Let’s face it, we expect our domesticated microorganisms to do many things evolution never asked of them. From isolated growth on agar plates to extended fermentation runs in massive metal tanks, our production strains are exposed to multiple stressors, many of which we may not fully appreciate or understand. Your ability to rapidly engineer your strain for robustness under stress gives you a leg up on your competition.
We used Onyx technology to methodically target every open reading frame in the S. cerevisiae genome for a total of 40,000 precise edits. By employing three different edit types — gene knockouts, transcription factor binding site replacements, and terminator substitutions — we were able to impart functionally distinct genetic changes at every locus in an otherwise unbiased manner.
To test our genome-wide libraries for stress tolerance, the populations were cultivated in the presence of either glucose or glycerol as the lone carbon source. The combination of three technical replicates and three harvest time points resulted in 720,000 data points for all 40,000 library designs. Data analysis revealed almost 900 designs across all three edit types enriched for growth on glycerol, rapidly populating a stable of potential hits for Forward Engineering of robust growth under the example stressor condition of glycerol as a lone carbon source. With so many variants in the edited cell population to genotype and track, Inscripta’s barcode sequencing kits and InscriptaResolver™ were critical tools in the workflow.