Engineering the bacteria present in animal microbiomes promises to lead to breakthroughs in medicine and agriculture, but progress is hampered by a dearth of tools for genetically modifying the diverse species that comprise these communities. Here we present a toolkit of genetic parts for the modular construction of broad-host-range plasmids built around the RSF1010 replicon. Golden Gate assembly of parts in this toolkit can be used to rapidly test various antibiotic resistance markers, promoters, fluorescent reporters, and other coding sequences in newly isolated bacteria. We demonstrate the utility of this toolkit in multiple species of Proteobacteria that are native to the gut microbiomes of honey bees (Apis mellifera) and bumble bees (Bombus sp.). Expressing fluorescent proteins in Snodgrassella alvi, Gilliamella apicola, Bartonella apis, and Serratia strains enables us to visualize how these bacteria colonize the bee gut. We also demonstrate CRISPRi repression in B. apis and use Cas9-facilitated knockout of an S. alvi adhesion gene to show that it is important for colonization of the gut. Beyond characterizing how the gut microbiome influences the health of these prominent pollinators, this bee microbiome toolkit (BTK) will be useful for engineering bacteria found in other natural microbial communities.

中文翻译：

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蜜蜂肠道微生物组细菌的基因工程与广泛的宿主范围质粒的模块化组装的工具包。

对动物微生物区系中存在的细菌进行工程改造有望在医学和农业领域取得突破，但是由于缺乏对这些群落组成的多种物种进行基因改造的工具而阻碍了进展。在这里，我们介绍了围绕RSF1010复制子构建的宽宿主范围质粒的模块化构建的遗传部分工具包。该工具包中的零件的Golden Gate组件可用于快速测试新分离的细菌中的各种抗生素抗性标记，启动子，荧光报告基因和其他编码序列。我们展示了此工具包在多种变形杆菌中的效用，而变形杆菌是蜜蜂（Apis mellifera）和大黄蜂（B ombus）肠道微生物区系原生的sp。）。在Snodgrassella alvi，Gilliamella apicola，Bartonella apis和Serratia菌株中表达荧光蛋白使我们能够直观地看到这些细菌如何在蜂肠中定居。我们还证明了B. apis中的CRISPRi抑制，并使用Cas9促进的al。S. alvi粘附基因的敲除，表明它对肠道定植很重要。除了表征肠道微生物组如何影响这些著名的传粉媒介的健康外，该蜜蜂微生物组工具包（BTK）对于工程改造其他天然微生物群落中发现的细菌也很有用。