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Authors: Raghavan, Alankrita
Advisors: Hecht, Michael
Department: Molecular Biology
Class Year: 2015
Abstract: The design of functional de novo proteins has always been a great challenge in the field of protein design. The Hecht Lab has discovered a series of de novo proteins that are found to rescue certain E. coli auxotrophs. This work focuses on the sequence dependence of the rescue of one particular auxotroph (ΔserB) by de novo proteins. ΔserB cells are missing SerB – an enzyme that catalyzes the conversion of phosphoserine to serine. From the Hecht Lab library, 4 sequences were found that rescued the ΔserB deletion. However, even though the deletion is rescued, the relationship between the structure and the sequence of the proteins is still unknown. Two approaches were used to investigate the sequence dependence of these proteins: directed evolution and sitesaturation mutagenesis of the SynSerB proteins. Notably, two of the rescue sequences differed only by 6 residues but presented different phenotypes. Site-saturation mutagenesis was used to change the 6 residues in SynSerB3 that are different from SynSerB1 in order to see why the two SynSerB proteins display different characteristics despite having almost identical sequences. The directed evolution experiments in SynSerB4 suggest that growth time is an insufficient phenotype to screen for serine synthesis and that it is difficult to see a sufficient improvement in serine synthesis in order to select for beneficial mutations. The site-saturation mutagenesis experiments revealed some mutations that rescued in the same time on plates but appeared to enhance activity in liquid media. Conversely, many mutations were never observed – suggesting these mutations do not allow rescue. Taken together, this research offers indications of which residues are important for the rescue of the ΔserB auxotrophy by the SynSerB proteins and gives us information about the potential structure of the SynSerB3 protein.
Extent: 85 pages
Type of Material: Princeton University Senior Theses
Language: en_US
Appears in Collections:Molecular Biology, 1954-2017

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