(A) Escherichia coli BW25113-Wt cells with pCyn-v2-GFP plasmid were induced with varying azide concentrations and GFP fluorescence of cell lysate was measured over 24 hours. (B) E. coli BL21 contaning control pEC-GFP plasmid was induced with IPTG (isopropyl β-d-1-thiogalactopyranoside). (C). BW25113-Wt cells with pCyn-v2-GFP plasmid was incubated with 1-Glc-N3 vs. 2-Glc-N3 and induced GFP fluorescence of cell lysates due to azide release is shown here.
Azides are used as functional groups in fine chemicals and pharmaceuticals synthesis, and recently azide-functionalized reagents as molecular-imaging probes for disease detection, therapeutics delivery, glycans synthesis precursors for CAZymes, and '-omics' applications (e.g., glycomics, metabolomics) have been developed. However, in vivo use of azido-based drugs or reagents requires ability to monitor organic azide stability for release of toxic azide ions. Current methods for azide ion detection involving spectrophotometry or fluorescence requires laborious sample derivation that are not suited for high-throughput. Therefore, availability of a synthetic biology toolkit for in vivo azide ion detection can broadly enable the fields of chemical-biology, glycoengineering, and biotechnology in general.
Rutgers scientists have developed an in-vivo synthetic promoter based azide biosensor toolkit to selectively detect azide anions. A promiscuous cyanate-specific native promoter was engineered to detect azide and rapidly induce model green fluorescent protein (GFP) expression in Escherichia coli.
- Low-cost heterologous protein expression system
- Real-time and in-vivo bioorthogonal reaction products (e.g., azides, azido sugars) biosensing
- High-throughput CAZyme & glycoengineering
- Synthetic azide operon allows highly-tunable GFP expression over the classic IPTG lac-operon
- Selective detection of inorganic azide ions inside living cells
- Biosensing system can be evolved for use with other prokaryotic or eukaryotic cells
Intellectual Property & Development Status:
Patent pending. Available for licensing and/or collaboration.
Bandi et al. Synthetic promoter based azide biosensor toolkit to advance chemical-biology. BioRxiv. Preprint. DOI: 10.1101/2020.07.08.193060 .