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Biosensor-enabled droplet microfluidic system for the rapid screening of 3-dehydroshikimic acid produced in Escherichia coli.
Journal of Industrial Microbiology & Biotechnology ( IF 3.4 ) Pub Date : 2020-09-27 , DOI: 10.1007/s10295-020-02316-1
Ran Tu 1, 2 , Liangpo Li 1 , Huiling Yuan 1 , Ronglin He 1, 3 , Qinhong Wang 1, 2
Affiliation  

Genetically encoded biosensors are powerful tools used to screen metabolite-producing microbial strains. Traditionally, biosensor-based screening approaches also use fluorescence-activated cell sorting (FACS). However, these approaches are limited by the measurement of intracellular fluorescence signals in single cells, rather than the signals associated with populations comprising multiple cells. This characteristic reduces the accuracy of screening because of the variability in signal levels among individual cells. To overcome this limitation, we introduced an approach that combined biosensors with droplet microfluidics (i.e., fluorescence-activated droplet sorting, FADS) to detect labeled cells at a multi-copy level and in an independent droplet microenvironment. We used our previously reported genetically encoded biosensor, 3-dehydroshikimic acid (3-DHS), as a model with which to establish the biosensor-based FADS screening method. We then characterized and compared the effects of the sorting method on the biosensor-based screening system by subjecting the same mutant library to FACS and FADS. Notably, our developed biosensor-enabled, droplet microfluidics-based FADS screening system yielded an improved positive mutant enrichment rate and increased productivity by the best mutant, compared with the single-cell FACS system. In conclusion, the combination of a biosensor and droplet microfluidics yielded a more efficient screening method that could be applied to the biosensor-based high-throughput screening of other metabolites.



中文翻译:

具有生物传感器功能的微滴微流控系统,用于快速筛选大肠杆菌中产生的3-dehydroshikimicacid。

遗传编码的生物传感器是用于筛选产生代谢物的微生物菌株的强大工具。传统上,基于生物传感器的筛选方法也使用荧光激活细胞分选(FACS)。然而,这些方法受到单个细胞中细胞内荧光信号而不是与包含多个细胞的群体相关的信号的测量的限制。由于单个细胞之间信号水平的差异,该特性降低了筛选的准确性。为了克服这一局限性,我们引入了一种将生物传感器与微滴微流控技术相结合的方法(即荧光激活的微滴分选,FADS),以多拷贝水平和独立的微滴微环境检测标记的细胞。我们使用了先前报道的基因编码生物传感器,3-dehydroshikimicacid(3-DHS),作为建立基于生物传感器的FADS筛选方法的模型。然后,我们通过对相同的突变文库进行FACS和FADS表征并比较了分选方法对基于生物传感器的筛选系统的影响。值得注意的是,与单细胞FACS系统相比,我们开发的基于生物传感器的,基于微滴微滴的FADS筛选系统可产生最佳突变体,从而提高了阳性突变体的富集率,并提高了生产率。总之,生物传感器和液滴微流体的结合产生了一种更有效的筛选方法,该方法可用于基于生物传感器的其他代谢产物的高通量筛选。然后,我们通过对相同的突变文库进行FACS和FADS表征并比较了分选方法对基于生物传感器的筛选系统的影响。值得注意的是,与单细胞FACS系统相比,我们开发的基于生物传感器的,基于微滴微滴的FADS筛选系统可产生最佳突变体,从而提高了阳性突变体的富集率,并提高了生产率。总之,生物传感器和液滴微流体的结合产生了一种更有效的筛选方法,该方法可用于基于生物传感器的其他代谢产物的高通量筛选。然后,我们通过对相同的突变文库进行FACS和FADS表征并比较了分选方法对基于生物传感器的筛选系统的影响。值得注意的是,与单细胞FACS系统相比,我们开发的基于生物传感器的,基于微滴微滴的FADS筛选系统可产生最佳突变体,从而提高了阳性突变体的富集率,并提高了生产率。总之,生物传感器和液滴微流体的结合产生了一种更有效的筛选方法,该方法可用于基于生物传感器的其他代谢产物的高通量筛选。与单细胞FACS系统相比,基于液滴微流控的FADS筛选系统可产生最佳突变体,从而提高了阳性突变体的富集率,并提高了生产率。总之,生物传感器和液滴微流体的结合产生了一种更有效的筛选方法,该方法可用于基于生物传感器的其他代谢产物的高通量筛选。与单细胞FACS系统相比,基于液滴微流控的FADS筛选系统可产生最佳突变体,从而提高了阳性突变体的富集率,并提高了生产率。总之,生物传感器和液滴微流体的结合产生了一种更有效的筛选方法,该方法可用于基于生物传感器的其他代谢产物的高通量筛选。

更新日期:2020-09-28
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