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Gene CloningBiotechnology
Gene CloningMaking multiple copies of a singlegeneStep 1: Forming recombinant DNAStep 2: Transformation (In vivoamplifi...
AnimationIntroduces gene cloning includinginformation on forming arecombinant and transformationhttp://highered.mcgraw-h...
Cloning VectorPlasmid: small circular DNAfound in bacterial cells that isnot the chromosomal DNACloning vector:a plasmi...
Cloning vector componentsOriampR genelacZ generestriction sites
Activity: Gene CloningIn this activity you will determine the function ofthe ampR and lacZ gene and explain its use ingen...
Cloning vector componentsReplication origin (ori):allows plasmid toreplicate in the host cell
Cloning vector componentsAntibiotic resistance(ampR) gene: allowscells to be resistance toampicillin (an antibiotic)Sele...
Cloning vector componentsβ-galactosidase (LacZ)gene: enzyme produced willchange a clear substratecalled X-gal into a blue...
β-galactosidase Reactionβ-gal acts on X-Gal (a clear soluble substrate) toproduce a blue precipitateX-gal(colourless)5-br...
LacZ question The cloning vector on theright has a functioning lacZgene. What will be the colour of thebacterial cell if...
Cloning vector componentsCloning site:Where gene of interest willbe inserted (ligated)Where transcription canoccur becau...
Step 1: Forming Recombinant DNAWhere would you insertthe DNA of interest so thatyou can “see” it in thebacterial cell (as...
Step 1: Forming Recombinant DNALigate the gene ofinterest into the vectorsuch that it interrupts thelacZ geneThus β–gala...
Step 2: TransformationTransform recombinantDNA into bacterial cellAs bacterial cells multiply,the gene of interest will ...
Step 2: TransformationBacteria grown inflasks of liquidmediumIncubate atoptimal growingtemperaturehttp://photos.news.wis...
Step 3: SelectionSelection: Identify colonies of bacteria containingthe recombinant DNA with gene of interestPossible ba...
Step 3: SelectionPlating: taking a sampleof the bacteria andgrowing them on platesPlates have a mediumcontaining:Antibi...
Selection Mechanism: AntibioticResistance Select for bacterial clonesthat contain a vector(select for propertransformatio...
Antibiotic ResistanceVector confersantibiotic resistant tobacteria because thevector contains anantibiotic resistantgene ...
Selection for successful transformationhttp://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to...
Selection Mechanisms: β-galactosidase ScreeningSelect for bacterial clonesthat contain a vector withgene of interest (sel...
Selection for successful ligation Vectors contain lac Zgene that codes for theβ-galactosidase (β-gal) Vectors that have ...
Selection for successful ligationBacteria whichaccepted a vectorWITHOUT theDNA of interestwill have a workinglacZ geneGe...
Possible Transformation ResultsLB MediumadditionsNo vector CloningvectorRecombinantDNAAmp No growth White WhiteX-gal White...
Fig. 20.1
Animation: Gene cloninghttp://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html (includesantibiotic re...
Cloning Application: Flavr SavrTomatoes First genetically modified produce Genetically modified tomatoes that suppressed...
Cloning Application: Bt PlantsBacillusThuringiensis abacterium usedas a biologicalpesticideBt gene iscloned intoplants s...
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Biotechnology - Gene Cloning

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Biotechnology - Gene Cloning

