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Genetic Engineering.docx

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Biology 3593B
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Genetic EngineeringJan 1012Lecture 1IntroWhat is genetic engineeringThe use of DNA technologies to alter genes for practical purposeGenerate DNA fragmentsjoin to vectorintroduce into host cellselectRestriction enzyme and ligase step is requiredAfter transformation need to select transformed cells using markerArose from microbial and molecular genetics and first achieved 1973 at StanfordBase of modern biotechnology using organisms to modify product to improve themBiotechnology TimelineEvidence from 10000 years ago of selecting plants with best qualitieso4000BCpotatoes cultivatedo2000BCbiotechnology used to leaven bread ferment beer cheese wineo500BCmoldy soybean curds tofu first antibiotic used to treat boils1797Jenner first vaccination from cowpox lesion1857Pasteur microbe fermentation and germ theory of disease1859Darwin evolution by natural selection1865Mendel laws of inheritance science of genetics begins1953Watson and Crick DNA double helix1955Sanger insulin first Biotech drug insulin approved in 19821966Each amino acid is encoded by 3 nucleotides1973Cohen and Boyer start genetic engineering with restriction enzymes 1977 is first expression of human gene in bacteriaoStart of genetic engineering using plasmid from E coli and cloningoInsert foreign DNA fragment into plasmid using restriction enzymes and ligase to create recombinant plasmidoFrequency of transformation is 1 in 10000oUnderstand this diagram1981First transgenic animal produced1983PCR discovered by Kary Mullis thermostable Tag DNA polymerase needed1987Approval for testing of resistant tomatoes sold in 1994 Bt cotton in 19951990Human genome project launched by Collins draft published in 20021997Dolly is first animal cloned from adult cell1998Stem cells for regenerative medicine established2003Tools for sequencing viruses SARS sequenced 3 weeks after discovery2010Venter creates first synthetic cellJan 1212Lecture 2Plant Cell CulturePlant Tissue CultureWhole plant can be regenerated from isolated plants cells or tissuesoAny plant cell is plastic can survive in different conditions animal cells only at specific temperature and totipotent plant cells have whole genomic potential to regenerate whole plantPlant transformation experiments rely on tissue culture and environmentoPhysical factors light temperature gases and chemical factors cell culture mediaBasic components of Culture MediaEssential elements mineral ionsmacroelements NPKMgCaS microelements cofactors iron source ETCOrganic supplementssome vitamins and amino acidsCarbon sourcesucroseDifferent than animal cell cultureoAnimal cells require all essential amino acids plants can synthesize amino acidsoAnimal cells use glucoseMS mediumTypical composition of plant tissue culture mediumPay attention to difference between macro high level that constitute most of medium eg nitrogen in nitrates for amino acids calcium magnesium phosphate and microelements much lower concentration almost 100x less they serve as cofactors for enzymesoIron is very important because of electron transport chain cytochromesNeed it with EDTA because it is a chelator that binds iron ions so they release slowly and there is continuous supplyoOrganic supplementsvitamins and covitamins to serve as cofactors for enzymesoSucrose as carbon sourcePlant growth regulatorsNeed to add additional growth regulators or hormonesoAuxins and cytokinins promote cell division and growthoABA inhibits cell division helps formation of seedsoGibberellins promote stem elongationoEthylene contributes to fruit ripening plant growth regulator can inhibit growthDifferent types of these hormones but only one ABA and ethyleneAuxin to cytokinin ratioHigh auxin to cytokinin ratio root formationLow auxin to cytokinin ratioshoot formationIntermediate ratiocallus formationoCallus is undifferentiated tissue that develops on or around an injured or cut plant surface which can be growth in cultureCulture typesTop left picture is callus culture unorganized undifferentiated growing mass of cellsoSolid agar based medium with MSCell suspension culture top rightoCells in flasksProtoplast cultureif you degrade cell wall you get protoplast which is easier for transformation of transgene because no protectionoCell wall degraded by polysaccharidedegrading enzymes cellulase pectinase xylanaseRoot shoot tip embryo and microspore pollen or anther explants culturesInitiation and maintenance of plant suspension culturesMajor steps to establish plant suspension cultureSelect tissue leaf root etcsterilizecut and put on solid medium agar based containing MS and let it grow for 26 weeks you will get callus cultureCut the callus and put in liquid medium no agar but still MS in flasks to get cell suspensions culturegrow from 12 weeksRemove aggregates to prepare single celled suspension by filtering pipetting or pectinase which will destroy cellcell interactions
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