【作者】 黄琛；

【导师】 冯雁；

【作者基本信息】 上海交通大学 ， 生物学， 2013， 硕士

【摘要】 定向进化作为蛋白质工程领域的重要方法，通过构建随机突变库，并用具有针对性的筛选方式对突变体的性质进行评估和分选，从而实现酶功能在分子水平上的改造。目前限制定向进化成功的关键仍是缺少真正高通量、通用型的高效筛选方法。荧光激活微液滴分选（FluorescenceActivated Droplet Sorting, FADS）方法是在微型液滴中进行酶反应，并通过荧光信号对酶活性进行高速定量分析和分选，因此成为目前最有潜力的超高通量筛选方法之一，并由于其通用性较好受到关注。然而，现有技术所生成的微液滴均一性较差，影响FADS体系筛选的准确性和效率，从而严重限制了该体系的广泛应用。为了解决这一问题，本论文在膜挤出仪和微流控芯片两种不同的微液滴生成技术中进行了探索，重点研究了生成均一微液滴的方法，并考察了其用于FADS系统的可行性。一、对“膜挤出仪法-FADS”体系进行了系统优化与表征，并测试了其在酯酶筛选中的应用。本论文将互不相溶的两相液体（轻石蜡油和水）在膜挤出仪内反复挤压通过聚碳酸酯滤膜，实现乳化过程并得到微液滴。具体研究包括：（1）乳化次数的优化。确定了一级乳化次数为15.5-20.5次，二级乳化次数为25.5-30.5次，同步提高了单层和双层微液滴的均一性。与常用的匀浆仪乳化法比较，膜挤出仪法保持了操作简便、液滴生成迅速的特点，而且在微液滴的均一程度上有着显著优势。验证了膜挤出仪法生成的微液滴中包裹单细胞的效率符合泊松分布；（2）酶反应的表征。确定了在皮升（10-12L）级别的微液滴中能够进行酶反应，且荧光信号稳定。筛选系统能够分辨不同底物浓度、不同酶浓度和不同酶活力形成的荧光信号差异，证明了此系统用于筛选的可行性；（3）酯酶活力的模式筛选实验。以表面展示酯酶AFEST的大肠杆菌细胞作为阳性个体，含有空质粒pGF101的大肠杆菌作为阴性对照，证明可在含大量阴性对照的群体中对少量有活性的阳性个体（如1/1000）进行富集，单次分选最高富集效率达到333倍以上，显示了该体系较高的筛选效率。经过优化和表征，“膜挤出仪法-FADS”体系的效率和稳定性得到了显著提升，拥有进行实际突变库筛选的巨大潜力。二、对“微流控芯片-FADS”体系进行了初步探索。本论文基于微流控芯片，通过连续相液流对分散相液流的剪切，模仿乳化过程从而制备得到微液滴。研究包括：（1）聚二甲基硅氧烷（PDMS）芯片的设计和优化。成功对芯片通道的流动聚焦结构、内部通道、接口与滤器等处进行了改良，提高了微液滴均一性、操作稳定性和实验重复性；（2）溶剂系统的选择。确定了适合生成“水-油”（waterin oil, w/o）微液滴的油相为轻石蜡油，而适合“油-水”（oil in water, o/w）微液滴的油相为碳氟油；（3）单层微液滴的制备。通过对芯片通道宽度、液流速度等条件的摸索，稳定地生成了不同直径大小（10μm和30μm）的单层微液滴，证明了影响液滴大小的关键因素为流动聚焦的尺寸；（4）液滴性质表征。经过系统优化的液滴具有极高的均一度和稳定性（在37℃下能够稳定超过24小时）；（5）单细胞包裹的尝试。针对进样过程中的细胞沉降现象，开发了使用羟乙基纤维素作为增稠剂的有效解决方案，并发现了影响包裹效率的另一关键参数——液滴直径。初步建立的微流控实验平台为继续发展超高通量筛选方法奠定了基础。本论文面对建立通用型超高通量酶活性筛选方法这一目标，在FADS系统中进行了方法学上的探索，构建了两种不同的液滴体系以应对不同的需求。膜挤出仪法简单灵活，易于操作，但受到设备精度的限制，其误差仍相对较大；微流控芯片技术高度准确，有利于筛选效率的进一步提升，是未来重要的发展方向。通过对新方法的实际应用，将会大大缩短用于突变库筛选的时间，显著减少所需成本，高效率地获得新功能酶。更多还原

【Abstract】 As an important method for protein engineering, directed evolution is widelyused to modify enzymes at molecular level. Lack of genuine high-throughput,high-versatility screening methods limits the success of directed evolution.Fluorescence Activated Droplet Sorting (FADS) is one of the most promisingscreening methods with high potential. The core to FADS is the preparation of microreactor, or micro droplets, which reaction happens inside. Due to the technicallimitation in droplet generation, the poor uniformity is always a bottleneck, whichaffects the accuracy and efficiency of FADS.To solve this problem, this thesis focused on exploring two droplet generationmethods, known as membrane-extrusion and microfluidic chips. And their feasibilityfor FADS was investigated, respectively.On one hand, based on the previous study in our laboratory, an “extrusion-FADS”system was deeply optimized and explored. After optimization for emulsificationtimes, the ideal generation process was established, including15.5-20.5for w/odroplets and25.5-30.5for w/o/w droplets, and the uniformity was significantlyimproved. Then this method was compared with homogenizing, which was the mostcommonly used for droplet generation, and the advantages were proved. The statusfor single cell encapsulation in droplets generated by mini-extruder was given asPoisson distribution. To grope for the suitable conditions of enzyme reactions withindroplets, several experiments including different substrate concentrations, differentenzyme concentrations, etc. were implemented. The results showed that this systemcould make a distinction between enzymes with different substrate concentrations, enzyme concentrations, and activities, thereby indicated the feasibility for mutantscreening. Finally, the model screening assay (using esterase AFEST as positiveindividual and pGF101blank as negative one) revealed that the esterase-displayingcells could be enriched out of a large excess of non-active ones (positive: negative=1:1000) in a single round of sorting (with the highest enrichment factor of330).On the other hand, a microfluidic-based experimental platform was initiallyestablished for FADS. Through careful design of the internal channel, and search forappropriate oil phase, highly-uniformed droplets (w/o and o/w) could be generatedusing PDMS chips. Different droplets size could be obtained as well by changing thewidth of orifice. The stability of droplets was tested at37℃and the integralityremained good overnight. A “trick” to prevent the E.coli cells from sedimentationduring encapsulation was also developed by using hydroxyethyl cellulose asthickening agent.In this thesis, facing the target of establishing a high-throughput andhigh-versatility enzymatic screening system, we set up two different droplet systemsby implement several explorations in methodology. Membrane extrusion was simple,flexible and easy to operate, but with relatively large deviation. Microfluidic chip wasaccurate, and being the direction of future development. However, with highcomplexity the system remains under discussion. Practical application of the newmethod, it will greatly shorten the time for directed evolution, significantly reduce thecost required to facilitate enzymatic properties modification.更多还原