Scientists have studied osmotic stress in different contexts for many years to understand topics such as how climate change can affect both ocean currents and agriculture. In bacteria, researchers have explored how cells deal with pressure from increased salt concentrations in their environment. In a recent webinar, Katherine Krouse, a senior research associate in the applications development group at Inscripta, described how she used our OnyxTM genome engineering platform to study the osmotic stress response in E. coli. If you don’t have time to watch the full webinar, here are some highlights from the talk.
Previous studies focused on genome-wide responses to osmotic stress in bacteria, a common limitation has been relying on DNA libraries with little variety of edit types and imprecise editing.
This is where the Onyx genome engineering platform can make a difference. Its algorithms are optimized to select the best designs for the proposed edit and accommodate different types of edits.
For her experiments, Krouse designed seven libraries that targeted roughly 4,000 E. coli genes. Specifically, she designed five promoter and ribosome binding site insertion libraries of varying strengths, plus two triple-stop knockout libraries. In total, Krouse generated about 25,000 designs. Next, she mixed the seven libraries volumetrically with one another so she could perform a pooled selection. This allowed Krouse to study each edit individually as well as how the edits performed in relationship to one another under osmotic stress in a range of sodium chloride conditions. She allowed the pooled engineered cell libraries to grow for 48 hours and then extracted DNA for sequencing using Inscripta's Barcode Amplicon assay.
The project generated about 300,000 data points from the sequencing step and offered scores of insights. For example, one of the interesting findings around specific genes included the waaZ gene, which is involved in lipopolysaccharide core biosynthesis. There were multiple locations in the gene where a knockout mutation resulted in a depleting edit in the population.
At the same time, Krouse found several different promoters that resulted in population enrichment, but some promoters were better than others for regulating the gene’s response to osmotic stress. Lastly, results indicated that several genes involved in things like membrane integrity and generalized cell stress responses were associated with resistance to osmotic stress.
Krouse also described how her work shed light on genes involved in salt tolerance for which there is little to no information about their functionality or role in response to osmotic stress. She noted that these genes could be included in a second round of editing to generate even more optimal designs for salt tolerance.
Learn more about how the libraries and experiments were designed and dive deeper into the results, watch the webinar recording.