Methods for DNA Transfer BT 201 Biotechnology Techniques I

Transferring Genes Vectors are used to move genes around Plasmids, Bacteriophage, Cosmids, YACs, BACs, Viruses are used E. coli often used to express genes that have been transferred Transformation is a common method for gene transfer

Transformation Recipient cells take up foreign DNA from surrounding media Often accomplished using plasmid vectors Artificially induced in laboratories Allows introduction of unrelated genes into bacterial expression systems Products of interest can be produced, extracted, and purified for use

Bacterial Plasmids Circular pieces of DNA that can be replicated outside the bacterial chromosome Occur in varying sizes Capable of carrying varying sizes and types of genes May produce several hundred copies in a single cell

Vector Creation Restriction enzymes are used to cut DNA to be inserted into small fragments Plasmids are cut open with REs so DNA fragments may be inserted Plasmids, DNA fragments, and DNA ligase are mixed to put it all back together: cloning Ready for transformation now

Restriction enzyme recognition sequence Restriction enzyme cuts the DNA into fragments Addition of a DNA fragment from another source DNA GCAATT T T C G G C C C G G A A A A A A T T T T A A T T Sticky end Two (or more) fragments stick together by base-pairing DNA ligase pastes the strand Recombinant DNA molecule G G G GC C C C AAAA A A TT TT TT TT AA C T TA AG

Vector Creation

Transformation Transformation was discovered in 1928 by Frederick Griffith using S. pneumoniae in mice: “Transforming Principle” In 1944 a genetic basis was for process was discovered by Avery, McLeod, and McCarty –They named the process “Transformation”

Transformation of E. coli Cells capable of taking up foreign DNA are competent –Some cells are naturally competent, some cells have to be made competent –E. coli not naturally competent Artificial competence induced using cold and cationic solutions (cold CaCl 2 ) Plasmid introduced into iced solution Cells heat shocked to force plasmid uptake Solution allowed to return to ambient temperature, pores close

Isolate DNA from two sources Cut both DNAs with the same restriction enzyme Mix the DNAs; they join by base-pairing Add DNA ligase to bond the DNA covalently Recombinant DNA plasmid E. coli Plasmid Human cell Sticky ends Gene V DNA Gene V Put plasmid into bacterium by transformation Recombinant bacterium Clone the bacterium Bacterial clone carrying many copies of the human gene

Detecting Transformation Many plasmids carry antibiotic resistance genes: R factors Used to select transformants pBestLuc plasmid contains amp r and luc genes Ampicillin resistance, Luciferase production Potential transformants plated on ampicillin-containing media Colonies exposed to luciferin solution and observed for luminescence

Manipulating Genes for Biotechnology To make products To improve crops To improve livestock To improve quality of life To treat disease

Biotechnology Applications Recombinant pharmaceutical products Transgenic animals as pharmaceutical “factories” and organ sources Transgenic crops

Bacterial chromosome Plasmid Bacterium Plasmid isolated Recombinant DNA (plasmid) Recombinant bacterium Copies of gene Clone of cells Gene for pest resistance inserted into plants Gene used to alter bacteria for cleaning up toxic waste Protein used to dissolve blood clots in heart attack therapy Copies of protein Cell multiplies with gene of interest Protein used to make snow form at higher temperature Plasmid put into bacterial cell DNA Gene of interest Gene inserted into plasmid DNA isolated Cell containing gene of interest

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