Until now, the ability to comprehensively study gene sequence variants at the native locus has been unavailable to researchers. That all changes with the introduction of the Onyx platform. With Onyx, every residue of every protein of interest can be systematically probed and evaluated in detail like never before.
In collaboration with one of our Early Access customers, we used Onyx technology to design and build complete scanning site-saturation mutagenesis libraries on four E. coli cell envelope biosynthesis genes.
By methodically substituting every wild-type codon with one encoding each of the other 19 amino acids along the full length of each gene, we rapidly generated a library of approximately 23,000 members for a complete mapping of all possible single amino acid variants of the four proteins of interest. And don’t forget, all edits were made on the genes at their native loci, in the context of wild-type gene copy number and regulatory control.
Site-saturation mutagenesis identified essential residues around the active site of LpxC
For this application — saturation mutagenesis of essential genes — any edit resulting in compromised cell viability would fail to appear in the population at the conclusion of a pooled library cultivation. With deep sequencing of the four targeted loci, this is exactly what we observed, in exquisitely high resolution! Known catalytic and substrate-binding residues proved especially sensitive to this readout and served to validate the experiment.
Even more exciting was the discovery of a set of additional positions in these four proteins not previously identified as functionally essential.
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