The Use of Bacteriophage in the Prevention, Diagnosis, a

病原体检测。我们开发了一种简单快速的基于噬菌体的系统，用于检测临床或环境样品中的少量细菌病原体。这是通过用生物素化肽标记测试噬菌体衣壳来实现的。通过这种方式，噬菌体在细菌样本感染和生长后被生物素化。生物素化后的噬菌体颗粒通过特定量子点偶联进行监测，然后在显微镜下测量荧光。荚膜表面是噬菌体感染的屏障。产生K1多糖胶囊的大肠杆菌菌株长期以来与发病机制有关。这种胶囊被认为可以增加细胞的侵袭性，使细菌避免被补体吞噬和失活。它也被一些噬菌体识别为受体，如K1F和K1-5，它们具有降解多糖的病毒粒子相关酶。在本报告中，我们发现K1胶囊的表达是大肠杆菌通过T7物理阻断感染，T7是一种识别脂多糖为主要受体的噬菌体。酶从细胞中去除K1抗原允许T7吸附和复制。这一观察结果表明，胶囊在防御某些识别其下方结构的噬菌体方面起着重要作用，而k1特异性噬菌体进化以对抗这种物理屏障。蛋白质组学的双杂交纳米技术。鉴于全基因组注释的不断实现，蛋白质-蛋白质相互作用的全面描述是现代研究的一个日益增长的挑战。基于噬菌体lambda展示和简单的抗生素耐药性选择，建立了一种遗传双杂交系统，用于研究蛋白质-蛋白质相互作用。通过分析蛋白质分选信号泛素与Vsp9p的CUE结构域以及人工酸性和碱性适配体的特异性相互作用和竞争，证明了该方法的有效性。与酵母2-Hybrid系统、基于肽固定化的筛选程序以及T7和M13噬菌体展示系统相比，这里描述的方法是在没有细胞蛋白（离体）的情况下进行的，没有膜通道，不偏向小结构域，也没有苛刻的化学处理，可以实现高密度的展示。该lambda 9-hybrid系统为独立验证通过其他方法获得的蛋白质相互作用提供了一种简单的方法，并且是研究蛋白质-蛋白质相互作用的一个有吸引力的补充，旨在以最少的背景量识别大量的蛋白质相互作用。它还应该提供一个灵活的平台，以单一或多个诱饵进行文库筛选，在缺乏高浓度细胞蛋白质成分的情况下研究蛋白质- dna关联，药物发现以及选择和分析依赖于特定调节性的大分子相互作用。分泌抗hiv肽的活共生菌的构建和测试（与Dean Hamer合作）。大多数HIV传播发生在胃肠道和宫颈阴道粘膜表面，这两者通常都被一层非致病性共生细菌的生物膜所覆盖。我们建议对这种自然产生的细菌进行基因工程改造，通过分泌抗病毒肽来保护其免受HIV感染。在这里，我们描述了Nissle 1917的发展和特性，这是一种高度定植的大肠杆菌益生菌菌株，分泌HIV-gp41-溶血素a杂交肽，阻止HIV融合并进入靶细胞。通过使用顺式和交易分泌和调节信号的适当组合，实现了抗hiv肽的微摩尔分泌水平。

Detection of pathogens.We have developed a simple and fast bacteriophage-based system for detection of small number of bacterial pathogens in clinical or environmental samples. This was achieved by tagging tester phage capsids with biotinylating peptides. This way phage gets biotinylated after infection and growth in specimen bacterial samples. Biotinylated output phage particles are then monitored by specific quantum dots conjugation and then measuring fluorescence in microscope.Capsular surface is a barrier to bacteriophage infection.Escherichia coli strains that produce the K1 polysaccharide capsule have long been associated with pathogenesis. This capsule is believed to increase the cell's invasiveness, allowing the bacteria to avoid phagocytosis and inactivation by complement. It is also recognized as a receptor by some phages, such as K1F and K1-5, which have virion-associated enzymes that degrade the polysaccharide. In this report we show that expression of the K1 capsule is E. coli physically blocks infection by T7, a phage that recognizes lipopolysaccharide as the primary receptor. Enzymatic removal of the K1 antigen from the cell allows T7 to adsorb and replicate. This observation suggests that the capsule plays an important role as a defense against some phages that recognize structure beneath it and the K1-specific phages evolved to counter this physical barrier.A two-hybrid nanotechnology for proteomics.In light of the continued realization of full genome annotations, a comprehensive description of protein-protein interactions is a growing challenge of modern research. A genetic two-hybrid system has been developed for studying protein-protein interactions that is based on bacteriophage lambda display and simple antibiotic resistance selection. The validity of this approach was demonstrated by analyzing the specific interaction and competition of both the protein sorting signal ubiquitin with the CUE domain of Vsp9p, and of artificial acidic and basic aptamers. In contrast to the yeast 2-Hybrid system, peptide immobilization-based panning procedures, and T7 and M13 phage display systems, the approach described here is carried out in the absence of cellular proteins (ex vivo), there is no membrane passage, no bias towards small domains and no harsh chemical treatment, and a high density of display can be achieved. This lambda 9-hybrid system provides a simple way for independent verification of protein interactions obtained through other means, and is a attractive complement for studying protein-protein interactions that aim to identify the large number of protein interactions with lowest amount of background. It should also provide a flexible platform to perform library panning with singular or multiple bait(s), to study protein-DNA association in the absence of high concentration of cellular protein components, drug discovery and to select for and analyze macromolecular interactions that are dependent upon specific modulators.Construction and test of a live commensal bacteria secreting an anti-HIV peptide (collaboration with Dean Hamer).Most HIV transmission occurs on the mucosal surfaces of the gastrointestinal and cervicovaginal tracts, both of which are normally coated by a biofilm of nonpathogenic commensal bacteria. We propose to genetically engineer such naturally occurring bacteria to protect against HIV infection by secreting antiviral peptides. Here we describe the development and characterization of Nissle 1917, a highly colonizing probiotic strain of Escherichia coli, secreting HIV-gp41-hemolysin A hybrid peptides that block HIV fusion and entry into target cells. By using an appropriate combination of cis- and transacting secretory and regulatory signals, micromolar secretion levels of the anti-HIV peptides are achieved.