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Title: Searching for de novo Proteins Capable of Performing Non-natural, Life-sustaining Reactions in E. coli
Authors: Yost, Colin
Advisors: Hecht, Michael H
Department: Chemistry
Class Year: 2019
Abstract: Synthetic biology seeks to push the boundaries of nature by importing non-native structures and functions into living organisms. Previous work in the Hecht lab has pursued this objective by using synthetic de novo proteins to perform natural, life-sustaining reactions in E. coli. However, the ability of de novo proteins to enable growth through non-natural functions remains largely unexplored. In order to investigate de novo proteins’ ability to catalyze non-natural, life-sustaining reactions, a system of genetic selections and a growth enhancement screen were developed. Using “pro-nutrients” – molecules that are not normally metabolizable by wild-type E. coli but can be converted into metabolizable nutrients through chemically facile reactions – these tests tied survival ability to non-natural activity by de novo proteins. Initial testing of the Hecht lab’s current libraries of de novo protein sequences was unsuccessful in identifying non-natural, life-sustaining activity. As a result, two “positive control plasmids” – which encoded natural proteins known to convert two pro-nutrients to their corresponding nutrients – were constructed and used to test the efficacy of the selections and screen developed. The current methodology was unable to identify the non-natural, life-sustaining activity of the positive control plasmids, indicating that the current approach for choosing pro-nutrients and designing the selections and screen must be further refined. Once the pro-nutrient-based selections and growth enhancement screen are optimized enough to identify a positive control plasmid, they can be used as a standard methodology for efficiently testing current and future de novo protein libraries for a variety of non-natural, life-sustaining functions.
Type of Material: Princeton University Senior Theses
Language: en
Appears in Collections:Chemistry, 1926-2019

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