微生物流式分选的单细胞样品制备及存活率提高的方法优化

Preparation of single cell suspension and survival rate improvement of microorganisms in fluorescence-activated cell sorting

摘要:

【背景】以流式细胞技术为代表的高通量筛选技术能够高效筛选具有目标性状的微生物工程菌株。在流式分选中微生物的粘连会造成分析数据不准确，分选纯度降低，因此快速简便的单细胞样品制备是流式检测的关键。优势菌大多是通过筛选偶联荧光蛋白的随机突变库获得，阳性率低，杂质和死细胞的自发荧光较强，容易混入分选门内造成存活率降低，亟须提高分选存活率的方法。【目的】建立一种简便的微生物流式分选的单细胞样品制备方法，并通过碘化丙啶(propidium iodide, PI)染色提高分选样品存活率。【方法】分别在大肠杆菌、枯草芽孢杆菌、谷氨酸棒状杆菌和酵母菌4种底盘细胞中探索超声波、消化酶、表面活性剂及超声-表面活性剂联合作用4种方式对单细胞制备效率的影响。提高微生物流式分选存活率，用常压室温等离子诱变(atmospheric and room temperature plasma, ARTP)技术处理含有绿色荧光蛋白(green fluorescent protein, GFP)的酿酒酵母HZ848 (简称HZ848-GFP)，形成不同强度GFP文库后，按照GFP强度分选全细胞和PI染色阴性细胞的前0.5%，统计单细胞存活率。【结果】酵母细胞分散条件为：0.01% Tween-80联合超声1 min，单细胞率达到88%以上，PI染色细胞破损率＜1.4%。谷氨酸棒状杆菌单细胞分散条件为：0.01% Tween-80联合超声5 min，单细胞率达到97%以上，PI染色细胞破损率＜1%。分选存活率结果表明，未用PI染色的酿酒酵母分选后单细胞存活率是4.3%，用PI染色去除死细胞后再分选单细胞存活率是18.3%，后者是前者的4.3倍，且具有显著性差异。【结论】本研究为微生物流式分选建立了一套简单快捷的单细胞样品制备方法，证实了PI染色法能够显著提高分选样品存活率。

Abstract:

[Background] High-throughput screening technologies represented by flow cytometry can efficiently screen the microbial strains with target characteristics. In fluorescence-activated cell sorting (FACS), the adhesion of microorganisms will lead to inaccuracy results and low purity. Therefore, a rapid and simple method for preparing single cell suspension is crucial for FACS to obtain reliable results. Desired mutants are usually screened out in fluorescence-based manners from the libraries generated by random mutagenesis with low positive mutation rates. However, the impurities and dead cells with strong spontaneous fluorescence tend to be included into the sorting gate, which will lead to false-positive results and reduce the survival rates of samples. It is urgent to improve the survival rate of microorganisms in FACS. [Objective] To establish a simple method for preparing single cell suspensions for FACS and improve the survival rate of sorted microorganisms. [Methods] Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, and Saccharomycetes were used as the samples. The morphological characteristics and cell adhesion degrees of the live microorganisms were observed by microscopy. Four treatments, including ultrasound, digestive enzyme, surfactant, and ultrasound-surfactant, were then employed to alleviate cell adhesion. To improve the survival rate of microorganisms in FACS, we treated Saccharomyces cerevisiae HZ848 expressing green fluorescent protein by atmospheric and room temperature plasma (ARTP) to generate a mutant library. The top 0.5% of the whole cells and the negative cells after propidium iodide (PI) staining were separately sorted based on their GFP intensities, and the single-cell survival rates under both conditions were calculated. [Results] Yeast cells were effectively monodispersed by the treatment of 0.01% Tween-80 combined with ultrasound for 1 min, where the single cell rate was over 88%, and the breakage rate of PI-stained cells was lower than 1.4%. The single cell dispersion of C. glutamicum was achieved by the treatment with 0.01% Tween-80 combined with ultrasound for 5 min, where the single cell rate was over 97% and the breakage rate of PI-stained cells was lower than 1%. The survival rate of S. cerevisiae without PI staining was 4.3%, while this rate was raised to 18.3% for the PI-stained sample after removal of dead cells. [Conclusion] This study established a simple and rapid method to prepare single cell suspensions and provided a PI-staining strategy to significantly improve the survival rates of sorted samples in microbial FACS.