Optimized CRISPR Interference System for Investigating <i>Pseudomonas alloputida</i> Genes Involved in Rhizosphere Microbiome Assembly.

ABSTRACT: Pseudomonas alloputida KT2440 (formerly P. putida) has become both a well-known chassis organism for synthetic biology and a model organism for rhizosphere colonization. Here, we describe a CRISPR interference (CRISPRi) system in KT2440 for exploring microbe-microbe interactions in the rhizosphere and for use in industrial systems. Our CRISPRi system features three different promoter systems (XylS/P_m, LacI/P_lac, and AraC/P_BAD) and a dCas9 codon-optimized for Pseudomonads, all located on a mini-Tn7-based transposon that inserts into a neutral site in the genome. It also includes a suite of pSEVA-derived sgRNA expression vectors, where the expression is driven by synthetic promoters varying in strength. We compare the three

SUBMITTER: Roghair Stroud MN 

PROVIDER: S-EPMC11421427 | biostudies-literature | 2024 Sep

REPOSITORIES: biostudies-literature