Gene CloningMaking multiple copies of a singlegeneStep 1: Forming recombinant DNAStep 2: Transformation (In vivoamplification)Step 3: Selectionhttp://tainano.com/Molecular%20Biology%20Glossary.files/image053.gif
AnimationIntroduces gene cloning includinginformation on forming arecombinant and transformationhttp://highered.mcgraw-hill.com/olc/dl/120078/micro10.swfhttp://tainano.com/Molecular%20Biology%20Glossary.files/image053.gif
Cloning VectorPlasmid: small circular DNAfound in bacterial cells that isnot the chromosomal DNACloning vector:a plasmid into which the gene ofinterest is introducedContains a number of specificgenes useful in selection
Activity: Gene CloningIn this activity you will determine the function ofthe ampR and lacZ gene and explain its use ingene cloning
Cloning vector componentsReplication origin (ori):allows plasmid toreplicate in the host cell
Cloning vector componentsAntibiotic resistance(ampR) gene: allowscells to be resistance toampicillin (an antibiotic)Selection for host cellsthat have resistanceThus, selecting fortransformation
Cloning vector componentsβ-galactosidase (LacZ)gene: enzyme produced willchange a clear substratecalled X-gal into a blueproduct
β-galactosidase Reactionβ-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
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?
Cloning vector componentsCloning site:Where gene of interest willbe inserted (ligated)Where transcription canoccur because contains anupstream promoter
Step 1: Forming Recombinant DNAWhere would you insertthe DNA of interest so thatyou can “see” it in thebacterial cell (assume cellsare grown in X-gal)?
Step 1: Forming Recombinant DNALigate the gene ofinterest into the vectorsuch that it interrupts thelacZ geneThus β–galactosidase isnot madeQuestion: What colourwould the bacterial cellsbe if grown in X-gal?
Step 2: TransformationTransform recombinantDNA into bacterial cellAs bacterial cells multiply,the gene of interest will bereplicated with each cell
Step 2: TransformationBacteria grown inflasks of liquidmediumIncubate atoptimal growingtemperaturehttp://photos.news.wisc.edu/photos/8402/original/Shen_bacteria_flask08_8579.jpg?1286762168
Step 3: SelectionSelection: Identify colonies of bacteria containingthe recombinant DNA with gene of interestPossible bacterial clone products:A. bacteria without vectorB. bacteria with vector without the geneC. bacteria with vector with the gene of interest
Step 3: SelectionPlating: taking a sampleof the bacteria andgrowing them on platesPlates have a mediumcontaining:AntibioticsX-gal
Selection Mechanism: AntibioticResistance Select for bacterial clonesthat contain a vector(select for propertransformation) Bacteria are grown onPetri plate containing aspecific antibiotic (e.g.ampicillin)
Antibiotic ResistanceVector confersantibiotic resistant tobacteria because thevector contains anantibiotic resistantgene (ampR)Only bacterial cellsthat properlytransformed thevector will live andgrow on the plate
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
Selection Mechanisms: β-galactosidase ScreeningSelect for bacterial clonesthat contain a vector withgene of interest (select forproper ligation)Bacteria are grown onPetri plates containing X-Gal
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
Selection for successful ligationBacteria whichaccepted a vectorWITHOUT theDNA of interestwill have a workinglacZ geneGene codes forworking β-galenzyme which willprocess X-gal intoa blue product
Possible Transformation ResultsLB MediumadditionsNo vector CloningvectorRecombinantDNAAmp No growth White WhiteX-gal White Blue WhiteAmp + X-gal No growth Blue White
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
Cloning Application: Bt PlantsBacillusThuringiensis abacterium usedas a biologicalpesticideBt gene iscloned intoplants so thatthey will beresistant topests
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