Biotechnology 2007-2008

DNA Technology Recombinant DNA- DNA sequences from two different sources Genetic engineering- artificial manipulation of the genetic material of organisms, including the creation of novel genetic material Biotechnology is the development of new biological products

A Brave New World

Biotechnology today Genetic Engineering Our tool kit… manipulation of DNA if you are going to engineer DNA & genes & organisms, then you need a set of tools to work with this topic is a survey of those tools… Our tool kit…

1. Bacteria Bacteria review one-celled prokaryotes reproduce by binary fission generation every ~20 minutes 108 (100 million) colony overnight! dominant form of life on Earth incredibly diverse

How have these little guys gotten to be so diverse?? 2. Bacterial genome Single circular chromosome naked DNA no histone proteins ~4300 genes 1/1000 DNA in eukaryote Genome = all the DNA of an organism Eukaryotes • 1000 times more DNA • only 10 times more genes - introns, spacers, inefficiency How have these little guys gotten to be so diverse??

3. Transformation Bacteria are opportunists pick up naked foreign DNA wherever it may be hanging out have surface transport proteins that are specialized for the uptake of naked DNA incorporate the DNA bits into their own chromosome mix heat-killed pathogenic & non-pathogenic bacteria While 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

4. Plasmids Small supplemental circles of DNA carry extra genes 5000 - 20,000 base pairs self-replicating carry extra genes 2-30 genes genes for antibiotic resistance can be exchanged between bacteria rapid evolution can be imported from environment Mini-chromosomes

How can plasmids help us? insert new gene into plasmid insert plasmid into bacteria = vector bacteria now expresses new gene bacteria make new protein transformed bacteria gene from other organism recombinant plasmid vector plasmid cut DNA + glue DNA

 5. How do we cut DNA? Restriction enzymes restriction endonucleases evolved in bacteria to cut up foreign DNA “restricts” the action of the attacking organism protection against viruses & other bacteria bacteria protect their own DNA by methylation 

Restriction enzymes Madam I’m Adam Action of enzyme cut DNA at specific sequences restriction site symmetrical “palindrome” produces protruding ends sticky ends will bind to any complementary DNA Many different enzymes named after organism they are found in EcoRI, HindIII, BamHI, SmaI  CTGAATTCCG GACTTAAGGC  CTG|AATTCCG GACTTAA|GGC

chromosome want to add gene to 6. 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

7. Cloning Genes Fast! PCR polymerase chain reaction alternative method for cloning a gene used primarily when the source of DNA is scanty or impure applications: ancient DNA amplify DNA from fingerprints or tiny amounts of blood, tissue, or semen found at crime scenes amplify DNA from embryonic cells to test for genetic disorders

8. Gel Electrophoresis-find the gene

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Applications of Biotechnology Forensics Environmental use- oil- eating bacteria Gene Therapy- replace damaged cells with healthy ones Transgenic Animals -Pharm Animals- have animals produce human products Genetically Modified Organisms- more meat, disease and pest resistant etc

Blood from defendant’s blood (D) Blood from defendant’s clothes Victim’s blood (V) LE 20-17 DNA Blueprints are bands of RFLP markers Looks like defendant is heading for jail- why? What blood evidence likely convicted him/her?

Environmental Uses Genetically microbes can extract heavy metals to clean up toxic wastes in mining Bacteria can degrade oil in oil spills Bacteria can degrade plastics

Transgenic Organisms Desired gene is implanted in fertilized egg “Pharm” animals can produce large amounts of desired gene

Examples of Trangenic Animals E. coli bacteria producing insulin Spider silk in goat’s milk Knock Out mice to study human disease GFP ( green flourescent protein) as a marker

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 For 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.

Regular zebras Glo- fish zebras

Jelly fish GFP-green fluorescent Transformed vertebrates protein markers Green with envy?? Transformed vertebrates