Genetic Engineering Biotechnology What is Genetic Engineering The
Genetic Engineering Biotechnology
What is Genetic Engineering? The manipulation of a trait in an organism to create a desired change
We have been manipulating DNA for generations! § Artificial breeding u creating new breeds of animals & new crop plants to improve our food
Animal breeding
Breeding food plants § “Descendants” of the wild mustard u the “Cabbage family”
Breeding food plants Evolution of modern corn (right) from ancestral teosinte (left).
A Brave New World
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
TACGCACATTTACGCGGATGCCGCGACT ATGATCACATAGACATGCTGTCAGCTCTAGTA human genome GACTAGCTGACTCGACTAGCATGATCAG CTACATGCTAGCACACYCGTACATCGATCCTG 3. 2 billion bases ACATCGACCTGCTCGTACATGCTACTAGCTAC TGACTCATGATCCAGATCACTGAAACCCTAGA TCGGGTACCTATTACAGTACGATCATCCGATC AGATCATGCTAGTACATCGATACTGCTA CTGATCTAGCTCAAACTCTTTTTGCATCA TGATACTAGCTGATCATGACTCT GATCCCGTAGATCGGGTACCTATTACAGTACG ATCATCCGATCATGCTAGTACATCGATACTGCTACTGATCTAGCTCAAACT CTTTTTGCATCATGATACTAGCTGACT
Can we mix genes from one creature to another? YES! Green Fluorosceint Protein (GFP)
How do we do mix genes? § Genetic engineering find gene u _______ DNA in both organisms u _______ gene from one creature into other creature’s DNA u _______ new chromosome into organism u organism _______ new gene as if it were its own u organism _______ gene as if it were its own u ___________________: Remember: we all use the same genetic code! u
Uses of genetic engineering § Genetically modified organisms (GMO) u enabling plants to produce new proteins § ______________: BT corn w corn produces a bacterial toxin that kills corn borer (caterpillar pest of corn) § ______________: fishberries w strawberries with an anti-freezing gene from flounder § ______________: golden rice w rice producing vitamin A improves nutritional value
Basic steps in genetic engineering 1. Isolate the gene 2. Insert it in a host using a vector 3. Produce as many copies of the host 4. as possible Separate and purify the product of the gene
Gene Cloning Techniques 2. Extract/isolate DNA 1 - Grow the target microorganism 4 - Insert DNA fragments in a plasmid cloning vector Recombinant DNA target 3 - Digest fragment DNA with restriction enzymes
Continued 5 Transform E. coli with library “Vibrio DNA library” Each bacteria will receive a single plasmid from the library Each bacteria will grow to form an individual colony
Tools 1. 2. 3. 4. DNA you want to clone Restriction endonucleases (molecular scissors) Cloning vector (e. g. p. GEM, p. BR 322…) Ligase enzyme (molecular glue)
Step 1: Isolating the gene
Step 1: Isolating the gene
Cutting DNA § DNA “scissors” u ____________________________ § used by bacteria to cut up DNA of attacking viruses § Eco. RI, Hind. III, Bam. HI u cut DNA at specific sites § enzymes look for specific base sequences GTAACG|AATTCACGC GTAACGAATTCACGC TT CATTGCTTAA|GTGCG CATTGCTTAAGTGCG AA
Restriction enzymes § Cut DNA at specific sites u ______________ restriction enzyme cut site GTAACGAATTCACGC TT CATTGCTTAAGTGCG AA restriction enzyme cut site GTAACG AATTCACGCTT CATTGCTTAA GTGCGAA
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 gene you want GGACCTG AATTCCGGATA CCTGGACTTAA GGCCTAT chromosome want to add gene to GGACCTG AATTCACGCTT CCTGGACTTAA GTGCGAA combined DNA
Restriction Endonucleases • Restriction endonucleases, a. k. a. “restriction enzymes” or “enzymes” by molecular biologists. • Type II restriction enzymes recognize and cut specific DNA sequences 5’-NNNAAGCTTNNN-3’ 3’-NNNTTCGAANNN-5’
