We deploy an array of platforms that can survey libraries of engineered strains with altered levels of protein abundance and/or function.

Targeted proteomics 

Targeted proteomics is a mass spectrometry-based protein quantification technique that can accurately quantify protein abundance, stoichiometry of protein isoforms and protein post-translational modifications.

The targeted proteomics approach is hypothesis driven, and the proteins in the assay are already known.  This approach has been applied to determine pathway bottlenecks, compare enzyme homologs for a specific enzyme steps, and to track dynamic regulation of protein levels in engineered microbes.

Potential applications of targeted proteomics:

• Identifying protein-associated bottlenecks, resulting in improved final product levels
• Quantifying protein levels for genetic tool development studies 
• Characterization and quantification of stable post-translational modifications for engineered microbes

The most common approach in targeted proteomics is liquid chromatography-selected reaction monitoring (LC-SRM), utilizing triple quadrupole mass spectrometers. The LC-SRM assay has high sensitivity, accuracy, reproducibility, and throughput. The method can quantify tens to hundreds of proteins in a single analysis and requires unique peptides for a specific protein that can be targeted. In addition, isotope-labeled internal peptide standards can be added to further improve the accuracy. 

This capability uses automation to reduce sample preparation variability and leverages integration with our ICE sequence/strain repository platform and Experiment Data Depot. 

Our targeted proteomics capability supports the Agile BioFoundry through rapid testing of engineered microbes to reduce the number of DBTL cycles required to successfully produce target molecules. 

Biocatalyst optimization 

We use a microfluidic platform to identify improved and/or missing catalysts in production pathways.  This approach is compatible with aerobic or anaerobic enzyme screening (in vivo or cell-free). The system can utilize a wide-array of reporter systems to rank the efficiency of pathways, including transcription-factor-based biosensors, FRET biosensors, or enzyme-linked sensors. 

Once candidate genes have been identified, variants are synthesized using rational design/directed evolution approaches. Appropriate sensors are linked to monitor reaction improvements, and in-depth characterization of candidate enzymes is carried out in vitro and in target-host systems. 

This pairing of microfluidics and biosensor design/engineering for molecular adaptation in the Agile BioFoundry can only be found in a few unique research environments.