10-15 - Transforming an Organism Transforming an Organism...

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Unformatted text preview: Transforming an Organism Transforming an Organism Find your gene of interest Make lots of copies of your gene­ cloning Insert it into genome of organism Determine if gene is functional Regenerate organism Genetic Engineering Genetic Engineering Find your Gene of Interest Protein/mRNA—reverse transcriptase and cDNA Reverse Transcriptase­ viral enzyme makes DNA from RNA­called cDNA Related Gene/ from other organism Find your gene in a library Make a Library Make a Library Library­ collection of fragments of genome Genomic Library—Restriction enzymes cDNA Library Insert genes into Plasmids and grow in bacteria Find your gene of interest in fragments Clone it in bacteria­ make lots of copies Restriction Enzymes Restriction Enzymes Cut DNA with sticky ends­ useful for splicing fragments together Genomic Library Genomic Library Cut DNA into lots of pieces Insert Pieces into plasmids Insert plasmids into Bacteria Grow Bacteria and Multiply fragments Probe cloned fragments to find your gene Bacterial Genome Bacterial Genome Bacteria have a single circular chromosome Also contain plasmids­ small circular DNA which are transferred during conjugation­ genetic recombination Genomic Library Genomic Library Cut DNA into lots of pieces Insert Pieces into plasmids Insert plasmids into Bacteria Grow Bacteria and Multiply fragments Probe cloned fragments to find your gene Bacterial Genome Bacterial Genome Bacteria have a single circular chromosome Also contain plasmids­ small circular DNA which are transferred during conjugation­ genetic recombination Once you have your gene reinsert into Bacteria and make lots of copies Use a reporter gene to identify recombinant colonies Usually antibiotic resistance or a reporter gene Polymerase Chain Reaction­ another method to Polymerase Chain Reaction­ another method to amplify your gene Uses a special DNA polymerase which works at high temperatures to multiply DNA quickly without Bacteria No selection needed Transform your Organism Transform your Organism Promoters/ Reporter genes Bacterial systems—Protein/enzyme production Electroporation/ChemoPoration/lipisomes Biolistics Microinjection Retroviruses—mRNA Agrobacterium tumefaciens –Plant System for transformation Agrobacterium tumefaciens Agrobacterium tumefaciens Pathogenic bacterium that infects plant tissue and induces tumors Incorporates portions of its genome into the plant genome­ causes uncontrolled growth of infected cells Desirable genes can be inserted using non­ virulent strains, along with a reporter gene ...
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