OCT 14, 2021 7:00 AM PDT

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

Sponsored by: Inscripta
C.E. Credits: Florida CE P.A.C.E. CE
Speaker

Abstract
Date:  October 14, 2021
Time: 7:00am (PDT),  10:00am (EDT)
 
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 OnyxTM 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.
 
Learning Objectives
 
  • Generate massively parallel genomic libraries for heterologous protein engineering in E.coli
  • Apply fluorescence-based assays for high-throughput novel variant discovery and investigations into genomic regulatory elements
  • Identify novel green fluorescent protein (GFP) variants with altered spectral characteristics and improved activity
  • Evaluate expression of native promoter and ribosomal binding site (RBS) sequences using a stable chromosomal GFP reporter system
 
 
Webinars will be available for unlimited on-demand viewing after live event.
 
 
LabRoots is approved as a provider of continuing education programs in the clinical laboratory sciences by the ASCLS P.A.C.E. ® Program. By attending this webinar, you can earn 1 Continuing Education credit once you have viewed the webinar in its entirety.

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