerized vector recircularized vector + etc linear vector no reaction vector containing insert transform into host

• rganism-host

repairs the single nick in each strand B A B A A B A B ligase p OH p OH OH OH OHOH OH OH OHOH OH OH p p USE OF ALKALINE PHOSPHATASE TO PREVENT RECIRCULARISATION OF A VECTOR

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• LINKERS AND ADAPTORS

LINKERS AND ADAPTORS

• short

short synthetic synthetic DNA DNA fragments fragments that that add add new new restriction restriction sites sites to to the the end end of

• f a

a fragment fragment

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• 5’-CCGGATCCGG-3’

3’-GGCCTAGGCC-5’ 5’-CCC-3’ 3’-GGG-5’ 5’-CCCCCGGATCCGG-3’ 3’-GGGGGCCTAGGCC-5’ 5’-CCCCCG-3’ 3’-GGGGGCCTAG-5’ 5’-CCGGATCCGG-3 3’-GGCCTAGGCC-5 Self-complementary

• ligonucleotide

Blunt-ended (SmaI) Fragment (in insert) Anneal Ligate (T4 ligase) Cut with BamHI Fragments with sticky ends (ready to ligate to compatible end vector)

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• 5’-GATCCCGGG-3’

3’-GGCCCTTAA-5’ 5’-GATCCCGGG-3’ 3’-GGCCCTTAA-5’ 5’-G-3’ 3’-CCTAG-5’ 5’-GGAATCCCGGG-3’ 3’-CCTTAGGGCCCTTAA-5’ Two synthetic, partly complementary,

• ligonucleotides

Anneal Ligate Fragment with BamHI sticky ends Fragment with EcoRI compatible ends

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• GGGGGG

CCCCC GGGG CCCCCC CCCCCC CCCCC GGGGGG GGGG

VECTOR INSERT Terminal transferase dGTP dCTP Mix and anneal Cloning using homopolymer tailing Use DNA pol Ito fill unequal Homopoymer ends HOMOPOLYMER TAILING

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• TYPES OF CLONING VECTOR

TYPES OF CLONING VECTOR

Cloning limit Cloning limit Purpose Purpose

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• TYPES OF CLONING VECTOR

TYPES OF CLONING VECTOR ( (CLONING LIMIT) CLONING LIMIT)

Plasmid : Plasmid : 3 3-

• 5

5 Kb Kb Phage : Phage : 8 8-

• 23

23 Kb Kb Cosmids : up to Cosmids : up to 40 40 Kb Kb Bacterial Artificial Chromosomes :Up to Bacterial Artificial Chromosomes :Up to 1 1 Mb Mb Yeast Artificial Chromosomes : Up to Yeast Artificial Chromosomes : Up to 1 1 Mb(eukaryot) Mb(eukaryot) Retroviral : ( for animal cells) Retroviral : ( for animal cells) Phasmids (combination of PBR Phasmids (combination of PBR322 322 & Cosmid of & Cosmid of lambda phage) lambda phage)

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• TYPES OF CLONING VECTOR

TYPES OF CLONING VECTOR (PURPOSE) (PURPOSE)

• Cloning vector (

Cloning vector ( )

– – reproduction of the DNA fragment(for gene study

reproduction of the DNA fragment(for gene study due to stability and easy ) due to stability and easy )

• Expression vector

Expression vector

– – expression of certain gene in the DNA fragment

expression of certain gene in the DNA fragment

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• Stable

Easily detected Easily isolated Self-replicating Multiple cloning sites Small CHARACTERISTICS OF A GOOD CHARACTERISTICS OF A GOOD VECTOR VECTOR

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• PLASMIDS

PLASMIDS

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• Bacterial cells may contain extra-chromosomal DNA called plasmids.

Plasmids are usually represented by small, circular DNA. (engineered plasmids can not transfer by itself)

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• Schematic drawing of a bacterium with plasmids enclosed

(1) Chromosomal DNA (2) Plasmids.

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• Selective marker is required for maintenance of plasmid in the cell. Because
• f the presence of the selective marker the plasmid becomes useful for the
• cell. Under the selective conditions, only cells that contain plasmids with

selectable marker can survive. Commonly, genes that confer resistance to various antibiotics are used as selective markers in cloning vectors. For example, genes that render cells resistant to ampicillin, neomycin, or chloramphenicolare among commonly used selectivemarkers.

