1. Use of Quantum Dots in detection of pathogens. With current concerns of antibiotic-resistant bacteria and biodefense, it has become important to rapidly identify infectious bacteria. Traditional technologies involving isolation and amplification of the pathogenic bacteria are time-consuming. We report a rapid and simple method that combines in vivo biotinylation of engineered host-specific bacteriophage and conjugation of the phage to streptavidin-coated quantum dots. The method provides specific detection of as few as 10 bacterial cells per milliliter in experimental samples, with an approximately 100-fold amplification of the signal over background in 1 h. We believe that the method can be applied to any bacteria susceptible to specific phages and would be particularly useful for detection of bacterial strains that are slow growing, e.g., Mycobacterium, or are highly infectious, e.g., Bacillus anthracis. The potential for simultaneous detection of different bacterial species in a single sample and applications in the study of phage biology are discussed. In summary, we established a detection system for bacteria and are planning to use similar concepts for pathogenic bacteria and other targets in whole animals. 2. A mutant bacteriophage that survives mammalian innate immune system for bacteriophage therapy.In experiments with germ free mice, free from adaptive antibodies to the bacterial virus lambda phage, titers of the virus in the circulatory system have been reported to decrease by more than 10(9)pfu within 48 h of intraperitoneal intravenous or oral administration. Based on these observations, serial passage techniques have been used to select lambda phage mutants, with 13,000-16,000-fold greater capacity to remain in the mouse circulatory system 24h after intraperitoneal injection. In these prior studies the "long-circulating" phage, designated lambdaArgo phage, had at least three mutations including one in the major phage capsid (E) protein, which resulted in the change of glutamic acid to a lysine at residue 158. In the current study, we demonstrate that this single specific substitution in the E protein is sufficient to confer the "long-circulating" phenotype. The isogenic pair of phage developed in this study consisting of the long-circulating marker-rescued lambdaArgo phage, and the parental wild type phage can be used for studies of viral recognition mechanisms of the innate immune system and for the development of more effective antibacterial therapeutic phage strains.3. A two-hybrid nanotechnology for proteomics.Our goal is to develop lambda D display as a novel 2-Hybrid system that is inherently broad in application and very low in false positives. A central challenge intrinsic to the rapidly growing field of proteonomics is the establishment of an accurate and comprehensive description of those protein-protein interactions responsible for governing complex communication networks as well as those alterations that are responsible for the development and/or maintenance of disease states. In order to identify the components and study their interactions, we have developed a 2-Hybrid system that is based on bacteriophage lambda display tools. Since lambda display has experienced great functional successes in binding site prediction and bio-panning, the development of a lambda-2Hybrid strategy to study protein interactions and inhibition of association is expected to provide a simple way to independently verify interactions between proteins obtained through other means, as well as being an attractive complement for easily identifying new interactions with a flexibility and sensitivity unmatched by other 2Hybrid systems.
NIH ExPorter
NIH ExPorter