Gene CloningBiotechnology

Gene CloningMaking multiple copies of a single gene

Step 1: Forming recombinant DNAStep 2: Transformation (In vivo amplification)Step 3: Selectionhttp://tainano.com/Molecular%20Biology%20Glossary.files/image053.gif

AnimationIntroduces gene cloning including information on forming a recombinant and transformationhttp://highered.mcgraw-hill.com/olc/dl/120078/micro10.swfhttp://tainano.com/Molecular%20Biology%20Glossary.files/image053.gif

Cloning VectorPlasmid: small circular DNA found in bacterial cells that is not the chromosomal DNACloning vector: a plasmid into which the gene of interest is introduced Contains a number of specific genes useful in selection

Cloning vector componentsOriampR genelacZ generestriction sites

Activity: Gene CloningIn this activity you will determine the function of the ampR and lacZ gene and explain its use in gene cloning

Cloning vector componentsReplication origin (ori): allows plasmid to replicate in the host cell

Cloning vector componentsAntibiotic resistance (ampR) gene: allows cells to be resistance to ampicillin (an antibiotic)Selection for host cells that have resistanceThus, selecting for transformation

Cloning vector components-galactosidase (LacZ) gene: enzyme produced will change a clear substrate called X-gal into a blue product

-galactosidase Reaction-gal acts on X-Gal (a clear soluble substrate) to produce a blue precipitate

LacZ questionThe cloning vector on the right has a functioning lacZ gene.What will be the colour of the bacterial cell if it has this plasmid and is grown in X-gal?What would be the colour of the bacterial cell if it does not have this plasmid and is grown in X-gal?

Cloning vector componentsCloning site:Where gene of interest will be inserted (ligated)Where transcription can occur because contains an upstream promoter

Step 1: Forming Recombinant DNAWhere would you insert the DNA of interest so that you can see it in the bacterial cell (assume cells are grown in X-gal)?

Step 1: Forming Recombinant DNALigate the gene of interest into the vector such that it interrupts the lacZ geneThus galactosidase is not madeQuestion: What colour would the bacterial cells be if grown in X-gal?

Step 2: TransformationTransform recombinant DNA into bacterial cellAs bacterial cells multiply, the gene of interest will be replicated with each cell

Step 2: TransformationBacteria grown in flasks of liquid mediumIncubate at optimal growing temperaturehttp://photos.news.wisc.edu/photos/8402/original/Shen_bacteria_flask08_8579.jpg?1286762168

Step 3: SelectionSelection: Identify colonies of bacteria containing the recombinant DNA with gene of interest Possible 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 sample of the bacteria and growing them on plates Plates have a medium containing:AntibioticsX-gal

Selection Mechanism: Antibiotic ResistanceSelect for bacterial clones that contain a vector (select for proper transformation)Bacteria are grown on Petri plate containing a specific antibiotic (e.g. ampicillin)

Antibiotic ResistanceVector confers antibiotic resistant to bacteria because the vector contains an antibiotic resistant gene (ampR)Only bacterial cells that properly transformed the vector will live and grow 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: -galactosidase ScreeningSelect for bacterial clones that contain a vector with gene of interest (select for proper ligation)Bacteria are grown on Petri plates containing X-Gal

Selection for successful ligationVectors contain lac Z gene that codes for the -galactosidase (-gal)Vectors that have the DNA insert wont have a functional -gal enzymeThese bacteria, when grown in X-gal, cannot process it and stays white

Selection for successful ligationBacteria which accepted a vector WITHOUT the DNA of interest will have a working lacZ gene Gene codes for working -gal enzyme which will process X-gal into a blue product

Possible Transformation Results

Fig. 20.1

Animation: Gene cloninghttp://www.sumanasinc.com/webcontent/animations/content/plasmidcloning.html (includes antibiotic resistance info)

Cloning Application: Flavr Savr TomatoesFirst genetically modified produceGenetically modified tomatoes that suppressed a gene responsible for fruit ripeningProcess required cloning the gene and transforming a reverse-orientation copy which would have inhibitory effects.

Video CBC: The science and controversy behind the worlds first genetically modified produce (watch first 2 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 PlantsBacillus Thuringiensis a bacterium used as a biological pesticideBt gene is cloned into plants so that they will be resistant to pests