Genetic Engineering and Biotechnology

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2 So, what is biotechnology?? The use of living organisms to carry out defined chemical processes for industrial or commercial application. The office of Technology Assessment of the U.S. Congress defines biotechnology as "any technique that uses living organisms or their products to make or modify a product, to improve plants or animals, or to develop microorganisms for specific uses." GENETIC ENGINEERING However Today, Biotechnology often implies and organism which has been manipulated for carrying out a process What type of manipulation -- isolation, manipulation and sequencing of DNA, regulation of and control of gene products Use of in vitro techniques, especially molecular cloning, which result in genetically modified organisms is often termed GENETIC ENGINEERING 2

3 Molecular Aspect Tools which allow -- isolation, identification, verification, purification and manipulation of the gene of interest. Applied Aspect Production of useful products 2 MAIN ASPECTS TO THIS DISCUSSION. Useful products insulin, growth hormone, frost free strawberries, bacteria which eat oil, vaccines, industrial enzymes Tools -- Vectors Enzymes polymerase Plasmids, Cosmids, YAC, Bacmids, Transposons Reverse Transcriptase, Ligase, Restriction Endonuclease, DNA 3

4 Restriction Endonuclease WHAT? WHY? Three types Type I, Type II, and Type III Type I and III ATP dependent Don t cut at recognition site Type II No ATP dependence Cut within the recognition site Molecular Scissors WHY IN THE WORLD WOULD A BACTERIA HAVE SOMETHING THAT CUTS UP DNA? HOW DO THEY PROTECT THERE OWN DNA? --- METHYLASE MODIFICATION OF OWN DNA ENZYME YOU HAVE -- HAVE METHYLASES FOR EACH?? RESTRICTION 4

5 Type II Restriction Enzymes Cut within recognition sequence symmetrical palindromes (mostly) sticky end or blunt end 5

6 Sticky Ends Blunt ends 6

7 Cloning Vectors Early cloning vector WHY GOOD CLONING VECTOR???? Small 4.3kb Sequence known High Copy Number copies/cell stably maintained Accept up to 10kb inserts Many single cleavage sites 2 Antibiotic Resistance Markers Easily Transformed Tet resistance is INSERTIONALLY INACTIVATED 7

8 A cloning we will go Creating Recombinant DNA SPECIFIC GENE CLONING (ALREADY KNOW WHAT YOU WANT) VS. CREATION OF A DNA LIBRARY (FISHING FOR STUFF) DON T FORGET TO GO ALL THE WAY TO TRANSFORMATION == INCREASE THE NUMBER OF COPIES BY REPLICATION WITHIN THE HOST BACTERIUM. CAN ALSO INCREASE THE NUMBER OF COPIES BY ---- PCR 8

9 PCR Polymerase Chain Reaction Shared the noble prize in chemistry in 1993 for his invention $10,000 bonus $300,000,000 sale to Roche 9

10 POINT OUT TEMPERATURES --- DENATURING 94-96oC ANNEALING 45-64oC DEPENDING ON PRIMERS EXTENSTION 68 TO 72oC DEPENDING ON TAQ USED TAQ POLYMERASE THERMOPHILE THERMOSTABLE POLYMERASE Thermus aquaticus REPEAT TIMES Hot Springs in Yellowstone 1976 first reported LOGARRITHMIC AMPLIFICATION 10

11 But how do you know if you got the right clone?? How to pick from a plate full of colonies enzyme detect activity -- assays protein detect with an antibody to the protein gene will it hybridize with DNA probe? 11

12 Practical Application of Genetic Engineering What we do with what we can do Therapeutic products blood proteins TPA, Factors VII, VIII, IX hormones Insulin, Somatotropin, FSH immune products Interferons (a/b) vaccines hepatitis B Transgenic Plants and Animals Bt corn, flavor saver tomatoes, vaccines(?), vitamin A Gene Therapy augmentation or replacement(?) of a non or dys functional gene with a functional one Bioremediation Mammalian proteins can be produced cheaper, more safely, and in larger amounts than isolating from tissue TPA == Tissue plasminogen activator dissolved clots Factors == Clotting agents Somatotropin == growth hormone VACCINES CLONE GENE FOR PROTECTIVE VIRAL COAT (THIS IS THEN THE ANTIGEN) OR ATTENUATED VACCINES THOSE CREATED BY RECOMBINANT DNA TECHNOLOGIES THAT DO NOT HAVE THE VIRULENCE FACTORS. GENETICALLY MODIFIED ORGANISMS (EUROPE DOESN T ACCEPT MANY OF THESE) GMO S OR GM PLANTS NO DISIGNATION IF DONE IN VIVO, (BEEN GENETICALLY ALTERING PLANTS AND ANIMALS FOR OVER 10,000YRS) BUT IN VITRO USUALLY ADDS GENES FROM OTHER ORGANISMS SO CALLED TRANSGENIC ORGANISMS. BT == BACILLUS THURINGIENSIS ROUNDUP READY == RESISTANCE TO GLYPHOSATE FROM AGROBACTERIUM FLAVOR SAVOR TOMATOES == BLOCKS PROUCTION OF POLYGALACTMORASE THIS BREAKS DOWN CELL WALL AND LEADS TO MUSHY TOMATOES Vaccine GM petunia and honey parvo vaccine Hepatitis B and bananans GOLDEN RICE GENE THERAPY -- NOT REALLY REPLACED Introduce functional copy of a gene using retroviruses or adenovirus(dna) Problems mostly center on vectors being used give stable integration, but site of insertion is unpredictable amount of cloned DNA is limited protein expression is often transient vectors are rapidly limited infectivity and rapidly inactivated adenovirus can also present adverse reactions to the host 12