Heterologous Protein Engineering and characterization of native regulatory elements in E. coli using Massively Parallel CRISPR Editing

Heterologous protein expression in model organisms has many applications, including protein engineering, production of industrial enzymes and therapeutics, and structure-function discovery. Heterologous proteins are commonly expressed from plasmids to facilitate cloning and genetic manipulation. However, plasmid-based expression has many drawbacks, such as reduced stability and limited control over gene copy number. With CRISPR editing, chromosomal gene integration and subsequent editing has become more accessible, enabling thorough examination of protein expression and function. In this webinar, we demonstrate how we generated an E. coli saturation mutagenesis library for a chromosomally expressed green fluorescent protein (GFP) using the Onyx^TM platform. We identify several engineered variants with increased fluorescence or altered spectral characteristics, including literature validated and novel variants. We also generate a library of all predicted E. coli promoters and ribosome binding sites (RBS) and quantify their strength using the engineered high-expression GFP reporter system. These high-throughput experiments yield insights into native regulatory sequences and demonstrate the advantages of heterologous protein engineering directly in the host genome.

 

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