W5-2 Biotechnology Past Paper PDF

II. Making Products from Genetically Engineered Microbes: Biotechnology 12.6 Somatotropin and Other Mammalian Proteins 12.7 Transgenic Organisms in Agriculture and Aquaculture 12.8 Engineered Vaccines and Therapeutics 12.9 Mining Genomes and Engineering Pathways 12.10 Engineering...

II. Making Products from Genetically Engineered Microbes: Biotechnology 12.6 Somatotropin and Other Mammalian Proteins 12.7 Transgenic Organisms in Agriculture and Aquaculture 12.8 Engineered Vaccines and Therapeutics 12.9 Mining Genomes and Engineering Pathways 12.10 Engineering Biofuels 12.6 Somatotropin and Other Mammalian Proteins Biotechnology Use of living organisms for production of valuable products Many mammalian proteins have high pharmaceutical value but are costly to purify because of low amounts in normal tissue. Genetically engineered microorganisms used instead  Genentech founders: venture capitalist Robert A. Somatotropin: Swanson (right) and biochemist Herbert Boyer (left) Insulin was the first human protein made commercially by genetic engineering. Human somatotropin (growth hormone) is a single polypeptide encoded by a single gene. treats stunted growth cloned as cDNA from mRNA Mutated human somatotropin targets only growth, not milk production. The history of Genentech: https://www.gene.com/topics/defining-moments  Recombinant bovine somatotropin (rBST) is commonly used in the dairy industry (Figure 12.18); stimulates milk production in cows. Figure 12.18 Table 12.1  12.7 Transgenic Organisms Transgenic organism genetically engineered organism that contains a gene (transgene) from another organism The Ti (tumor inducing) plasmid and transgenic plants Galls on the root caused Agrobacterium tumefaciens (plant by A. tumefaciens pathogen) contains the Ti plasmid responsible for virulence. Ti plasmid contains genes that mobilize DNA for transfer to plant. T-DNA: plasmid segment transferred to plant; sequences at ends essential for transfer Ti plasmid contains genes that mobilize wiki DNA for transfer to the plant. Gram-negative and rod-shaped  The Ti plasmid and transgenic plants binary vector: common Ti-vector system for gene transfer to plants and consisting of cloning vector plus helper plasmid Cloning vector contains multiple cloning site flanked by T-DNA ends, two origins of replication for E. coli and A. tumefaciens, two antibiotic resistance markers for plants and bacteria. foreign DNA inserted into vector vector transformed into E. coli and conjugated into A. tumefaciens (Figure 12.19) Helper plasmid (D-Ti) allows for transfer to plant. Ti system works well with broadleaf plants (dicots); does not work with monocots, which need alternative methods (e.g., transfection by microprojectile bombardment with particle gun or called gene gun)  Herbicide- and insect-resistant plants Targets for improvement include herbicide, insect, and microbial disease resistance and improved product quality. Main genetically modified (GM) crops are soybeans, corn, cotton, canola. herbicide resistance engineered to protect crop plants (e.g., soybeans) from herbicides that kill weeds, for example, glyphosate (RoundupTM) inhibition of aromatic amino acid biosynthesis (Figure 12.21). Also caused huge controversy due to carcinogen potential. New York Times  Insect-resistant plants Resistance to damage by some insects (Figure 12.22) Example: Bt toxin from Bacillus thuringiensis is toxic to moth, butterfly, beetle, and/or fly larvae and mosquitoes. wild-type Transgenic plant that expresses Bt toxin in its chloroplasts. Bt toxin structure https://www.permaculturenews.org/2012/08/14/bt-toxicity-confirmed-flawed-studies-exposed/ https://www.mrc-lmb.cam.ac.uk/genomes/madanm/articles/dnashuff.htm 12.8 Engineered Vaccines and Therapeutics Vaccines elicit immunity when injected. Recombinant vaccines, vaccinia virus, and subunit vaccines can modify a pathogen with genetic engineering to delete virulence factors and retain those that elicit immune responses: recombinant, infective, attenuated vaccine can engineer genes from a pathogenic virus to genome of a harmless carrier virus: vector vaccine induces immunity to pathogen polyvalent vaccine: a single vaccine that immunizes against two different diseases subunit vaccines contain only a specific protein or proteins from a pathogenic organism (e.g., coat protein of a virus). – popular because large amounts of immunogenic proteins can be administered at high dosage with less risk than attenuated or killed vaccines Pathogens as engineered anticancer therapeutics How to specifically target drugs/radiation to tumor cells? Listeria monocytogenes: intracellular pathogenic bacterium that causes listeriosis (foodborne illness) Weakly pathogenic recombinant strains are cleared by healthy cells but not by tumor cells Such strains have been turned into anticancer vehicles to deliver drugs or radioisotopes to tumor cells by coupling 188Rh (a radiopharmaceutical), killing pancreatic tumor cells. (Figure 12.25)  Intracellular antibody delivery by bacterial toxin Bacillus anthracis (Figure 12.26) protective antigen has been engineered to carry a synthetic anticancer antibody. Binding of antibodies to intracellular targets in cancer cells can trigger immune system to kill those cells. Figure 12.26  12.9 Mining Genomes and Engineering Pathways Environmental gene mining metagenome: genomes of an environment gene mining: process of identifying and isolating potentially useful genes from the environment without culturing the organisms that contain them To do so, DNA (or RNA, then cDNA) is directly isolated from the environment and cloned into appropriate expression vectors to construct a metagenomic library. (Figure 12.27) Screening has identified novel genes encoding pollutant-degrading and antibiotic biosynthetic enzymes, which can then be put into use.  Many enzymes with industrial applications have been isolated. examples: enzymes with resistance to industrial conditions (high temperature, high or low pH, oxidizing conditions), enzymes with combinations of properties (heat stable lipases) example: heat- and acid-stable enzymes for cleaning food- processing equipment (Figure 12.28) Application of CinderBio hyperstable enzymes for the cleaning of creamery equipment. These heat-stable enzymes clean industrial food-processing equipment as well as or better than traditional cleaning methods and do not generate large amounts of toxic wastewater. Pathway engineering (or ‘Synthetic biology’) to improve synthesis and production pathway engineering: assembling a new or improved biochemical pathway using genes from one or more organisms engineered microbes used to make many products (e.g., alcohols, food additives, antibiotics, etc.). Engineered microbes can degrade toxic and/or unwanted materials. Major challenges include controlling resulting pathway and producing sufficient yields cost-effectively. Example: Indigo synthesis in E. coli