PDF(Pubmed)
Abstract：
BACKGROUND: Single-cell droplet microfluidics is an important platform for high-throughput analyses and screening because it provides an independent and compartmentalized microenvironment for reaction or cultivation by coencapsulating individual cells with various molecules in monodisperse microdroplets. In combination with microbial biosensors, this technology becomes a potent tool for the screening of mutant strains. In this study, we demonstrated that a genetically engineered yeast strain that can fluorescently sense agonist ligands via the heterologous expression of a human G-protein-coupled receptor (GPCR) and concurrently secrete candidate peptides is highly compatible with single-cell droplet microfluidic technology for the high-throughput screening of new agonistically active peptides.
RESULTS: The water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists.
CONCLUSIONS: We established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.
RESULTS: The water-in-oil microdroplets were generated using a flow-focusing microfluidic chip to encapsulate engineered yeast cells coexpressing a human GPCR [i.e., angiotensin II receptor type 1 (AGTR1)] and a secretory agonistic peptide [i.e., angiotensin II (Ang II)]. The single yeast cells cultured in the droplets were then observed under a microscope and analyzed using image processing incorporating machine learning techniques. The AGTR1-mediated signal transduction elicited by the self-secreted Ang II peptide was successfully detected via the expression of a fluorescent reporter in single-cell yeast droplet cultures. The system could also distinguish Ang II analog peptides with different agonistic activities. Notably, we further demonstrated that the microenvironment of the single-cell droplet culture enabled the detection of rarely existing positive (Ang II-secreting) yeast cells in the model mixed cell library, whereas the conventional batch-culture environment using a shake flask failed to do so. Thus, our approach provided compartmentalized microculture environments, which can prevent the diffusion, dilution, and cross-contamination of peptides secreted from individual single yeast cells for the easy identification of GPCR agonists.
CONCLUSIONS: We established a droplet-based microfluidic platform that integrated an engineered yeast biosensor strain that concurrently expressed GPCR and self-secreted the agonistic peptides. This offers individually isolated microenvironments that allow the culture of single yeast cells secreting these peptides and gaging their signaling activities, for the high-throughput screening of agonistic peptides. Our platform base on yeast GPCR biosensors and droplet microfluidics will be widely applicable to metabolic engineering, environmental engineering, and drug discovery.
摘要：
背景：单细胞液滴微流体是高通量分析和筛选的重要平台，因为它通过将单个细胞与各种分子共封装在单分散的微液滴中，为反应或培养提供了独立且分隔的微环境。结合微生物生物传感器，该技术成为筛选突变菌株的有力工具。在这项研究中,我们证明了通过人G蛋白偶联受体（GPCR）的异源表达可以荧光感知激动剂配体并同时分泌候选肽的基因工程酵母菌株与用于高通量筛选新的激动活性肽的单细胞液滴微流控技术高度兼容。
结果：使用流动聚焦微流控芯片封装共表达人类GPCR的工程酵母细胞[即，血管紧张素II受体1型（AGTR1）]和分泌激动肽[即，血管紧张素II（AngII）]。然后在显微镜下观察在液滴中培养的单个酵母细胞，并使用结合机器学习技术的图像处理进行分析。通过在单细胞酵母液滴培养物中表达荧光报告基因，成功检测到了由自分泌的AngII肽引起的AGTR1介导的信号转导。该系统还可以区分具有不同激动活性的AngII类似物肽。值得注意的是,我们进一步证明，单细胞液滴培养的微环境能够检测模型混合细胞库中很少存在的阳性（分泌AngII）酵母细胞，而使用摇瓶的常规分批培养环境却无法做到这一点。因此，我们的方法提供了分隔的微培养环境，可以防止扩散，稀释，以及从单个单个酵母细胞分泌的肽的交叉污染，以便于鉴定GPCR激动剂。
结论：我们建立了基于液滴的微流体平台，该平台整合了同时表达GPCR和自分泌激动肽的工程化酵母生物传感器菌株。这提供了单独分离的微环境，允许培养分泌这些肽的单个酵母细胞并测量其信号活动，用于激动肽的高通量筛选。我们基于酵母GPCR生物传感器和液滴微流体的平台将广泛适用于代谢工程，环境工程,和药物发现。
结果：使用流动聚焦微流控芯片封装共表达人类GPCR的工程酵母细胞[即，血管紧张素II受体1型（AGTR1）]和分泌激动肽[即，血管紧张素II（AngII）]。然后在显微镜下观察在液滴中培养的单个酵母细胞，并使用结合机器学习技术的图像处理进行分析。通过在单细胞酵母液滴培养物中表达荧光报告基因，成功检测到了由自分泌的AngII肽引起的AGTR1介导的信号转导。该系统还可以区分具有不同激动活性的AngII类似物肽。值得注意的是,我们进一步证明，单细胞液滴培养的微环境能够检测模型混合细胞库中很少存在的阳性（分泌AngII）酵母细胞，而使用摇瓶的常规分批培养环境却无法做到这一点。因此，我们的方法提供了分隔的微培养环境，可以防止扩散，稀释，以及从单个单个酵母细胞分泌的肽的交叉污染，以便于鉴定GPCR激动剂。
结论：我们建立了基于液滴的微流体平台，该平台整合了同时表达GPCR和自分泌激动肽的工程化酵母生物传感器菌株。这提供了单独分离的微环境，允许培养分泌这些肽的单个酵母细胞并测量其信号活动，用于激动肽的高通量筛选。我们基于酵母GPCR生物传感器和液滴微流体的平台将广泛适用于代谢工程，环境工程,和药物发现。
