Duarte José M, Barbier Içvara, Schaerli Yolanda
Department of Evolutionary Biology and Environmental Studies, University of Zurich , Winterthurerstrasse 190, 8057 Zürich, Switzerland.
Department of Fundamental Microbiology, University of Lausanne , Biophore Building, 1015 Lausanne, Switzerland.
ACS Synth Biol. 2017 Nov 17;6(11):1988-1995. doi: 10.1021/acssynbio.7b00111. Epub 2017 Aug 21.
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.
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识别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。特殊挑战包括细胞间的显著变异性以及检查多种状态的要求（例如，根据输入处于开启或关闭状态）。在这里，我们提出了一种解决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控琼脂糖凝胶珠中。孵育后，它们含有单克隆细菌微菌落（例如，表达一种合成构建体），并且可以通过荧光激活细胞分选（FACS）根据其荧光进行分选。我们确定了富集率，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，从而消除了细胞间变异性的问题。最后，我们应用这种方法对处于开启和关闭状态的pBAD启动子文库进行分选。
