Whole Cell Biosensing of Bacterial Chemotaxis

描述（由申请人提供）：美国面临着清理危险废物的艰巨任务。野生型（Wt）和基因工程微生物（GEMs）的生物修复具有完全降解废物的潜力，几乎没有有毒副产物。细菌粘附和向污染物移动（称为“趋化性”）是可能影响细菌在污染物生物降解中的作用的两个重要因素。然而，野生型（Wt）和基因工程微生物（GEMs）控制这些因素的基本机制仍然知之甚少，并且由于无法测量在化学引诱剂存在下细菌趋化行为的基本物理化学性质，因此尚未得到很好的定义。在这个项目中，我们提出开发一个全细胞生物传感系统，以实时测量细菌的趋化行为，基于一种新的双模电阻抗测量设计。具体目标是：1)集成的全细胞生物芯片设计和微制造，能够测量电阻抗（ECIS）和声阻抗（AIA）响应；2)实时、同步测量Wt和基因工程恶臭假单胞菌KT 2440在集成微加工工作电极上的粘附和趋化行为；测量Wt和GEM的形态学（ECIS）、粘弹性（AIA）和速度（延时视频显微镜）特性。3)将提出的全细胞生物传感系统与目前可用的趋化性检测技术在灵敏度和通用性方面进行比较；4)量化Wt和GEM（各种趋化基因的改变）在各种趋化物质存在下的趋化行为，并通过分析所提出的生物传感系统比较参数响应；5)通过解决NIH区域拨款计划的目标，加强犹他州立大学的生物工程项目。

DESCRIPTION (provided by applicant): The U.S. faces an enormous task in cleaning up hazardous wastes. Bioremediation via wild-type (Wt) and genetically engineered microorganisms (GEMs) has the potential of completely degrading waste material with little or no toxic byproducts. Bacterial adhesion and movement towards contaminants (termed "Chemotaxis") are two important factors that may affect the role of bacteria in the biodegradation of pollutants. However, the fundamental mechanisms governing these factors for wild type (Wt) and genetically engineered microorganisms (GEMs) are still poorly understood and have not been well defined because of the inability to measure basic physico-chemical properties of bacterial chemotaxic behaviors in the presence of chemoattractants. In this project, we propose to develop a whole cell biosensing system to measure chemotaxic behaviors of bacteria in real-time based on a novel design of dual mode electric impedance measurements. The specific aims are: 1) integrated whole cell biochip design and microfabrication that is capable of measuring electric impedance (ECIS) and acoustic impedance (AIA) responses; 2) real-time and simultaneous measurement of adhesion and chemotaxic behaviors of Wt and genetically engineered Pseudomonas putida KT 2440 on the integrated microfabricated working electrode; measurements of morphological (by ECIS), viscoelastic (by AIA), and velocity (by time-lapse video microscopy) properties of Wt and the GEM. 3) comparison of the proposed whole cell biosensing system with currently available chemotaxis detection techniques in terms of sensitivity and versatility; 4) quantification of the chemotaxic behaviors of Wt and GEM (alteration of various chemotaxis genes) in the presence of various chemotaxic substances, and comparison of parameter responses by analysis of the proposed biosensing system; 5) enhancement of the Biological Engineering program at Utah State by addressing the goals of the NIH AREA grant program.