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• An example of a typical plasmid vector

(PUC18 has Amp resistant and Beta Gal. marker)

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• Production of Whole Genomic library by phages or plasmids

cDNA library can also be produced

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• promoter site

multiple cloning site terminator site

• ri

bla Expression vector 4.85 kb

An expression vector. The diagram

illustrates the principal components of an expression vector

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• Bacteriophage

Bacteriophage

• (

(as a cloning vector) as a cloning vector)

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• Bacteriophage lambda (l)

Bacteriophage lambda (l)

Transcriptional switches can Transcriptional switches can regulate cellular decisions regulate cellular decisions

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• protein coat

(phage head) DNA Bacteriophage

• the phage lambda virion

the phage lambda virion

– –an icosahedral head containing the

an icosahedral head containing the 48 48. .5 5 kb linear double kb linear double-

• stranded DNA

stranded DNA genome genome

– –a long flexible tail

a long flexible tail

• the phage binds to specific receptor on the outer membrane of

the phage binds to specific receptor on the outer membrane of E. coli

• E. coli

and the viral genome is injected through the phage and the viral genome is injected through the phage’ ’s tail into the cell s tail into the cell phage tail

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• Genes are clustered by function in the

Genes are clustered by function in the lambda genome lambda genome

Recombination Control regionReplication Lysis Virus head & tail

• rigin
• R

Pint

• L

PL PRM PR PRE PR‘ tR3 tL1 tR1 tR2 t6S

att int xis red gam cIII N cI cro cII O P Q S R A…J

promoter

• perator

terminator Late control cos Not to scale!

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• Cos site for circularization of genome whithin the E.coli

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• Lysis or Lysogeny

Lysis or Lysogeny

Lysis Lysis: Infection by phage produces many

Infection by phage produces many progeny and breaks open (lyses) the host progeny and breaks open (lyses) the host bacterium bacterium

Lysogeny Lysogeny: After infection, the phage DNA

After infection, the phage DNA integrates into the host genome and resides integrates into the host genome and resides there passively there passively

No progeny No progeny No lysis of the host No lysis of the host Can subsequently lyse (lyso Can subsequently lyse (lysogeny geny) )

Bacteriophage lambda can do Bacteriophage lambda can do either.

either.

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• 43.3 kb

Immunity region EcoRI Left arm 32.7 kb Right arm 10.6 kb Lambda insertion vector gt10

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• In vitro packaging by cell lysate

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• (Used for chromosomal walking)

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• COSMIDS

COSMIDS

• a plasmid containing a

a plasmid containing a cos cos site site

– – cos

cos site: the sequence of bases of bacteriophage site: the sequence of bases of bacteriophage lambda that is cut asymmetrically during lambda that is cut asymmetrically during packaging, generating an unpaired sequence of packaging, generating an unpaired sequence of 12 12 bases at each end of the phage DNA bases at each end of the phage DNA

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• cos

ampr

• ri

BamHI pJB8 5.4 kb Structure of a cosmid

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• YEAST ARTIFICIAL CHROMOSOMES

YEAST ARTIFICIAL CHROMOSOMES (YACs) AND BACTERIAL ARTIFICIAL (YACs) AND BACTERIAL ARTIFICIAL CHROMOSOMES (BACs) CHROMOSOMES (BACs)

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• YACs AND BACs

YACs AND BACs

• vectors capable of carrying very large cloned

vectors capable of carrying very large cloned fragments fragments

– – can accommodate genomic DNA fragments of

can accommodate genomic DNA fragments of more than more than 1 1 Mb ( Mb (1 1 Mb= Mb=1000 1000kb) kb)

• cloning the entire human genes

cloning the entire human genes

• example: the cystic fibrosis gene ~

example: the cystic fibrosis gene ~ 250 250 kb kb

– – mapping the large

mapping the large-

• scale structure of large

scale structure of large genome genome

• example: the human genome project

example: the human genome project

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• Vector transfer in yeast

Vector transfer in yeast

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• HOST

HOST

1 1. . E. coli

• E. coli

well understood and conveniently well understood and conveniently manipulable manipulable organism

• rganism

2 2. . Bacillus Bacillus subtillis subtillis 3 3. . Saccharomyces Saccharomyces cerevisiae cerevisiae

– –

frequently frequently used used eukaryotic eukaryotic host host

– –

retains retains many many of

• f the

the convenient convenient features features of

• f

E E. . coli coli 4 4. . Picchia Picchia pastoris pastoris (another (another yeast) yeast) 5 5. . Cultured cells of Cultured cells of Spodoptera Spodoptera frugiperda frugiperda (an insect) (an insect) 6 6-

• Dorosophila

Dorosophila for for use use with with baculoviruses baculoviruses 7 7-

• cell

cell lines lines

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• Summary of E.coli Elite host

Summary of E.coli Elite host Cells Cells

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• Introducing vectors into host cells

Introducing vectors into host cells

• Transformation

Transformation

• transfection

transfection

• Electroporation

Electroporation

• Nucleic acid gun

Nucleic acid gun

• Protoplast fusion

Protoplast fusion

• Micro injection of DNA

Micro injection of DNA

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• TRANSFORMATION

TRANSFORMATION

• the most common hosts for simple cloning

the most common hosts for simple cloning experiment: strains of experiment: strains of E. coli

