ses5_ln

ses5_ln - MIT OpenCourseWare http:/ocw.mit.edu 7.344...

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MIT OpenCourseWare http://ocw.mit.edu 7.344 Directed Evolution: Engineering Biocatalysts Spring 2008 For information about citing these materials or our Terms of Use, visit: http://ocw.mit.edu/terms .
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Session 5 Lecture Notes 1. What are the authors trying to do in the aspartate aminotransferase paper? They are trying to give aspartate 2-oxoglutarate aminotransferase (AspAT) a new substrate specificity. Typically this enzyme prefers acidic substrates and aromatic substrates. They want to make it tolerate branching at the beta position. Show the picture of the aminotransferase reaction. Explain how PLP (pyridoxal phosphate) transfers amino groups (typically extracted from the gamma position of lysine residues) to the carbonyl carbon of the substrate. Why do they want to do this? Mostly just to show that they can, but one could imagine synthetic targets that would benefit from this too. 2. What process do they use to conduct the evolution? Get students to explain the process written in the materials and methods. It is very nicely done! First they have to make an auxotrophic strain that can’t make any branched AAs. Then they make their library and do selection in between rounds of mutagenesis. 3.
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ses5_ln - MIT OpenCourseWare http:/ocw.mit.edu 7.344...

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