Temperature-sensitive gene modules for synthetic biology applications

拟议的研究建立在我们最近的成功基础上，即将北极必要基因替换为中温致病菌，使后者对温度稳定敏感(DuPlantis等人，PNAS，2010年)。这项工作被广泛认为是开创性的，我们是世界上开展这一领域研究的领导小组。对于这项提议，我们概述了一项研究计划，在该计划中，我们将发现或开发一系列使微生物对温度敏感的基因盒，我们收集的基因盒将使我们能够应用我们的技术来解决生物技术以及人类和动物健康方面的许多问题。将使用两种基本方法。我们目前的方法依赖于生物勘探来发现嗜冷(嗜寒)细菌基因组中编码的必要基因。我们将继续使用这种方法来显著扩大我们对温度敏感的基本基因的收集。我们还将测试嗜热菌(喜暖)的必需基因是否可以设定中温菌(喜温)的生长温度下限。在第二种方法中，我们将使用各种合成生物学方法来开发温度敏感型基因盒。这些盒式磁带将被用来诱导致命基因的表达或消除一种杀死微生物的毒素的解毒剂。我们希望温度敏感型必需基因或盒的家族具有足够的通用性，可以用于任何细菌，其中一些盒将在真核微生物工程中有用。

The proposed research builds on our recent successes of substituting Arctic essential genes into mesophilic pathogenic bacteria to make the latter stably temperature-sensitive (Duplantis, et al., PNAS, 2010). This work was widely recognized as ground-breaking, and we are the leading group in the world carrying out this area of research. For this proposal we outline a research program in which we will discover or develop a broad family of gene cassettes that render microbes temperature-sensitive, and our collection of gene cassettes will allow us to apply our technology to solve many problems in biotechnology and in human and animal health. Two basic approaches will be used. Our current approach relies on bioprospecting to discover essential genes encoded in the genomes of psychrophilic (cold-loving) bacteria. We will continue to use this approach to significantly expand our collection of temperature-sensitive essential genes. We will also test if essential genes from thermophiles (warm-loving) can set the lower temperature growth limit of a mesophile (medium temperature-loving). In the second approach we will develop temperature-sensitive gene cassettes using a variety of synthetic biology approaches. These cassettes will be used to induce the expression of lethal genes or the elimination of an antidote to a toxin that kills a microbe. We expect the family of temperature-sensitive essential genes or cassettes to be versatile enough to be used in any bacterium and some of the cassettes will be useful in the engineering of eukaryotic microbes.