Optimizing a protein-RNA aptamer gene regulatory system using an engineered peptide library

[摘要] (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.

[发布机构] Massachusetts Institute of Technology