Biotechnology

A Brave New World

TACGCACATTTACGTACGCGGATGCCGC GACTATGATCACATAGACATGCTGTCAG CTCTAGTAGACTAGCTGACTCGACTAGC ATGATCGATCAGCTACATGCTAGCACAC YCGTACATCGATCCTGACATCGACCTGC TCGTACATGCTACTAGCTACTGACTCATG ATCCAGATCACTGAAACCCTAGATCGGG TACCTATTACAGTACGATCATCCGATCAG ATCATGCTAGTACATCGATCGATACTGCT ACTGATCTAGCTCAATCAAACTCTTTTTG CATCATGATACTAGACTAGCTGACTGATC ATGACTCTGATCCCGTAGATCGGGTACCT ATTACAGTACGATCATCCGATCAGATCAT GCTAGTACATCGATCGATACTGCTACTGA TCTAGCTCAATCAAACTCTTTTTGCATCA TGATACTAGACTAGCTGACTGATCATGAC TCTGATCCCGTAGATCGGGTACCTATTAC AGTACGATCATCCGATCAGATCATGCTAG TACATCGATCGATACT human genome 3.2 billion bases ~30,000 genes

Biotechnology Genetic engineering ◦ Manipulation of DNA ◦ If you are going to engineer DNA, genes, & organisms, then you will need a set of tools to work with

Bacteria Bacteria review ◦ One-celled prokaryotes ◦ Reproduce by mitosis  Binary fission ◦ Rapid growth  Generation every ~20 minutes  10 8 (100 million) colony population overnight ◦ Dominant form of life on Earth ◦ Incredibly diverse

Bacterial genome Single, circular chromosome ◦ Haploid ◦ Naked DNA  No histone proteins ◦ ~4 million base pairs  ~4300 genes  1/1000 DNA in eukaryotes How have these little guys gotten to be so diverse??

Transformation mix heat-killed pathogenic & non-pathogenic bacteria mice die 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 ◦ Import bits of chromosomes from other bacteria ◦ Incorporate the DNA bits into their own chromosome  Express new genes  Called transformation  A form of genomic recombination

Plasmids Small supplemental circles of DNA  5000 – 20,000 base pairs  Self-replicating ◦ Carry extra genes  2 – 30 genes  Genes for antibiotic resistance ◦ Can be exchanged between bacteria  Bacterial sex!  Rapid evolution ◦ Can be imported from the 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 expressed new gene!  Bacteria will make the new protein + transformed bacteria gene from other organism plasmid cut DNA recombinant plasmid vector glue DNA

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

How do we cut DNA? Restriction enzymes ◦ Restriction endonucleases  Discovered in 1960s ◦ Evolved in bacteria to cut up foreign DNA  “Restrict” the action of the attacking organism  Protection against viruses & other bacteria  Bacteria protect their own DNA by methylation & by not using the base sequences recognized by the enzymes in their own DNA  d d

What do you notice about these phrases? Radar Racecar Madam I’m Adam A man, a plan, a canal, Panama Was it a bar or a bat I saw? Taco cat palindromes

Restriction enzymes 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  

Restriction enzymes 1 st restriction enzyme found = EcoRI ◦ Found in E. coli Werner Arber Daniel Nathans Hamilton O. Smith

Restriction enzymes Cut DNA at specific sites ◦ Leave “sticky ends” GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA GTAACGAATTCACGC TT CATTGCTTAAGTGCG AA restriction enzyme cut site sticky ends

Sticky ends Cut other DNA with same enzyme ◦ 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 Glue = ligase

Sticky ends help glue genes together TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCACGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGTGCGAA gene you wantcut sites TCAACGCTTTAACG AATTCTACGATCCCGGATCTT AGTTGCGAAATTGCTTAA GATGCTAGGGCCTAGAA chromosome want to add gene to cut sites AATTCTACGAATGGTTACATCGCCG GATGCTTACCAATGTAGCGGCTTAA isolated gene sticky ends chromosome with new gene added TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTAGC sticky ends stick together DNA ligase joins the strands Recombinant DNA molecule

Why mix genes together? Gene produces specified protein in different organism (i.e. bacteria) TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACGATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATGCTA GC aa “new” protein from organism ex: human insulin from bacteria human insulin gene in bacteria bacteriahuman insulin How can bacteria read human DNA?