• E. coli
• treatment with solutions containing Ca

treatment with solutions containing Ca2

2+ + ions

ions (sometimes Rb (sometimes Rb+

+ and Mn

and Mn2

2+ +)

– – susceptible to take up exogenous DNA:

susceptible to take up exogenous DNA:

competent cells competent cells

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• heat shock

plasmids Ca++ Introduction of plasmid DNA into competent cells treated with calcium chloride. The CaCl2 around the cells promotes the binding of DNA to the cell’s surface and makes the cell more permeable to DNA. Upon heat shock (40ºC for 60-90 seconds) some of the bound DNA enters the cell Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++ Ca++

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• the diagram shows an electricalcircuit diagram for a simple electroporationdevice.
• a cell suspension,such as of plant protoplasts or bacteria, is placed in the cuvette.
• the capacitor is charged by closing the right-hand switch. When the capacitor has

been charged, the direct current pulse is discharged in the cuvette suspension by closing the left-hand switch.

• the DC pulse is thought both to disrupt temporarily the membrane and to

electrophoreseDNA into cells.

ELECTEROPORATION

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• TRANSFECTION

TRANSFECTION

• equivalent

equivalent to to transformation transformation

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• SELECTION OF

SELECTION OF TRANSFORMANTS/MUTANTS TRANSFORMANTS/MUTANTS

• sensitivity

sensitivity and and resistance resistance to to chemicals chemicals

• requirement

requirement for for certain certain compounds compounds for for growth growth (nutrient (nutrient requirements) requirements)

• ability

ability to to use use (breakdown) (breakdown) compounds compounds

• plaque

plaque formation/type formation/type

• specific

specific (by (by nucleic nucleic acid acid hybridization hybridization ((southern ((southern blotting)) blotting)) or

• r antibodies

antibodies (western (western blotting))) blotting)))

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• INSERTIONAL INACTIVATION

INSERTIONAL INACTIVATION

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• pBR

pBR322 322

• BamHI

BamHI

• pBR

pBR322 322

• DNA

DNA

• .

.

• Pst I

Pst I

• .

.

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• pBR322

4.4 kb recombinant Religated vector Ampicillin resistance (ampr) tetracycline resistance (tetr) B B B B B

• ri

X X Ampicillin resistant? Yes Yes Tetracycline resistance? No Yes Screening by insertional inactivation of a resistance gene

INSERTIONAL INACTIVATION INSERTIONAL INACTIVATION

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• REPLICA PLATING

REPLICA PLATING

Transfer (using an absorbent pad) Plate + ampicillin Plate + ampicillin and tetracycline These colonies have bacteria with recombinant plasmid

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• SELECTION OF TRANSFORMANTS

SELECTION OF TRANSFORMANTS IN BACTERIOPHAGE IN BACTERIOPHAGE

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• PLAQUE FORMATION/TYPE

PLAQUE FORMATION/TYPE

• bacterial

bacterial lysis lysis and and phage phage release release occurs

• ccurs:

: a a clear clear area area is is formed formed (clear (clear plaque) plaque)

• lysogeny

lysogeny: : turbid turbid plaques plaques

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• turbid

plaques clear plaques lawn of bacteria

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• Western blotting

Western blotting

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• INSERTIONAL INACTIVATION OF LAC Z GENE

(in Lac_. Neg Bacteria)

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• Lac selection

Lac selection ) )

Lac Operon Lac Operon

( (

• LacZ

LacZ

` `

• )

)

• (

(

• .

.

• IPTG

IPTG )(Isopropyl )(Isopropyl-

• thiogalactoside)

thiogalactoside)

• (

( X X-

• gal

gal

) )

• (

(

• .

.

• )

)

• (

(

• )

)

• (

(

• .

.

• LacZ

LacZ

` `

• Inserted DNA

Inserted DNA

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• blue colonies

(cells that have an intact lacZ gene) white colonies (recombinant cells with inactive lacZ genes)

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• promoter site (lac, Trp, Tac, T7)

multiple cloning site terminator site

• ri

bla Expression vector 4.85 kb An expression vector. The diagram illustrates the principal components of an expression vector

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• If lacZ is replaced by

the gene encoding the protein of interest, lactose or IPTG will stimulate the expression

• f desired proteins.

The expression vector contains the lac promoter and its neighboring lacZ gene encoding -

• galactosidase. Lactose
• r its analog IPTG will

stimulate the expression

• f -galactosidase.

PRODUCTION OF RECOMBINANT PROTEIN

(other controller regions :T7 promoter, Trp promoter,shine-Dalgarno, CI, signal peptide,…)

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• Recombinant INSULIN production

Recombinant INSULIN production

1-prepro insulin method 2- separate A & B fragment production

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