Engineered revision of the E. coli Nissle 1917 Genome

描述（由申请人提供）：遗传修饰的益生菌（有益）细菌递送治疗化合物的能力是一种有前途的新实验方法，用于在不注射的情况下递送脆弱和复杂的生物分子。这项研究的长期目标是提高益生菌生物体的遗传稳定性，以用作活的药物递送载体。大肠杆菌Nissle 1917是一株非致病性大肠杆菌。目前用于肠道疾病的益生菌治疗的大肠杆菌是一种有吸引力的候选药物递送系统。虽然重组DNA方法最初是在E.大肠杆菌，并且该生物体仍然是用于细菌生产重组产物的最常见宿主，但是仍然出现一些问题。菌株性能的降低在工业发酵中并不罕见，并且通常是由于源自细菌染色体的移动的元件引起的遗传不稳定性的结果。该提案概述了从E中去除移动的元素的方案。coliNissle 1917染色体上的重组。待删除的序列根据它们与致病性大肠杆菌中的区域的相似性来选择。大肠杆菌中存在许多移动的元件和可识别的原噬菌体序列，以及与将用于该菌株的治疗应用的基因的相似性。该研究计划的主要目标是生产E. coli Nissle 1917，其缺乏所有致病性和移动的遗传元件。将检测单个缺失菌株的活力及其在小鼠肠道中持续存在的能力。将记录未表现出活力或持久性丧失的缺失菌株，并将缺失累积合并。活力或持久性降低的缺失菌株将作为基因添加研究的宿主，以确定恢复活力和/或持久性需要哪些基因。

DESCRIPTION (provided by applicant): The ability of genetically modified probiotic (beneficial) bacteria to deliver therapeutic compounds is a promising new experimental method for delivering fragile and complex bio-molecules without injection. The long-term goal of this study is to improve the genetic stability of a probiotic organism for use as a living drug delivery vehicle. Escherichia coli Nissle 1917, a non-pathogenic E. coli, currently used in probiotic treatment of intestinal disorders, is an attractive candidate drug delivery system. Although recombinant DNA methods were originally pioneered in E. coli and this organism remains the most common host for bacterial production of recombinant products, some problems still occur. Degradation of strain performance is not unusual in industrial fermentations and is often the result of genetic instability due to mobile elements originating from the bacterial chromosome. This proposal outlines a program for removing mobile elements from the E. coli Nissle 1917 chromosome using well characterized recombination methods. Sequences to be deleted are chosen on the basis of their similarity to regions in pathogenic E. coli, the presence of many mobile elements and identifiable prophage sequences and similarity to genes that will be used in therapeutic applications of this strain. The primary goal of the proposed research program is to produce variants of E. coli Nissle 1917 that lack all pathogenic and mobile genetic elements. Individual deletion strains will be tested for viability and their ability to persist in the mouse gut. Deletion strains that exhibit no loss of viability or persistence will be noted and the deletions cumulatively combined. Deletion strains with diminished viability or persistence will serve as hosts for gene addition studies, to determine which genes are required to restore viability and/or persistence.