29Biotechnology - Biotechnology Introduction s pecies has a...

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1 Biotechnology Introduction Biotechnology involves the use of living organisms, or products from living organisms, as a way of benefiting humans. It began more than 12,000 years ago with the domestication of animals and plants. It is now often associated with molecular genetics. Organisms that have received genetic material via recombinant DNA technology are called genetically modified organisms (GMOs); if an organism has received genetic material from another species, it is called a transgenic organism and the gene is called a transgene. Mammalian cloning and stem cell research are associated with biotechnology. The introduction of cloned genes into individuals to treat genetic diseases is called gene therapy. Uses of Microorganisms in Biotechnology Experiment 19A. Somatostatin was the first human peptide hormone produced by recombinant bacteria. Somatostatin is a human hormone that functions to inhibit the secretion of other hormones, including growth hormone, insulin, and glucagon. It was chosen by Swanson and Boyer (1976) as the first test because of its small size (14 amino acids) and ease of detection using radioimmunoassay. They were able to construct it synthetically by making eight small single-stranded oligonucleotides, allowing them to hydrogen bond due to their complementarity, and then ligating them together to form the double-stranded DNA fragment that would code for somatostatin. Note: Today the entire fragment could be made at one time. Its middle portion encoded the amino acid sequence for the human somatostatin peptide hormone. It also had an Eco RI site and an extra methionine site at one end, and two stop triplets and a Bam HI site at the other end. The single-stranded ends allowed its insertion into EcoRI and BamHI restriction sites in plasmid DNA. The plasmid had a lac Z gene, an amp R gene, a unique restriction site for Eco RI, and a unique restriction site for Bam HI.
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2 The methionine provided a link between somatostatin and ! -galactosidase to produce a fusion protein, which was necessary because somatostatin made in bacteria is rapidly degraded by cellular proteases – the fusion protein is not. The researchers could then separate somatostatin from ! -galactosidase by treatment with cyanogen bromide (CNBr), which cleaves polypeptides at the COOH terminal side of methionine. The goal. Produce human somatostatin in a recombinant bacterium. Achieving the goal (Figure 19.1). Synthesize a DNA fragment that codes for somatostatin. Using recombinant techniques, insert the fragment into a plasmid by digesting the plasmid at unique Eco RI and Bam HI sites. As a control, insert the somatostatin gene in the wrong orientation to produce plasmids that will not make somatostatin. Transform
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29Biotechnology - Biotechnology Introduction s pecies has a...

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