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Analysis Biomolecular Interactions of Lrp to Target Regions of the P.aeruginosa Chromosome by BLI (CAT#: STEM-MB-0231-CJ)

Introduction

Pseudomonas aeruginosa, a Gram-negative bacillus native to soil and aqueous environment, is capable of inhabiting a wide variety of environments. This bacteria has been shown to attach and grow in biofilms on PVC pipes, persist in distilled water, live on the surface of human skin or cause infection within many body tissues. It is an opportunistic pathogen of burn victims, immunocompromised individuals, and cystic fibrosis (CF) patients. Since the treatment of P. aeruginosa infections with antibiotics became common practice, antibiotic resistant strains have developed and pose serious complications in successful treatment. P. aeruginosa is particularly challenging for CF patients because it can inhabit the lungs (often as a co-infection with Staphylococcus aureus) and form a biofilm in the sticky mucus that cannot be penetrated by therapeutic antibiotics. Once their respiratory pathway is colonized, the biofilm is difficult to remove from these patients. The vast majority of CF patients will die from these infections.




Principle

Bio-Layer Interferometry (BLI) is an optical technique for measuring macromolecular interactions by analyzing interference patterns of white light reflected from the surface of a biosensor tip. BLI experiments are used to determine the kinetics and affinity of molecular interactions. In a BLI experiment, one molecule is immobilized to a Dip and Read Biosensor and binding to a second molecule is measured. A change in the number of molecules bound to the end of the biosensor tip causes a shift in the interference pattern that is measured in real-time.

Applications

Microbiology; Pharmacy

Procedure

1. Detect Buffers and prepare samples. BLI experiments are set up with one molecule immobilised on the surface of the biosensor (load sample) and a second molecule in solution (the analytical sample).
2. Fix the load sample on the biocompatible biosensor while the analytical sample is in solution.
3. The biosensor tip is immersed in the solution so that the target molecule begins to bind to the analysis sample.
4. Set up and run the BLI experiment. Molecules bound to or dissociated from the biosensor can generate response curves on the BLI system; unbound molecules, changes in the refractive index of the surrounding medium or changes in flow rate do not affect the interferogram pattern.
5. Collect and analyse data on the BLI's system.

Materials

• Equipment: Fortebio Bio-Layer Interferometry (BLI)
• Sample Type: DNA, RNA, Protein, Antibodies, Peptides, Small Molecules
• Optionals: Pseudomonas aeruginosa strain PAO1
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