Engineered revision of the E. coli Nissle 1917 Genome

描述（由申请人提供）：转基因益生菌（有益）细菌递送治疗化合物的能力是一种很有前途的新实验方法，可以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的遗传稳定性，使其成为一种活的药物传递载体。大肠杆菌Nissle 1917是一种非致病性大肠杆菌，目前用于肠道疾病的益生菌治疗，是一种有吸引力的候选给药系统。虽然重组DNA方法最初是在大肠杆菌中首创的，并且这种生物仍然是细菌生产重组产品的最常见宿主，但仍然存在一些问题。菌株性能的退化在工业发酵中并不罕见，通常是由于源自细菌染色体的可移动元素的遗传不稳定造成的。这一建议概述了一个程序，从大肠杆菌Nissle 1917染色体上去除移动元件，使用表征良好的重组方法。要删除的序列是根据其与致病性大肠杆菌区域的相似性、许多可移动元件和可识别的噬菌体序列的存在以及与将用于该菌株治疗应用的基因的相似性来选择的。拟议研究计划的主要目标是生产大肠杆菌尼塞尔1917的变种，这些变种缺乏所有致病性和可移动的遗传元素。将测试单个缺失菌株的生存能力及其在小鼠肠道内持续存在的能力。没有表现出生存力或持久性损失的缺失菌株将被记录下来，并将这些缺失累积起来。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，以确定哪些基因需要恢复活力和/或持久性。

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.