Researcher(s)
- Braden Rogers, Chemical Engineering, University of Delaware
Faculty Mentor(s)
- Aditya Kunjapur, Chemical and Biomolecular Engineering, University of Delaware
Abstract
Non-standard amino acids (nsAAs), especially para-nitro-phenylalanine (pN-Phe), offer a promising strategy to enhance immune recognition of bacterial epitopes, potentially enabling next-generation vaccines against antibiotic-resistant pathogens. However, preliminary studies suggest that factors such as the insertion site within a bacterial epitope and the surrounding codon context can significantly affect nsAA incorporation efficiency. To probe this, an amber codon was introduced at each position of a 15-amino-acid epitope displayed on the surface of E. coli via the Adhesin Involved in Diffuse Adherence (AIDA-I) anchor. Since translation of AIDA-I occurs after the amber codon, surface display served as a direct readout of nsAA incorporation efficiency. Using FLAG-tag labeling and flow cytometry in MOPS media lacking aromatic amino acids to suppress misincorporation, we assessed the percentage of epitopes that properly incorporated different supplied concentrations of pN-Phe. We observed that incorporation efficiency varied significantly by position, confirming that local sequence context plays a critical role.
To investigate codon-level effects more systematically, we sought a more quantitative and high-throughput readout. While prior studies used single fluorescent protein (FP) reporters to assess incorporation, we developed dual-fluorescence reporters to (1) normalize for expression variability by measuring both amber-dependent and amber-independent signals and (2) reduce false positives from leaky suppression or truncation. Using combinations of mCherry, mTurquoise, and mTagBFP, it was found that placing mCherry downstream of the amber codon resulted in the most consistent and reliable performance. Future work will involve generating a saturation mutagenesis library targeting the immediate upstream and downstream codons surrounding the amber codon.
To complement flow-based readouts, we also established orthogonal methods to isolate and characterize nsAA-containing surface proteins. Specifically, we optimized conditions for outer membrane isolation and TEV protease cleavage of surface-displayed constructs, enabling downstream analysis of incorporation outcomes.
Ultimately, this work will allow us to interpret nsAA-dependent epitope display data through the lens of both position and codon context. These insights will guide the rational design of immunogenic bacterial epitopes for incorporation into screening platforms and vaccine candidates.