RUI: Molecular Analysis of the Poly-Beta-Hydroxybutyrate Biosynthetic Pathway

本项目旨在分析聚-羟基丁酸酯生物合成途径（PHB）的分子结构。PHB生物合成途径已从富营养盐中克隆到大肠杆菌中表达。这种表达可以通过限氧或限氮诱导。该途径编码的蛋白质将在体内特异性翻译，然后通过变性和非变性聚丙烯酰胺凝胶电泳进行分析。Northern blot杂交和核酸酶保护实验将分别用于确定转录模式和转录开始。构成该途径的三种酶最初将利用大。快速蛋白液相色谱（FPLC）纯化技术。最后的提纯将通过制备等电聚焦进行。蛋白质将被表征为它们的大小，亚基结构，部分氨基酸序列。将利用PHB:lac融合和诱导实验来定位PHB转录控制区，并确定诱导PHB生物合成的药物。这是一个建议，奠定了分子和遗传研究的代谢途径，导致生产的可生物降解的聚合物在很少研究的土壤细菌物种的基础。细菌似乎产生这种聚合物，作为在压力时期储存能量的一种手段，但它调节聚合物生产的机制是完全不清楚的，就像大多数土壤细菌的调节机制一样（除了少数已经得到充分研究的细菌）。本提案中的工作可能导致发现以前未知的基因表达调节机制，因此具有普遍的兴趣。它还特别令人感兴趣，因为它可能告诉我们关于生物可降解聚合物的自然合成，这些信息可能具有重要的实际后果。这项工作是由RUI项目资助的，因为大量的工作实际上是由本科生完成的，否则他们就不容易参加一个严肃的研究项目。

The goal of this project is to analyze the molecular structure of the poly.beta.hydroxybutyrate biosynthetic pathway (PHB). The PHB biosynthetic pathway has been cloned from Alcaligenes eutrophus into Escherichia coli where it is expressed. This expression can be induced via oxygen limitation or nitrogen limitation. Proteins encoded by the pathway will be specifically translated in vivo and then analyzed by denaturing and non.denaturing polyacrylamide gel electrophoresis. Northern blot hybridization and nuclease protection assays will be employed to determine the mode of transcription and start of transcription, respectively. The three enzymes that constitute the pathway will be initially purified utilizing large.scale purification techniques followed by fast protein liquid chromatography (FPLC). Final purification will be by preparative isoelectric focusing. The proteins will be characterized as to their size, subunit structure, partial amino acid sequence. Putative PHB transcriptional control regions will be located using PHB:lac fusions in concert with an induction assay, and agents that induce PHB biosynthesis will be identified. This is a proposal to lay the groundwork for the molecular and genetic study of a metabolic pathway leading to the production of a biodegradable polymer in a little studied species of soil bacterium. The bacterium seems to produce this polymer as a means of storing energy during times of stress, but the mechanism by which it regulates polymer production is completely obscure, as are regulatory mechanisms in most species of soil bacteria (except for a small number which have been well studied). The work in this proposal could lead to the discovery of previously unknown mechanisms regulation of gene expression, and is therefore of general interest. It is also of particular interest because of what it may tell us about the natural synthesis of the biodegradable polymer, information which may have important practical consequences. This work is funded under the RUI program, since a great deal of the work is actually performed by undergraduates who would not otherwise easily be able to participate in a serious research program.