The code is universal Since all living organsims … ◦ Use the same DNA ◦ Codons code for the same amino acids ◦ Create the same polypeptide chains ◦ Read their genes the same way

Copy (& read) DNA Transformation ◦ Insert recombinant plasmid into bacteria ◦ Grow recombinant bacteria in agar cultures  Bacteria make lots of copies of plasmid  “cloning” the plasmid ◦ Production of many copies of inserted gene ◦ Production of “new” protein  Transformed phenotype DNA  RNA  protein  trait

Want more protein? Grow more bacteria grow bacteria harvest (purify) protein transformed bacteria plasmid gene from other organism + recombinant plasmid vector

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)  Extending growing season: fishberries  Strawberries with an anti-freeze gene from flounder  Improve quality of food: golden rice  Rice enriched with beta-carotene, which is converted into vitamin A (improved nutritional value)

Green with envy? Jelly fish “GFP” Transformed vertebrates Cells from these specimens are used to study transplants & grafts

Factor-IX Sheep Sheep produce human clotting factors in their milk Used in treatment for hemophilia

The ethical debate of GMO crops Benefits ◦ Increased crop yields ◦ Crops can be grown in harsher environments  Salt-resistant tomatoes ◦ Decreased needs for pesticides ◦ Nutrient-enhanced crops Potential harms ◦ Genetic pollution through fertilization  Pollen  Monsanto ◦ Unknown health risks ◦ Potential hybridization with other species

Gene therapy A virus vector is used to insert the recombinant plasmid into the genes of affected cells The virus is chosen and designed to target only those specific cells

Clones A group of genetically identical organisms A group of cells derived from a single parent cell Monozygotic twins are naturally occurring clones ◦ Why do appear different as they age? ◦ Epigenetics Plant cuttings are clones Asexual reproduction results in clones

Reproductive cloning Called nuclear transfer Remove a differentiated diploid nucleus from individual to be cloned Enucleate a donor egg cell ◦ Remove nucleus Insert the diploid nucleus into the enucleated cell Implant into the endometrium of a surrogate & gestate Newborn will be genetically identical to the parent that donated the diploid nucleus

Dolly the sheep First successful cloning of a mammal from a differentiated somatic cell She was the result of many attempts ◦ 277 attempts (276 failures) She died young ◦ Age-related illnesses  Severe arthritis & progressive lung disease  Had shortened telomers Human reproductive cloning is illegal

Therapeutic cloning Remove a differentiated diploid nucleus from the cell to be cloned Enucleate a donor egg cell Insert the diploid nucleus into the enucleated cell Stimulate it to divide & grow in vitro Resulting blastocyst is a rich source of stem cells Outer layer is removed, so only inner cell mass is used to culture tissues

Uses for therapeutic cloning Create stem cells for transplants ◦ Burn patients ◦ Leukemia Replace damaged tissues ◦ Nerves ◦ Pancreatic cells Significantly reduced risk of rejection of cells that are genetically identical to the recipient

Ethics of therapeutic cloning In favor ◦ Stem cells can be created without the destruction of human embryos ◦ Decreased risk of transplant rejection ◦ Transplants don’t require the death of another person ◦ Embryos are not allowed to develop to point where a nervous system forms  First organ to form in embryo development Against ◦ Religious/moral objections  “playing God” ◦ Embryo could be used in IVF  Develop into a fetus  Are we destroying a life? ◦ Even though illegal, potential attempts to clone first human

Any Questions??