| Autor: |
Drenkard E; Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital., Hibbler RM; Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital., Gutu DA; Department of Molecular Biology, Massachusetts General Hospital., Eaton AD; Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital., Silverio AL; Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital., Ausubel FM; Department of Molecular Biology, Massachusetts General Hospital; Department of Genetics, Harvard Medical School., Hurley BP; Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital; Department of Pediatrics, Harvard Medical School., Yonker LM; Department of Pediatrics, Mucosal Immunology and Biology Research Center, Massachusetts General Hospital; Department of Pediatrics, Harvard Medical School; lyonker@mgh.harvard.edu. |
| Abstrakt: |
Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants. |
