同期两篇Cell登上封面:肠道菌研究新工具
2017/04/24
在4月21日的Cell上,有两篇文章在单细胞和基因基础上提供了改进的遗传工具,用于剖析细菌群落的空间组织和时间动态。可调启动子的工程菌植入小鼠,可以区分单个细胞菌株的表达状况。


设计成功的菌群治疗需要对于这个微生物社区的生态基础有深入的了解。在4月21日的Cell上,有两篇文章在单细胞和基因基础上提供了改进的遗传工具,用于剖析细菌群落的空间组织和时间动态。

可调启动子的工程菌植入小鼠

肠道菌群影响健康和疾病的许多方面,将它们联系起来的挑战性是因为肠道厌氧菌的遗传工具是非常有限的。诱导型启动子是特别有价值的工具,因为这些平台允许对微生物的基因产品对菌群组成、生理和疾病的贡献进行实时分析。

耶鲁大学的研究人员开发的可调表达平台,让拟杆菌属(Bacteroide)中基因的表达由人工合成的诱导控制因子调节。拟杆菌属在西方人的微生物菌群中是最丰富的菌属。诱导剂不存在的情况下,启动子活性被完全抑制;当诱导剂迅速增加四至五个数量级,基因表达。由于小鼠体内和它们的饮食中诱导剂是不存在的,肠道内细菌基因的表达可以通过提供有诱导剂的饮用水调节。


启动子工作的原理

区分单个细胞菌株的表达工具

斯坦福大学的研究人员应用合成生物学为研究肠道微生物提供了新的途径,可以用来调查微生物宿主相互作用,进行诊断,并提供治疗。在文章中,他们描述了一个拟杆菌属工程菌平台。研究人员已经确定了一个新的噬菌体启动子和转录调节策略的实现:使表达荧光蛋白的拟杆菌工程菌在小鼠肠道稳定地被植入。

这些噬菌体启动子能在肠道内无负担的状况下,提供超过14天的跨越四个指数级别的不同强度。这些启动子的功能可以在拟杆菌属类别中在100万倍不同的表达。有了这些启动子,独特的荧光信号就被编码允许区分肠道内的六个种的分化。


不同启动子编码不同的拟杆菌菌种

微生物群落大型数据集的变化的研究需要在动物模型获得5个WS:谁、什么、何时、何地、为什么的基础知识。研究方案如下图所示,可以获得肠道菌菌群的丰富信息。


参考资料

Building a Translational Microbiome Toolbox

Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut

Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome

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  • Engineered Regulatory Systems Modulate Gene Expression of Human Commensals in the Gut

    The gut microbiota is implicated in numerous aspects of health and disease, but dissecting these connections is challenging because genetic tools for gut anaerobes are limited. Inducible promoters are particularly valuable tools because these platforms allow real-time analysis of the contribution of microbiome gene products to community assembly, host physiology, and disease. We developed a panel of tunable expression platforms for the prominent genus Bacteroides in which gene expression is controlled by a synthetic inducer. In the absence of inducer, promoter activity is fully repressed; addition of inducer rapidly increases gene expression by four to five orders of magnitude. Because the inducer is absent in mice and their diets, Bacteroides gene expression inside the gut can be modulated by providing the inducer in drinking water. We use this system to measure the dynamic relationship between commensal sialidase activity and liberation of mucosal sialic acid, a receptor and nutrient for pathogens.

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  • Tunable Expression Tools Enable Single-Cell Strain Distinction in the Gut Microbiome

    Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut. A detailed characterization of the phage promoter has provided a set of constitutive promoters that span over four logs of strength without detectable fitness burden within the gut over 14 days. These promoters function predictably over a 1,000,000-fold expression range in phylogenetically diverse Bacteroides species. With these promoters, unique fluorescent signatures were encoded to allow differentiation of six species within the gut. Fluorescent protein-based differentiation of isogenic strains revealed that priority of gut colonization determines colonic crypt occupancy.

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