Chapter review…Chapter review…Gene technologyGene technology

DNA DNA recombinant recombinant technologytechnology

Genetic Genetic engineerinengineerin

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Gene Gene cloningcloning

toolstools

Target Target genegene

Cloning Cloning vectorvector

RestrictioRestriction enzymen enzyme

Modifying Modifying enzymeenzyme

Host cellHost cell Polymerase Polymerase chain chain

reactionreaction

methods

Gene Gene librarylibrary

Genomic Genomic librarylibrary

cDNA cDNA librarlibrar

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Application Application

TransgenTransgenic ic

organismorganismGenetically Genetically modified modified organismorganism

DNA DNA finger finger

printingprinting

EthicsEthics

Tools…Tools…DNA DNA

(gene) (gene) sourcesource

DNA DNA cloning cloning vectorvector

Restriction Restriction enzymeenzyme

DNA DNA ligaseligase

Host Host cellcell

At the end of the lesson, you At the end of the lesson, you should be able to :should be able to :

Describe the steps in gene cloning by using plasmid as the vector.

Concepts of molecular cloning

The process of inserting a piece ofThe process of inserting a piece of DNA molecule of interestDNA molecule of interest into ainto a DNA DNA

carrier (vector) carrier (vector) in order to make in order to make multiple copies of the DNA of multiple copies of the DNA of interest interest in a host cell such asin a host cell such as

bacteria.bacteria.

Purposes of molecular Purposes of molecular cloning :cloning :◦Separate a gene from the other genes

◦Amplification of modified forms of genetic materials

◦Manipulation of a piece of DNA for further experiments

Concepts of molecular cloning

Methods in genetic engineering may be divided into three groups :◦Plasmid method◦Vector method◦Biolistic method

Plasmid method is the most familiar and frequently used techniques in cloning process.

Concepts of molecular cloning

Steps in Steps in gene gene

cloningcloningStep 1Step 1

Isolation Isolation of geneof gene

Step 2Step 2Cleave/cutCleave/cut

Step 3Step 3InsertionInsertion Step 4Step 4

Transformation Transformation and and

amplificationamplificationStep 5Step 5

ScreeninScreening g

Overall procedure

s

Step 1Step 1Isolation of geneIsolation of gene

Two different sources are selected :

a. Gene of interest.•( eg : gene coding for human insulin, etc)

b. Plasmid (as vector)

Isolation of geneIsolation of gene

Isolation of geneIsolation of gene

The source of plasmids typically E. coli.

This plasmid carries two useful genes :

◦Ampr - conferring resistance to the antibiotic ampicillin

◦LacZ - encoding the enzyme beta-galactosidase which catalyzes the hydrolysis of sugar.

Isolation of geneIsolation of gene

The plasmid has a single recognition sequence, within the lacZ gene, for the restriction enzyme used.

Isolation of geneIsolation of gene

Step 2Step 2CleaveCleave

Treat and splice DNA donor (human) and plasmid DNA bacterial using the same restriction enzyme (eg : Bam HI in this case).

Bam HI cuts the same site on both molecules (palindromic)5’- GGATCC - 3’

The ends of the cut have an over hanging piece of single-stranded DNA that called ‘sticky ends’.

Preparing plasmid vectorPreparing plasmid vector

CleaveCleave

5’

3’

G

C T T A A

A A T T C

G

G A A T T C

C T T A A G3’

5’

one DNA fragment another DNA fragment

3’

5’

CleaveCleave

Preparing plasmid vectorPreparing plasmid vector

Step 3Step 3Insertion of Insertion of

rDNArDNA

In this case, both DNA prepared have complimentary sticky ends and thus can pair with each other when mixed.

This process called ‘annealing’ and use DNA ligase (sealing enzyme) to bring these things together.

Insertion of rDNAInsertion of rDNAInsertion of rDNAInsertion of rDNA

Finally, this step formed a molecule of recombinant DNA/ recombinant plasmid (rDNA) / R-plasmid

Insertion of rDNAInsertion of rDNA

DNA fragments

+enzymes

recombinantplasmids

host cells containing recombinant plasmids

Insertion of rDNAInsertion of rDNA

Step 4Step 4Transformation Transformation

& & AmplificationAmplification

Genetically engineered plasmids are re-introduced into the bacterium.

E. coli bacteria are made to take up these plasmid, but only 1% are found to contain rDNA.

So, rDNA must be located.

Transformation & AmplificationTransformation & Amplification

Host cellHost cell Recombinant Recombinant plasmidplasmid

Calcium Calcium chloride (cold)chloride (cold)++ ++

To make the To make the bacterial cell wall bacterial cell wall

permeable to permeable to plasmidplasmid

heatheat

Treat the host cell with heat and Treat the host cell with heat and presence of CaClpresence of CaCl2 2 make the make the bacterial cell wall permeable to bacterial cell wall permeable to plasmidplasmid

Transformation & AmplificationTransformation & Amplification

Some of the recombinant plasmids Some of the recombinant plasmids enter the cytoplasm of the bacteria, enter the cytoplasm of the bacteria, this process formed genetically this process formed genetically engineered bacteria. engineered bacteria.

Bacterial cells take up the Bacterial cells take up the recombinant plasmids by recombinant plasmids by transformation.transformation.

Transformation & AmplificationTransformation & Amplification

Step 5 Step 5 ScreeningScreening

Genes are ‘expressed’ when their base sequence is being transcribed into mRNA for protein synthesis.

Many genes do so only when activated (‘switch on’).

ScreeningScreening

This creates a diverse pool of bacteria :

some bacteria that have taken up the desired r-plasmid DNA other bacteria that have taken up other DNA, both recombinant and non-recombinant.

ScreeningScreening

The vector used is a plasmid that carries two genes for antibiotic resistance.

Fortunately, plasmids often carry genetic information that makes bacterium resistant to certain antibiotics.

ScreeningScreening

So, we can grow or culture the bacterial population in the presence of that antibiotic.

Only bacteria that have taken up genetically altered plasmids will resist the antibiotic and survive.

The bacteria that have not taken up plasmid, will die (because they are not resistant to the antibiotic)

ScreeningScreening

In this case, plasmid that is used for this process is r-plasmid.

This r-plasmid has a gene for ampicillin resistance and a gene for tetracycline resistance. Bam HI is used to cut in half the tetracycline resistant gene whilst the ampicillin resistant gene is uncut.

So, resistance to tetracycline antibiotic is lost because it is inactivated.

ScreeningScreening

ScreeningScreening

Bacteria, E.coli (host cell) is cultured on nutrient agar.

Only bacteria containing recombinant plasmid/ rDNA can grow in a plated nutrient agar with antibiotic ampicillin.

This is because the ampicillin resistant gene in the bacteria is still intact (uncut) & cause the bacteria to grow in medium containing ampicillin.

ScreeningScreening

We can plate out the transformed bacteria on solid nutrient medium containing ampicillin and a sugar called X-gal.

Only bacteria that have the ampicillin-resistance plasmid will grow.

The X-gal in the medium is used to identify plasmids that carry foreign DNA.

ScreeningScreening

Bacteria with plasmids lacking foreign DNA stain blue when beta-galactosidase hydrolyzes X-gal.

Bacteria with plasmids containing foreign DNA are white because they lack beta-galactosidase.

These bacteria are LacZ - unable to hydrolyze lactose.

ScreeningScreening

In the final step, we will sort through the thousands of bacterial colonies with foreign DNA to find those containing our gene of interest.

ScreeningScreening

Carry gene of Carry gene of interestinterest Carry Carry

other other foreign foreign genegene

ScreeningScreening