Department of Biomedical Engineering, Duke University, Durham, 27705, USA.
Nanoscale. 2017 Mar 9;9(10):3485-3495. doi: 10.1039/c6nr08224f.
The rapid advances in synthetic biology and biotechnology are increasingly demanding high-throughput screening technology, such as screening of the functionalities of synthetic genes for optimization of protein expression. Compartmentalization of single cells in water-in-oil (W/O) emulsion droplets allows screening of a vast number of individualized assays, and recent advances in automated microfluidic devices further help realize the potential of droplet technology for high-throughput screening. However these single-emulsion droplets are incompatible with aqueous phase analysis and the inner droplet environment cannot easily communicate with the external phase. We present a high-throughput, miniaturized screening platform for microchip-synthesized genes using microfluidics-generated water-in-oil-in-water (W/O/W) double emulsion (DE) droplets that overcome these limitations. Synthetic gene variants of fluorescent proteins are synthesized with a custom-built microarray inkjet synthesizer, which are then screened for expression in Escherichia coli (E. coli) cells. Bacteria bearing individual fluorescent gene variants are encapsulated as single cells into DE droplets where fluorescence signals are enhanced by 100 times within 24 h of proliferation. Enrichment of functionally-correct genes by employing an error correction method is demonstrated by screening DE droplets containing fluorescent clones of bacteria with the red fluorescent protein (rfp) gene. Permeation of isopropyl β-d-1-thiogalactopyranoside (IPTG) through the thin oil layer from the external solution initiates target gene expression. The induced expression of the synthetic fluorescent proteins from at least ∼100 bacteria per droplet generates detectable fluorescence signals to enable fluorescence-activated cell sorting (FACS) of the intact droplets. This technology obviates time- and labor-intensive cell culture typically required in conventional bulk experiment.
合成生物学和生物技术的快速发展越来越需要高通量筛选技术，例如筛选合成基因的功能以优化蛋白质表达。将单细胞分隔在油包水（W/O）乳液滴中，可以对大量的个体化测定进行筛选，而最近自动化微流控设备的进展进一步有助于实现液滴技术在高通量筛选中的潜力。然而，这些单乳液滴与水相分析不兼容，并且内部液滴环境不易与外部相通信。我们提出了一种使用微流控技术生成的水包油包水（W/O/W）双重乳液（DE）液滴的高通量、微型化筛选平台，该平台克服了这些限制。使用定制的微阵列喷墨合成器合成荧光蛋白的合成基因变体，然后在大肠杆菌（E. coli）细胞中进行表达筛选。将带有单个荧光基因变体的细菌封装成单个细胞，在 DE 液滴中增殖 24 小时内，荧光信号增强 100 倍。通过筛选含有红色荧光蛋白（rfp）基因的荧光克隆细菌的 DE 液滴，证明了通过错误校正方法富集功能正确的基因。异丙基β-D-1-硫代半乳糖吡喃糖苷（IPTG）通过外部溶液中的薄油层渗透，引发目标基因表达。从至少约 100 个细菌/液滴中产生的合成荧光蛋白的诱导表达产生可检测的荧光信号，从而可以对完整的液滴进行荧光激活细胞分选（FACS）。这项技术避免了传统批量实验中通常需要的耗时且劳动密集型的细胞培养。
