SUGAR Program, X-star, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), 2-15 Natsushima-cho, Yokosuka 237-0061, Japan.
Department of Applied Chemistry, School of Engineering, The University of Tokyo, Tokyo 113-8656, Japan.
Sci Adv. 2019 Aug 21;5(8):eaav8185. doi: 10.1126/sciadv.aav8185. eCollection 2019 Aug.
We report a general strategy based on digital counting principle that enables an efficient acquisition of enzyme mutants with desired activities from just a few clones within a day. We prepared a high-density femtoliter droplet array, consisting of 1 million uniform droplets per 1 cm to carry out high-throughput protein synthesis and screening. Single DNA molecules were randomly distributed into each droplet following a Poisson process to initiate the protein synthesis with coupled cell-free transcription and translation reactions and then recovered by a microcapillary. The protein yield in each droplet was proportional to the number of DNA molecules, meaning that droplets with apparent intensities higher than the Poisson distribution-predicted maximum can be readily identified as the exact hits exhibiting the desired increased activity. We improved the activity of an alkaline phosphatase up to near 20-fold by using less than 10 nl of reagents.
我们报道了一种基于数字计数原理的通用策略，该策略可在一天内从仅几个克隆中高效获得具有所需活性的酶突变体。我们制备了高密度的皮升级液滴阵列，每个 1 厘米包含 100 万个均匀的液滴，以进行高通量蛋白质合成和筛选。单链 DNA 分子随机分布到每个液滴中，遵循泊松过程，以启动与无细胞转录和翻译反应偶联的蛋白质合成，然后通过微毛细管回收。每个液滴中的蛋白质产量与 DNA 分子的数量成正比，这意味着明显强度高于泊松分布预测最大值的液滴可以很容易地被识别为表现出所需活性增加的确切命中。我们使用不到 10nl 的试剂将碱性磷酸酶的活性提高了近 20 倍。
