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Authors: Korolev, Maria
Advisors: Hecht, Michael H
Contributors: Chemistry Department
Subjects: Chemistry
Issue Date: 2013
Publisher: Princeton, NJ : Princeton University
Abstract: Are functional proteins difficult to create in the laboratory from scratch? To probe this question, we created and tested a combinatorial library of de novo proteins in a gene knockout experiment. Bacterial strains lacking genes essential for survival on minimal media were transformed with the library and screened for growth. Six strains including ∆gltA and ∆fes were rescued by de novo sequences. ∆gltA is deficient of the enzyme citrate synthase in the citric acid cycle and ∆fes is deficient of the enzyme enterobactin esterase which is involved in iron acquisition. The mechanisms of rescue of these two auxotrophs by de novo sequences were investigated in this research. Chapter 2 focuses on the rescue protein for ∆gltA, named synGltA. synGltA was tested for its role in conferring life to a glutamate deficient cell. The sequence was determined to be important, based on alanine mutations, and it was also evolved into a more functional sequence. It was found that the protein was not bypassing the natural reaction of gltA, indeed, the expression of synGltA enabled citrate production. It was also found that purified protein was unable to catalyze citrate production alone, meaning that other proteins were involved in rescue in vivo. Protein partners were investigated for synGltA, and two proteins were found to be necessary for the rescue of ∆gltA by synGltA. Together, these findings lay the groundwork for showing that synGltA, a de novo protein, has a meaningful function in ∆gltA rescue. Chapter 3 focuses on one of the rescue proteins for ∆fes, named synFes2. synFes2 was probed directly for its ability to enable iron acquisition. It was found that synFes2 expression produced dramatically red cells. This red phenotype was found to be due to an accumulation of ferric enterobactin, a substrate of the wild type fes protein. Testing of different growth conditions and cell variants determined that this accumulation was due to more import of ferric enterobactin. Investigating the function of synFes2 suggested that the protein was not capable of hydrolyzing ferric enterobactin. synFes2 was also not found to significantly bind iron or enterobactin compared to a control. These findings suggest that synFes2 is functioning with other proteins to import more ferric enterobactin.
Alternate format: The Mudd Manuscript Library retains one bound copy of each dissertation. Search for these copies in the library's main catalog
Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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