Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology,ACS Synthetic Biology

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Bacterial Microcolonies in Gel Beads for High-Throughput Screening of Libraries in Synthetic Biology
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2017-08-21 00:00:00 , DOI: 10.1021/acssynbio.7b00111
José M. Duarte ₁ , Içvara Barbier ₂ , Yolanda Schaerli _{1,

2}

Affiliation

Synthetic biologists increasingly rely on directed evolution to optimize engineered biological systems. Applying an appropriate screening or selection method for identifying the potentially rare library members with the desired properties is a crucial step for success in these experiments. Special challenges include substantial cell-to-cell variability and the requirement to check multiple states (e.g., being ON or OFF depending on the input). Here, we present a high-throughput screening method that addresses these challenges. First, we encapsulate single bacteria into microfluidic agarose gel beads. After incubation, they harbor monoclonal bacterial microcolonies (e.g., expressing a synthetic construct) and can be sorted according their fluorescence by fluorescence activated cell sorting (FACS). We determine enrichment rates and demonstrate that we can measure the average fluorescent signals of microcolonies containing phenotypically heterogeneous cells, obviating the problem of cell-to-cell variability. Finally, we apply this method to sort a pBAD promoter library at ON and OFF states.

中文翻译：

凝胶珠中的细菌微菌落，用于合成生物学中图书馆的高通量筛选

合成生物学家越来越依赖定向进化来优化工程生物系统。应用适当的筛选或选择方法来鉴定具有所需特性的潜在稀有文库成员是这些实验成功的关键步骤。特殊的挑战包括很大的单元间差异性以及检查多个状态的要求（例如，根据输入为开或关）。在这里，我们提出了一种应对这些挑战的高通量筛选方法。首先，我们将单个细菌封装到微流琼脂糖凝胶珠中。孵育后，它们带有单克隆细菌微菌落（例如（表示合成的构建体），并可以通过荧光激活细胞分选（FACS）根据其荧光进行分选。我们确定富集率，并证明我们可以测量包含表型异质细胞的微菌落的平均荧光信号，从而避免了细胞间差异的问题。最后，我们使用此方法对处于ON和OFF状态的pBAD启动子库进行排序。

更新日期：2017-08-21

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