Sample 30
Pseudomonas aeruginosa
Protocols: Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections in immunocompromised individuals, such as patients with cystic fibrosis where it commonly forms biofilms. Ciprofloxacin is used extensively to treat P. aeruginosa infections, but its effectiveness can be significantly reduced due to biofilm formation. Although many individual genes associated with biofilm formation have been characterised, the genetic basis of P. aeruginosa biofilm fitness related to antibiotics challenge remain unexplored. In this study we employed a high-density TraDIS-Xpress library of P. aeruginosa PAO1 to assay the impact of gene disruptions or altered gene expression on biofilm formation at different concentrations of ciprofloxacin. Gene fitness was analysed by comparing the biofilm samples to planktonic samples harvested at 12h, 24h and 48h with and without ciprofloxacin. Gene determinants of survival for biofilms at different stages of maturity in the presence and absence of ciprofloxacin were identified. Genomic DNA (gDNA) of biofilm and planktonic samples was extracted using the Quick-DNA Fungal/Bacterial Microprep Kit and Miniprep Kit (Zymo research) respectively according to the manufacturer's instruction. A total of 100 ng of DNA from each sample was fragmented and tagged using the MuSeek Library Preparation Kit (Thermo Scientific). Following a purification step with AMPure XP beads, the DNA fragments containing transposon insertion junctions were selectively enriched by an initial PCR using a pair of custom primers. The first primer was biotinylated and annealed to the transposon sequence and a customed i7 primer annealed to the tagged sequence generated by the Mu transposase. The resulting PCR products were sequentially purified using AMPure XP beads and then streptavidin beads, ensuring high specificity and purification of the target amplicon. A second round of PCR was subsequently performed using custom i5 and i7 primers to incorporate adapter sequences for high-throughput sequencing using the Illumina method.
ENA first public
2026-06-12
INSDC center name
Quadram Institute
INSDC status
public
Submitter Id
E-MTAB-16928:Sample 30
broker name
ArrayExpress
collection date
not collected
geographic location (country and/or sea)
not collected
growth condition
Biofilm
isolate
not applicable
sample name
E-MTAB-16928:Sample 30
scientific_name
Pseudomonas aeruginosa
strain
Pseudomonas aeruginosa PAO1