Enzymes, nature's catalysts, are remarkable biomolecules capable of extraordinary specificity and selectivity. Directed evolution has been used to produce enzymes with many unique properties, including altered substrate specificity, thermal stability, organic solvent resistance, and enantioselectivity--selectivity of one stereoisomer over another. The technique of directed evolution comprises two essential steps: mutagenesis of the gene encoding the enzyme to produce a library of variants, and selection of a particular variant based on its desirable catalytic properties. In this course we will examine what kinds of enzymes are worth evolving and the strategies used for library generation and enzyme selection. We will focus on those enzymes that are used in the synthesis of drugs and in biotechnological applications. This course is one of many Advanced Undergraduate Seminars offered by the Biology Department at MIT. These seminars are tailored for students with an interest in using primary research literature to discuss and learn about current biological research in a highly interactive setting. Many instructors of the Advanced Undergraduate Seminars are postdoctoral scientists with a strong interest in teaching.
7.02 and 7.021 require simultaneous registration. Application of experimental techniques in biochemistry, microbiology, and cell biology. Emphasizes integrating factual knowledge with understanding the design of experiments and data analysis to prepare the students for research projects. Instruction and practice in written communication provided.
Subject uses molecular genetics to examine how Gram-positive and Gram-negative bacteria can be used in novel and relevant bioconversion processes, for example, to synthesize precursors to the drug Crixivan, a potent inhibitor of HIV replication, or to synthesize metabolites as food supplements. Students engage in independent research projects to address questions relating to these processess. Techniques used include plasmid manipulation, genetic complementation, mutagenesis, PCR, and DNA sequencing, enzyme assays, and gene expression studies. Instruction and practice in written and oral communication provided. Also referred to as the Microbial Genetics Project Lab, this is a hands-on research course designed to introduce the student to the strategies and challenges associated with microbiology research. Students take on independent and original research projects that are designed to be instructive with the goal of advancing a specific field of research in microbiology.
" In this class, students engage in independent research projects to probe various aspects of the physiology of the bacterium Pseudomonas aeruginosa PA14, an opportunistic pathogen isolated from the lungs of cystic fibrosis patients. Students use molecular genetics to examine survival in stationary phase, antibiotic resistance, phase variation, toxin production, and secondary metabolite production. Projects aim to discover the molecular basis for these processes using both classical and cutting-edge techniques. These include plasmid manipulation, genetic complementation, mutagenesis, PCR, DNA sequencing, enzyme assays, and gene expression studies. Instruction and practice in written and oral communication are also emphasized. WARNING NOTICE The experiments described in these materials are potentially hazardous and require a high level of safety training, special facilities and equipment, and supervision by appropriate individuals. You bear the sole responsibility, liability, and risk for the implementation of such safety procedures and measures. MIT shall have no responsibility, liability, or risk for the content or implementation of any of the material presented. Legal Notice "
This protocol represents a cost-effective modification of the Ames Test that allows students to investigate the mutagenic potential of various common substances. Potential mutagens are tested using well-characterized auxotrophic strains of Salmonella typhimurium. By analyzing the results, students determine if any of their compounds may be mutagenic. Follow-up experiments are designed to determine the dose response of these potential mutagens. Using this protocol, we have achieved reproducible results with several known mutagens, including sodium azide and ultraviolet irradiation. This approach enables faculty to control costs and results to improve student understanding of mutagenesis, biochemical pathways, experimental design, and data analysis.
Creation and characterization of mutants is the basis for any genetic analysis. This exercise demonstrates a simple, safe procedure for transposon mutagenesis of Rhodobacter sphaeroides, a purple non-sulfur photosynthetic bacteria. Students perform the mutagenesis by mating a transposon-carrying plasmid from Escherichia coli to R. sphaeroides and then selecting for the drug resistance carried on the transposon. Only R. sphaeroides carrying the transposon in the chromosome survive the selection. Transposon carrying mutants are then scored for various phenotypes. The metabolic diversity of R. sphaeroides allows the isolation of nutritional, photopigment, and photosynthetic mutants. Further analysis of mutants is possible.
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