콘텐츠로 건너뛰기
Merck

Metabolically-targeted dCas9 expression in bacteria.

Nucleic acids research (2023-01-12)
Gregory M Pellegrino, Tyler S Browne, Keerthana Sharath, Khaleda A Bari, Sarah J Vancuren, Emma Allen-Vercoe, Gregory B Gloor, David R Edgell
초록

The ability to restrict gene expression to a relevant bacterial species in a complex microbiome is an unsolved problem. In the context of the human microbiome, one desirable target metabolic activity are glucuronide-utilization enzymes (GUS) that are implicated in the toxic re-activation of glucuronidated compounds in the human gastrointestinal (GI) tract, including the chemotherapeutic drug irinotecan. Here, we take advantage of the variable distribution of GUS enzymes in bacteria as a means to distinguish between bacteria with GUS activity, and re-purpose the glucuronide-responsive GusR transcription factor as a biosensor to regulate dCas9 expression in response to glucuronide inducers. We fused the Escherichia coli gusA regulatory region to the dCas9 gene to create pGreg-dCas9, and showed that dCas9 expression is induced by glucuronides, but not other carbon sources. When conjugated from E. coli to Gammaproteobacteria derived from human stool, dCas9 expression from pGreg-dCas9 was restricted to GUS-positive bacteria. dCas9-sgRNAs targeted to gusA specifically down-regulated gus operon transcription in Gammaproteobacteria, with a resulting ∼100-fold decrease in GusA activity. Our data outline a general strategy to re-purpose bacterial transcription factors responsive to exogenous metabolites for precise ligand-dependent expression of genetic tools such as dCas9 in diverse bacterial species.

MATERIALS
제품 번호
브랜드
제품 설명

Sigma-Aldrich
Anti-β-Glucuronidase (N-Terminal) antibody produced in rabbit, ~1.5 mg/mL, affinity isolated antibody, buffered aqueous solution