Abstract
Synthetic perturbation of gene expression is central to our ability to reliably uncover genotype-phenotype relationships in microbes. Here, we present a novel transcription activation strategy that uses the Vibrio cholerae CRISPR-Associated Transposon (CAST) system to selectively insert promoter elements upstream of genes of interest. Through this strategy, we show robust activation of both recombinant and endogenous genes across the Escherichia coli chromosome. We then demonstrate the precise tuning of expression levels by exchanging the promoter elements being inserted. Finally, we demonstrate that CAST activation can be used to synthetically induce ampicillin-resistant phenotypes in E. coli.
| Original language | English |
|---|---|
| Pages (from-to) | 328-336 |
| Number of pages | 9 |
| Journal | ACS Synthetic Biology |
| Volume | 13 |
| Issue number | 1 |
| DOIs | |
| State | Published - Jan 19 2024 |
Funding
The authors acknowledge the Chappell Lab members for helpful discussion. They also acknowledge Daniel J. Haller for his assistance in the design and cloning of sJEC057, as well as Biki B. Kundu for his expertise in CRISPR-Cas9 editing that aided the creation of the reporter strains used in this study. This material is based on work supported by the National Science Foundation (grant nos. #2237512 and #1828869).
Keywords
- CRISPR
- CRISPR-associated transposon (CAST)
- gene activation
- transcriptional regulation
- transposons