4Biotechnology today Genetic Engineering Our tool kit… manipulation of DNAif you are going to engineer DNA & genes & organisms, then you need a set of tools to work withthis unit is a survey of those tools…Our tool kit…
5Bacteria Bacteria review one-celled prokaryotes reproduce by mitosis binary fissionrapid growthgeneration every ~20 minutes108 (100 million) colony overnight!dominant form of life on Earthincredibly diverse
6How have these little guys gotten to be so diverse?? Bacterial genomeSingle circular chromosomehaploidnaked DNAno histone proteins~4 million base pairs~4300 genes1/1000 DNA in eukaryoteGenome = all the DNA of an organismEukaryotes• 1000 times more DNA• only 10 times more genes- introns, spacers, inefficiencyHow have these little guys gotten to be so diverse??
7Transformation Bacteria are opportunists promiscuous!?Bacteria are opportunistspick up naked foreign DNA wherever it may be hanging outhave surface transport proteins that are specialized for the uptake of naked DNAimport bits of chromosomes from other bacteriaincorporate the DNA bits into their own chromosomeexpress new genestransformationform of recombinationmix heat-killed pathogenic & non-pathogenicbacteriaWhile E. coli lacks this specialized mechanism, it can be induced to take up small pieces of DNA if cultured in a medium with a relatively high concentration of calcium ions.In biotechnology, this technique has been used to introduce foreign DNA into E. coli.mice die
8Plasmids Small supplemental circles of DNA carry extra genes ,000 base pairsself-replicatingcarry extra genes2-30 genesgenes for antibiotic resistancecan be exchanged between bacteriabacterial sex!!rapid evolutioncan be imported from environmentMini-chromosomes
9How can plasmids help us? A way to get genes into bacteria easilyinsert new gene into plasmidinsert plasmid into bacteria = vectorbacteria now expresses new genebacteria make new proteintransformed bacteriagene from other organismrecombinant plasmidvectorplasmidcut DNA+glue DNA
10insert “gene we want” into plasmid... “glue” together BiotechnologyPlasmids used to insert new genes into bacteriacut DNAgene we wantlike what?…insulin…HGH…lactasecut plasmid DNACut DNA?DNA scissors?ligaseinsert “gene we want” into plasmid... “glue” togetherrecombinant plasmid
11 How do we cut DNA? Restriction enzymes restriction endonucleases discovered in 1960sevolved in bacteria to cut up foreign DNA“restrict” the action of the attacking organismprotection against viruses & other bacteriabacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA
12What do you notice about these phrases? radarracecarMadam I’m AdamAble was I ere I saw Elbaa man, a plan, a canal, PanamaWas it a bar or a bat I saw?go hang a salami I’m a lasagna hogpalindromes
13Restriction enzymes Madam I’m Adam Action of enzyme cut DNA at specific sequencesrestriction sitesymmetrical “palindrome”produces protruding endssticky endswill bind to any complementary DNAMany different enzymesnamed after organism they are found inEcoRI, HindIII, BamHI, SmaICTGAATTCCGGACTTAAGGCCTG|AATTCCGGACTTAA|GGC
14Discovery of restriction enzymes Werner ArberDaniel NathansHamilton O. SmithRestriction enzymes are named for the organism they come from:EcoRI = 1st restriction enzyme found in E. coliWerner Arber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences.Hamilton Smith verified Arber's hypothesis with a purified bacterial restriction enzyme and showed that this enzyme cuts DNA in the middle of a specific symmetrical sequence. Other restriction enzymes have similar properties, but different enzymes recognize different sequences. Ham Smith now works at Celera Genomics, the company who sequenced the human genome.Dan Nathans pioneered the application of restriction enzymes to genetics. He demonstrated their use for the construction of genetic maps and developed and applied new methodology involving restriction enzymes to solve various problems in genetics.
15restriction enzyme cut site restriction enzyme cut site Restriction enzymesCut DNA at specific sitesleave “sticky ends”restriction enzyme cut siteGTAACGAATTCACGCTTCATTGCTTAAGTGCGAArestriction enzyme cut siteGTAACG AATTCACGCTTCATTGCTTAA GTGCGAA
16chromosome want to add gene to Sticky endsCut other DNA with same enzymesleave “sticky ends” on bothcan glue DNA together at “sticky ends”GTAACG AATTCACGCTTCATTGCTTAA GTGCGAAgene you wantGGACCTG AATTCCGGATACCTGGACTTAA GGCCTATchromosome want to add gene toGGACCTG AATTCACGCTTCCTGGACTTAA GTGCGAAcombined DNA
17Sticky ends help glue genes together cut sitesgene you wantcut sitesTTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTTAACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAAAATTCTACGAATGGTTACATCGCCGGATGCTTACCAATGTAGCGGCTTAAisolated genesticky endsAATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTTTTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAAchromosome want to add gene tocut sitesRecombinant DNA moleculeDNA ligase joins the strandschromosome with new gene addedTAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGCsticky ends stick together
18How can bacteria read human DNA? human insulin gene in bacteria Why mix genes together?Gene produces protein in different organism or different individualhuman insulin gene in bacteriaTAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGCaa“new” protein from organismex: human insulin from bacteriabacteriahuman insulin
19The code is universal Since all living organisms… use the same DNA use the same code bookread their genes the same wayStrong evidence for a single origin in evolutionary theory.
20Copy (& Read) DNA Transformation insert recombinant plasmid into bacteriagrow recombinant bacteria in agar culturesbacteria make lots of copies of plasmid“cloning” the plasmidproduction of many copies of inserted geneproduction of “new” proteintransformed phenotypeDNA RNA protein trait
21Grow bacteria…make more transformed bacteriagene from other organism+recombinant plasmidvectorplasmidgrow bacteriaharvest (purify) protein
22Uses of genetic engineering Genetically modified organisms (GMO)enabling plants to produce new proteinsProtect crops from insects: BT corncorn produces a bacterial toxin that kills corn borer (caterpillar pest of corn)Extend growing season: fishberriesstrawberries with an anti-freezing gene from flounderImprove quality of food: golden ricerice producing vitamin A improves nutritional valueFor example, a transgenic rice plant has been developed that produces yellow grains containing beta-carotene.Humans use beta-carotene to make vitamin A.Currently, 70% of children under the age of 5 in Southeast Asia are deficient in vitamin A, leading to vision impairment and increased disease rates.
23Transformed vertebrates Green with envy??Jelly fish “GFP”Transformed vertebrates
24Cut, Paste, Copy, Find… Word processing metaphor… cut paste copy find restriction enzymespasteligasecopyplasmidsbacterial transformationis there an easier way??find????