Thesis (M. Eng.)--Harvard-MIT Division of Health Sciences and Technology, June 2011. / "June 2011." Cataloged from PDF version of thesis. / Includes bibliographical references (p. 75-79). / For this project, N-terminal and C-terminal peptide library fusions were designed,bconstructed, and screened in order to improve the repression achievable with a novel gene regulatory system. This system, based on the interaction between proteins and proteinbinding RNA aptamers, takes advantage of the reversible interaction between TetR and its RNA aptamer binding partner 5-1.2 to modulate gene expression. With no tetracyclines present, TetR preferentially binds to aptamer 5-1.2 in the mRNA of a gene of interest with low nanomolar affinity and represses translation. Tetracyclines such as aTc induce a conformation change in TetR, prevent TetR binding to aptamer 5-1.2, and induce gene expression. Therefore, TetR binds aptamer 5-1.2 in an aTc-dependent manner, allowing inducible control of gene expression through the TetR-aptamer system. Initial characterization showed a regulatory range of 78% or approximately 5 fold in S. cerevisiae. The aim of this project is to improve repression levels achievable with the TetR-aptamer system by creating libraries of N-terminal and C-terminal peptide fusions to TetR and screening for increased repression in S. cerevisiae. The N-terminal and C-terminal library fusions were constructed from synthesized oligonucleotide fragments and a baseline TetR vector containing library insertions sites at both the N-terminal and C-terminal ends. The library fragments contain 20 random amino acids and a standard SSG linker peptide flanked by both single-cutting restriction enzyme sites and 40 bases of homology to the library insertion sites on the baseline TetR vector, allowing for construction by both restriction/ligation cloning in bacteria and yeast homologous recombination. Both libraries were constructed using restriction/ligation cloning after initial experiments determined optimized conditions for PCR, digest, purification, ligation, and electrocompetent bacterial transformation to achieve a maximum efficiency, fidelity, and purity. The N-terminal and C-terminal libraries produced have a combined diversity of 2.5x 105 variants. These library variants were screened using a plate-based assay with URA3 as a reporter gene. A selection with 5-fluoroorotic acid (5-FOA) was performed to identify library variants with improved repression. Since 5-FOA is a competitive inhibitor of URA3, cells that have URA3 expression cannot live on media containing 5-FOA. Preliminary experiments determined that 0.035% 5-FOA is the threshold for growth for the baseline / (cont.) TetR-aptamer system. Library variants containing 5-1.2-URA3 were grown on media containing a gradient of 5-FOA concentrations between 0.03% and 0.06% and compared to baseline TetR growth. Thirty-one library variants grew at a 5-FOA concentration greater than the baseline threshold for growth. These library hits underwent testing to further characterize their repression, inducibility, and library sequence. The 31 colony hits were streaked on discrete concentrations of 5- FOA to determine colony-specific 5-FOA thresholds and on uracil dropout media in the presence and absence of aTc to screen for preserved inducibility. Of the original 31 colonies, 25 passed both plate assays, growing on 0.045% 5-FOA and in a Tc-dependent manner on uracil dropout media. These 25 colonies were sequenced and analyzed using MEME to detect any conserved motifs. Twenty of the 25 sequenced colonies contained correct and unique libraries, 3 with N-terminal libraries and 17 with C-terminal libraries. Both the N-terminal and C-terminal library sequences had significant motifs. For the Nterminal sequences, all three contained the same 14 nucleotide motif, and 16 of the 17 Cterminal sequences also contained a 21 nucleotide motif. However, the unbiased selection for improved TetR functionality likely returned hits that use multiple mechanisms of action to enhance repression. Therefore, out of a diverse library pool containing 2.5x 105 variants, 20 unique library variants conferred increased repression on the TetR-aptamer system while maintaining inducibility in the presence of tetracycline. / by Jessica Karen Wong. / M.Eng.
| Identifer | oai:union.ndltd.org:MIT/oai:dspace.mit.edu:1721.1/65513 |
| Date | January 2011 |
| Creators | Wong, Jessica Karen |
| Contributors | Jacquin Niles., Harvard University--MIT Division of Health Sciences and Technology., Harvard University--MIT Division of Health Sciences and Technology. |
| Publisher | Massachusetts Institute of Technology |
| Source Sets | M.I.T. Theses and Dissertation |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | 79 p., application/pdf |
| Rights | M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission., http://dspace.mit.edu/handle/1721.1/7582 |
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