Example • Hind III (Haemophilus influenza Rd) – Recognizes: AAGCTT – Cuts in between the two A’s AAGCTT TTCGAA A TTCGA AGCTT A
Types of Sticky Ends 5’ overhangs (Hind. III) 5’AAGCTT 3’ 5’A 3’TTCGAA 5’ 3’TTCGA 5’ 5’ AGCTT 3’ A 5’ 3’ overhangs (Kpn. I) 5’ GGTACC 3’ 5’ GGTAC 3’ 3’ CCATGG 5’ 3’ C C 3’ 3’ CATGG 5’
Types of Overhangs § Sticky ends u Examples include Hind. III & Kpn. I § Blunt Ends Example Sma. I u Recognize CCCGGG u Cut between C and G u CCCGGG GGGCCC GGG CCC
Sticky ends help glue genes together cut sites gene you want cut sites TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTC ACGCTT AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAG TGCGAA AATTCTACGAATGGTTACATCGCCG sticky ends GATGCTTACCAATGTAGCGGCTTAA isolated gene cut sites chromosome want to add gene to AATGGTTACTTGTAACG AATTCTACGATCGCCGATTCAACGCTT TTACCAATGAACATTGCTTAA GATGCTAGCGGCTAAGTTGCGAA DNA ligase joins the strands ________ DNA molecule sticky. TAACGAATTCTACGAATGGTTACATCGCCGAATTCTAC ends stick together chromosome with new gene added GATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGAT
How can bacteria read human DNA? Why mix genes together? § Gene produces protein in different organism or different individual human insulin gene in bacteria TAACGAATTCTACGAATGGTTACATCGCCGAATTCTAC GATC CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGAT GCTAGC “new” protein from organism ex: human insulin from bacteria aa aa aa bacteria human insulin
Step 2: Inserting gene into vector § Vector – molecule of DNA which is used to carry a foreign gene into a host cell
Plasmid Vector: p. BR 322 § First modern cloning vector (1976)
p. BR 322 • Contains: 1. col. E 1 origin of replication (ORI)
p. BR 322 • Contains: Bacteria plus plasmid Non-transformed bacteria 2. Selectable Markers: • Ampicillin Resistance (β-lactamase gene) • and Tetracycline Resistance (tet gene) Nutrient media plus antibiotic Overnight growth Only colonies from bacteria that have plasmid
p. BR 322 • Contains: 3. A few good restriction sites for inserting foreign DNA Bam. H 1 Your favorite DNA Pst. I Eco RI Bam HI Digest with Bam. H 1 and ligate Pst. I Eco RI Your favorite DNA Bam HI
p. BR 322 • Nice Features: √ √ √ 200 copies per E. coli cell Makes double stranded DNA All modern cloning vectors are based on p. BR 322
Next Generation: p. UC Plasmids • Advantages over p. BR 322 1. 2. Makes 1000’s of copies/cell Small size at 2. 7 kilobase pairs (kb) = easier uptake by E. coli
Step 3: inserting vector into host
Bacteria § Bacteria are great! one-celled organisms u reproduce by mitosis u § easy to grow, fast to grow w generation every ~20 minutes
§ A way to get genes into bacteria easily insert new gene into plasmid u insert plasmid into bacteria u bacteria now expresses new gene u § bacteria make new protein gene from other organism cut DNA plasmid recombinant plasmid + vector glue DNA transformed bacteria
Blue/White Selection Bacteria plus empty plasmid Bacteria with plasmid plus insert Non-transformed bacteria Nutrient media plus antibiotic plus X-Gal Overnight Colonies with insert - white Colonies w/o insert - blue growth Only colonies from bacteria that have plasmid
Grow bacteria…make more gene from other organism recombinant plasmid + vector plasmid grow bacteria harvest (purify) protein transformed bacteria
Applications of biotechnology
any Questions?
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