Determining Conserved Residues for OMeY Incorporation in Essential Genes

• Jae June Jang, Chemical Engineering, University of Delaware

• Aditya Kunjapur, Chemical & Biomolecular Engineering, University of Delaware

Because non-standard amino acids (nsAAs) are not readily available in nature, they present a promising opportunity for developing a biocontainment tool that prevents the accidental release of engineered organisms. Our project investigated this by researching whether a specific gene could successfully accommodate our chosen nsAA, O-Methyl-L-Tyrosine (OMeY), and still produce a functional protein. A key challenge, however, is that even when a protein successfully incorporates OMeY, the system can still fail due to “escape.” Therefore, we want to rationally pick sites for OMeY incorporation in essential genes to reduce the routes of escape via misincorporation or standard amino acids. Due to the similarity of OMeY to other aromatic amino acids, we looked at these residues within essential genes. We narrowed down potential sites by looking at highly conserved Tyrosine (Y) or Phenylalanine (F) residues using the ConSurf tool, which provides residue conservation from sequence alignments of homologues. To test this dependency, we utilize strains containing an orthogonal translation system in Bacillus Subtilis that allows OMeY to be incorporated into the proteins of essential genes for the bacteria to survive. We tested these criteria in the essential gene, dxs. Our results indicate that several genes we studied have the potential to incorporate OMeY and remain functional; however, we note higher escape with incorporation in certain positions. While more research is needed to ensure that the strains do not fully escape and incorporate other amino acids, our findings suggest that certain conserved residues can accommodate OMeY.