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Genetic Engineering Biotechnology 2006-2007 (c) define the term recombinant DNA; (d) explain that genetic engineering involves the extraction of genes from one organism, or the manufacture of genes, in order to place them in another organism (often of a different species) such that the receiving organism expresses the gene product; (e) describe how sections of DNA containing a desired gene can be extracted from a donor organism using restriction enzymes; (i) explain how isolated DNA fragments can be placed in plasmids, with reference to the role of ligase; (j) state other vectors into which fragments of DNA may be incorporated; (k) explain how plasmids may be taken up by bacterial cells in order to produce a transgenic microorganism that can express a desired gene product; (l) describe the advantage to microorganisms of the capacity to take up plasmid DNA from the environment; (m) outline how genetic markers in plasmids can be used to identify the bacteria that have taken up a recombinant plasmid; We have been manipulating DNA for generations! Artificial breeding creating new breeds of animals & new crop plants to improve our food Animal breeding Breeding food plants “Descendants” of the wild mustard the “Cabbage family” Breeding food plants Evolution of modern corn (right) from ancestral teosinte (left). A Brave New World The code is universal Since all living organisms… use the same DNA use the same code book read their genes the same way TACGCACATTTACGTACGCGGATGCCGCGACTATGATCACAT AGACATGCTGTCAGCTCTAGTAGACTAGCTGACTCGACTAGC ATGATCGATCAGCTACATGCTAGCACACYCGTACATCGATCC TGACATCGACCTGCTCGTACATGCTACTAGCTACTGACTCAT GATCCAGATCACTGAAACCCTAGATCGGGTACCTATTACAGT ACGATCATCCGATCAGATCATGCTAGTACATCGATCGATACT GCTACTGATCTAGCTCAATCAAACTCTTTTTGCATCATGATA CTAGACTAGCTGACTGATCATGACTCTGATCCCGTAGATCGG GTACCTATTACAGTACGATCATCCGATCAGATCATGCTAGTA CATCGATCGATACTGCTACTGATCTAGCTCAATCAAACTCTT TTTGCATCATGATACTAGACTAGCTGACTGATCATGACTCTG ATCCCGTAGATCGGGTACCTATTACAGTACGATCATCCGATC AGATCATGCTAGTACATCGATCGATACT human genome 3.2 billion bases Can we mix genes from one creature to another? YES! Mixing genes for medicine… Allowing organisms to produce new proteins bacteria producing human insulin bacteria producing human growth hormone How do we do mix genes? Genetic engineering find gene cut DNA in both organisms paste gene from one creature into other creature’s DNA insert new chromosome into organism organism copies new gene as if it were its own organism reads gene as if it were its own organism produces NEW protein: Remember: we all use the same genetic code! Cutting DNA DNA “scissors” enzymes that cut DNA restriction enzymes used by bacteria to cut up DNA of attacking viruses EcoRI, HindIII, BamHI cut DNA at specific sites enzymes look for specific base sequences GTAACG|AATTCACGCTT GTAACGAATTCACGCTT CATTGCTTAA|GTGCGAA CATTGCTTAAGTGCGAA Restriction enzymes Cut DNA at specific sites leave “sticky ends” restriction enzyme cut site GTAACGAATTCACGCTT CATTGCTTAAGTGCGAA restriction enzyme cut site GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA Sticky ends Cut other DNA with same enzymes leave “sticky ends” on both can glue DNA together at “sticky ends” GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA gene you want GGACCTG AATTCCGGATA CCTGGACTTAA GGCCTAT chromosome want to add gene to GGACCTG AATTCACGCTT CCTGGACTTAA GTGCGAA combined DNA Sticky ends help glue genes together cut sites gene you want cut sites TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA sticky ends AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAAisolated gene cut sites chromosome want to add gene to AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA DNA ligase joins the strands sticky ends stick together Recombinant DNA molecule chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC How can bacteria read human DNA? Why mix genes together? Gene produces protein in different organism or different individual human insulin gene in bacteria TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC “new” protein from organism ex: human insulin from bacteria aa aa aa aa aa aa aa aa aa aa bacteria human insulin Uses of genetic engineering Genetically modified organisms (GMO) enabling plants to produce new proteins Protect crops from insects: BT corn corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn) Extend growing season: fishberries strawberries with an anti-freezing gene from flounder Improve quality of food: golden rice rice producing vitamin A improves nutritional value Bacteria Bacteria are great! one-celled organisms reproduce by mitosis easy to grow, fast to grow generation every ~20 minutes Bacterial DNA Single circular chromosome only one copy = haploid no nucleus Other DNA = plasmids! bacteria chromosome plasmids There’s more… Plasmids small extra circles of DNA carry extra genes that bacteria can use can be swapped between bacteria bacterial sex!! rapid evolution = antibiotic resistance can be picked up from environment How can plasmids help us? A way to get genes into bacteria easily insert new gene into plasmid insert plasmid into bacteria = vector bacteria now expresses new gene bacteria make new protein gene from other organism cut DNA plasmid recombinant plasmid + vector glue DNA transformed bacteria Grow bacteria…make more gene from other organism recombinant plasmid + vector plasmid grow bacteria harvest (purify) protein transformed bacteria Applications of biotechnology Outline the use of genetic markers HGH gene inserted into plasmids that are resistant to certain antibiotics, for detail review p. 167 of OCR Biology 2. How ever, are there possible ‘issues’ with this type of marker DISCUSS. Suggest possible alternative marker… .. Insert a gene that cause fluorescence from jellyfish. I’m a very special pig! Got any Questions? 2006-2007