Chapter review…
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Transcript of Chapter review…
Chapter review…Chapter review…Gene technologyGene technology
DNA DNA recombinant recombinant technologytechnology
Genetic Genetic engineerinengineerin
gg
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
yy
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