  1. 1. Gene CloningBiotechnology
  2. 2. Gene CloningMaking multiple copies of a singlegeneStep 1: Forming recombinant DNAStep 2: Transformation (In vivoamplification)Step 3: Selectionhttp://tainano.com/Molecular%20Biology%20Glossary.files/image053.gif
  3. 3. AnimationIntroduces gene cloning includinginformation on forming arecombinant and transformationhttp://highered.mcgraw-hill.com/olc/dl/120078/micro10.swfhttp://tainano.com/Molecular%20Biology%20Glossary.files/image053.gif
  4. 4. Cloning VectorPlasmid: small circular DNAfound in bacterial cells that isnot the chromosomal DNACloning vector:a plasmid into which the gene ofinterest is introducedContains a number of specificgenes useful in selection
  5. 5. Cloning vector componentsOriampR genelacZ generestriction sites
  6. 6. Activity: Gene CloningIn this activity you will determine the function ofthe ampR and lacZ gene and explain its use ingene cloning
  7. 7. Cloning vector componentsReplication origin (ori):allows plasmid toreplicate in the host cell
  8. 8. Cloning vector componentsAntibiotic resistance(ampR) gene: allowscells to be resistance toampicillin (an antibiotic)Selection for host cellsthat have resistanceThus, selecting fortransformation
  9. 9. Cloning vector componentsβ-galactosidase (LacZ)gene: enzyme produced willchange a clear substratecalled X-gal into a blueproduct
  10. 10. β-galactosidase Reactionβ-gal acts on X-Gal (a clear soluble substrate) toproduce a blue precipitateX-gal(colourless)5-bromo-4-chloro-3-hydroxyindolegalactose5,5-dibromo-4,4-dichloro-indigo, an intensely blueproduct which is insolubleSpontaneousdimerization &oxidationhydrolysis
  11. 11. LacZ question The cloning vector on theright has a functioning lacZgene. What will be the colour of thebacterial cell if it has thisplasmid and is grown in X-gal? What would be the colour ofthe bacterial cell if it does nothave this plasmid and isgrown in X-gal?
  12. 12. Cloning vector componentsCloning site:Where gene of interest willbe inserted (ligated)Where transcription canoccur because contains anupstream promoter
  13. 13. Step 1: Forming Recombinant DNAWhere would you insertthe DNA of interest so thatyou can “see” it in thebacterial cell (assume cellsare grown in X-gal)?
  14. 14. Step 1: Forming Recombinant DNALigate the gene ofinterest into the vectorsuch that it interrupts thelacZ geneThus β–galactosidase isnot madeQuestion: What colourwould the bacterial cellsbe if grown in X-gal?
  15. 15. Step 2: TransformationTransform recombinantDNA into bacterial cellAs bacterial cells multiply,the gene of interest will bereplicated with each cell
  16. 16. Step 2: TransformationBacteria grown inflasks of liquidmediumIncubate atoptimal growingtemperaturehttp://photos.news.wisc.edu/photos/8402/original/Shen_bacteria_flask08_8579.jpg?1286762168
  17. 17. Step 3: SelectionSelection: Identify colonies of bacteria containingthe recombinant DNA with gene of interestPossible bacterial clone products:A. bacteria without vectorB. bacteria with vector without the geneC. bacteria with vector with the gene of interest
  18. 18. Step 3: SelectionPlating: taking a sampleof the bacteria andgrowing them on platesPlates have a mediumcontaining:AntibioticsX-gal
  19. 19. Selection Mechanism: AntibioticResistance Select for bacterial clonesthat contain a vector(select for propertransformation) Bacteria are grown onPetri plate containing aspecific antibiotic (e.g.ampicillin)
  20. 20. Antibiotic ResistanceVector confersantibiotic resistant tobacteria because thevector contains anantibiotic resistantgene (ampR)Only bacterial cellsthat properlytransformed thevector will live andgrow on the plate
  21. 21. Selection for successful transformationhttp://www.biotechlearn.org.nz/var/biotechlearn/storage/images/themes/from_genes_to_genomes/images/bacterial_transformation/4063-1-eng-AU/bacterial_transformation_large.jpg
  22. 22. Selection Mechanisms: β-galactosidase ScreeningSelect for bacterial clonesthat contain a vector withgene of interest (select forproper ligation)Bacteria are grown onPetri plates containing X-Gal
  23. 23. Selection for successful ligation Vectors contain lac Zgene that codes for theβ-galactosidase (β-gal) Vectors that have theDNA insert won’t have afunctional β-gal enzyme These bacteria, whengrown in X-gal, cannotprocess it and stayswhite
  24. 24. Selection for successful ligationBacteria whichaccepted a vectorWITHOUT theDNA of interestwill have a workinglacZ geneGene codes forworking β-galenzyme which willprocess X-gal intoa blue product
  25. 25. Possible Transformation ResultsLB MediumadditionsNo vector CloningvectorRecombinantDNAAmp No growth White WhiteX-gal White Blue WhiteAmp + X-gal No growth Blue White
  26. 26. Fig. 20.1
  27. 27. Animation: Gene cloninghttp://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html (includesantibiotic resistance info)
  28. 28. Cloning Application: Flavr SavrTomatoes First genetically modified produce Genetically modified tomatoes that suppressed agene responsible for fruit ripening Process required cloning the gene and transforming areverse-orientation copy which would have inhibitoryeffects. Video CBC: The science and controversy behind theworld’s first genetically modified produce (watch first2 minutes of 18:27): http://www.cbc.ca/archives/categories/economy-business/agriculture/genetically-modified-food-a-growing-debate/introducing-the-flavr-savr-tomato.htmlhttp://ucanr.org/repository/CAO/landingpage.cfm?article=ca.v054n04p6&fulltext=yes
  29. 29. Cloning Application: Bt PlantsBacillusThuringiensis abacterium usedas a biologicalpesticideBt gene iscloned intoplants so thatthey will beresistant topests

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