Plasmids Transformation AP Biology Bacteria Bacteria review onecelled

Plasmids & Transformation AP Biology

Bacteria § Bacteria review one-celled prokaryotes u reproduce by mitosis u § binary fission u rapid growth § generation every ~20 minutes § 108 (100 million) colony overnight! dominant form of life on Earth u incredibly diverse u AP Biology

Bacterial genome § Single circular chromosome haploid u naked DNA u § no histone proteins u ~4 million base pairs § ~4300 genes § 1/1000 DNA in eukaryote How have these little guys gotten to be so diverse? ? AP Biology

Transformation promiscuous!? § Bacteria are opportunists u pick up naked foreign DNA wherever it may be hanging out § have surface transport proteins that are specialized for the uptake of naked DNA u u mix heat-killed pathogenic & non-pathogenic bacteria import bits of chromosomes from other bacteria incorporate the DNA bits into their own chromosome § express new genes § transformation § form of recombination AP Biology mice die

Plasmids § Small supplemental circles of DNA § 5000 - 20, 000 base pairs § self-replicating u carry extra genes § 2 -30 genes § genes for antibiotic resistance u can be exchanged between bacteria § bacterial sex!! § rapid evolution u AP Biology can be imported from environment

How can plasmids help us? § A way to get genes into bacteria easily insert new gene into plasmid u insert plasmid into bacteria = vector u bacteria now expresses new gene u § bacteria make new protein gene from other organism cut DNA plasmid AP Biology recombinant plasmid + vector glue DNA transformed bacteria

Biotechnology § Plasmids used to insert new genes into bacteria cut DNA gene we want like what? …insulin …HGH …lactase cut plasmid DNA Cut DNA? DNA scissors? ligase recombinant AP Biology plasmid insert “gene we want” into plasmid. . . “glue” together

How do we cut DNA? § Restriction enzymes restriction endonucleases u discovered in 1960 s u evolved in bacteria to cut up foreign DNA u § “restrict” the action of the attacking organism § protection against viruses & other bacteria w bacteria protect their own DNA by methylation & AP Biology by not using the base sequences recognized by the enzymes in their own DNA

cut DNA at specific sequences CTGAATTCCG § restriction site symmetrical “palindrome” u produces protruding ends GACTTAAGGC u Restriction enzymes § Action of enzyme Madam I’m Adam u CTG|AATTCCG GACTTAA|GGC § sticky ends § will bind to any complementary DNA § Many different enzymes u named after organism they are found in § Eco. RI, Hind. III, Bam. HI, Sma. I AP Biology

1960 s | 1978 Discovery of restriction enzymes Werner Arber Daniel Nathans Restriction enzymes are named for the organism they come from: Eco. RI = 1 st restriction enzyme found in E. coli AP Biology Hamilton O. Smith

Restriction enzymes § Cut DNA at specific sites u leave “sticky ends” restriction enzyme cut site GTAACGAATTCACGCTT CATTGCTTAAGTGCGAA restriction enzyme cut site GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA AP Biology

Sticky ends § Cut other DNA with same enzymes u u leave “sticky ends” on both can glue DNA together at “sticky ends” GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA AP Biology 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 AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA sticky ends cut sites isolated gene 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 AP Biology 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 bacteria AP Biology human insulin

The code is universal § Since all living organisms… use the same DNA u use the same code book u read their genes the same way u AP Biology

Copy (& Read) DNA § Transformation insert recombinant plasmid into bacteria u grow recombinant bacteria in agar cultures u § bacteria make lots of copies of plasmid § “cloning” the plasmid production of many copies of inserted gene u production of “new” protein u § transformed phenotype AP Biology DNA RNA protein trait

Grow bacteria…make more gene from other organism recombinant plasmid + vector plasmid grow bacteria harvest (purify) protein AP Biology transformed bacteria

Uses of genetic engineering § Genetically modified organisms (GMO) u enabling plants to produce new proteins § Protect crops from insects: BT corn w corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn) § Extend growing season: fishberries w strawberries with an anti-freezing gene from flounder § Improve quality of food: golden rice w rice producing vitamin A AP Biology improves nutritional value

Green with envy? ? Jelly fish “GFP” AP Biology Transformed vertebrates

Cut, Paste, Copy, Find… § Word processing metaphor… u cut § restriction enzymes u paste § ligase u copy § plasmids w bacterial transformation § is there an easier way? ? u find § ? ? AP Biology

I’m a very special pig! Got any Questions? AP Biology 2007 -2008