Bernath Kalia, Hai Mingtan, Mastrobattista Enrico, Griffiths Andrew D, Magdassi Shlomo, Tawfik Dan S
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Anal Biochem. 2004 Feb 1;325(1):151-7. doi: 10.1016/j.ab.2003.10.005.
Water-in-oil (w/o) emulsions can be used to compartmentalize and select large gene libraries for a predetermined function. The aqueous droplets of the w/o emulsion function as cell-like compartments in each of which a single gene is transcribed and translated to give multiple copies of the protein (e.g., an enzyme) it encodes. While compartmentalization ensures that the gene, the protein it encodes, and the products of the activity of this protein remain linked, it does not directly afford a way of selecting for the desired activity. Here we show that re-emulsification of w/o emulsions gives water-in-oil-in-water (w/o/w) emulsions with an external (continuous) water phase through which droplets containing fluorescent markers can be isolated by fluorescence-activated cell sorting (FACS). These w/o/w emulsions can be sorted by FACS, while the content of the aqueous droplets of the primary w/o emulsion remains intact. Consequently, genes embedded in these water droplets together with a fluorescent marker can be isolated and enriched from an excess of genes embedded in water droplets without a fluorescent marker. The ability of FACS instruments to sort up to 40000 events per second may endow this technology a wide potential in the area of high-throughput screening and the directed evolution of enzymes.
油包水（w/o）乳液可用于对大型基因文库进行分隔，并筛选出具有预定功能的基因。w/o乳液的水滴起着类似细胞的分隔作用，每个水滴中单个基因被转录和翻译，从而产生其编码的蛋白质（如一种酶）的多个拷贝。虽然分隔确保了基因、其编码的蛋白质以及该蛋白质活性的产物保持关联，但它并不能直接提供一种筛选所需活性的方法。在此我们表明，w/o乳液的再乳化可得到水包油包水（w/o/w）乳液，其具有外部（连续）水相，通过荧光激活细胞分选（FACS）可分离出含有荧光标记的液滴。这些w/o/w乳液可通过FACS进行分选，而原始w/o乳液水滴的内容物保持完整。因此，嵌入这些水滴中的基因与荧光标记一起，可从大量嵌入无荧光标记水滴中的基因中分离和富集出来。FACS仪器每秒可分选多达40000个事件的能力，可能使该技术在高通量筛选和酶的定向进化领域具有广泛的潜力。
