Blue-shifting green fluorescent proteins using REPLACE

In the directed evolution experiments of EGFP or StayGold, the objective was to blue-shift the excitation light spectrum of GFP towards shorter wavelengths, resulting in an enhancement of fluorescence intensity upon 405 nm laser excitation. This facilitates the advancement of novel fluorescent proteins. These experiments represent a category of experiments that utilize REPLACE to achieve discontinuous directed evolution through FACS sorting (screening). Overall design: EGFP or StayGold was cloned into the repRNA-v4-derived vector. The vector was in vitro transcribed into RNA and delivered into host cells via electroporation. After 24 hours of electroporation, cells were subjected to continuous puromycin treatment (10 µg/ml) with regular passages. After another 5-7 days, a relatively stable and homogeneous population of polyclonal cells carrying replicative RNA with expression of EGFP or StayGold was generated (i.e., Parent). Approximately 1-2×106 cells were passaged into a new 10-cm culture dish and treated with molnupiravir (10 µM) for two consecutive days with daily media changes. Then, they were divided equally into five separate 10-cm dishes and subjected to flow cytometry sorting after an additional cultivation period of approximately 3-4 days. The sorting process involved drawing a triangular region along the diagonal and applying a gate to approximately 0.5% positive cells of the cell population. About 104 cells were sorted and cultured as positive population (i.e., Post_1st). After about 7 days, these cells are expected to proliferate to a quantity of around 107. Similarly, these cells were exposed to a two-day treatment with molnupiravir (10 µM) and subsequently transferred onto five new 10-cm dishes for cultivation over a period of 3-4 days. The positive population was then sorted using flow cytometry at a ratio of 0.5%. The sorted cells were cultured to a count of approximately 106 and utilized for cryopreservation and total RNA isolation (i.e., Post_2nd). The extracted RNA was reverse transcribed into cDNA and then utilized for mutation analysis and mutant identification.

在增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein, EGFP）或StayGold的定向进化实验中，研究目标为将绿色荧光蛋白（Green Fluorescent Protein, GFP）的激发光谱蓝移至更短波长，以提升405纳米激光激发下的荧光强度，为新型荧光蛋白的开发提供助力。本系列实验属于一类借助REPLACE技术、通过荧光激活细胞分选术（Fluorescence-Activated Cell Sorting, FACS）筛选实现不连续定向进化的实验体系。

实验整体设计如下：
将EGFP或StayGold克隆至repRNA-v4衍生载体中，该载体经体外转录为RNA后，通过电穿孔法转入宿主细胞。电穿孔24小时后，对细胞施以持续的嘌呤霉素（10 μg/mL）处理并定期传代。继续培养5~7天后，可获得携带复制型RNA且稳定表达EGFP或StayGold的相对均一多克隆细胞群（即亲本群，Parent）。
取1×10^6至2×10^6个该细胞传至新的10厘米培养皿，用莫努匹韦（10 μM）连续处理两天，期间每日更换培养基。随后将细胞均分至5个独立的10厘米培养皿，继续培养约3~4天后进行流式细胞术分选。分选过程中沿对角线划定三角区域，对细胞群中约0.5%的阳性细胞设门筛选，分选得到约1×10^4个阳性细胞并培养为首轮阳性细胞群（Post_1st）。
约7天后，该细胞群可增殖至约1×10^7个。重复上述处理流程：将这批细胞用10 μM莫努匹韦处理两天，随后转接至5个新的10厘米培养皿中培养3~4天，再次以0.5%的比例通过流式细胞术分选阳性细胞群。将分选得到的细胞培养至约1×10^6个，用于冻存及总RNA提取（即第二轮处理后细胞群，Post_2nd）。提取的RNA经逆转录得到互补DNA（cDNA）后，用于突变分析与突变体鉴定。