Isolation and Culture of Single Microbial Cells by Laser Ejection Sorting Technology

doi:10.1128/aem.01165-21

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	Isolation and Culture of Single Microbial Cells by Laser Ejection Sorting Technology
	P. Liang; B. Liu; Y. Wang; K. X. Liu; Y. P. Zhao; W. E. Huang and B. Li
	2022
发表期刊	Applied and Environmental Microbiology
ISSN	0099-2240
卷号	88 期号:3 页码:12
摘要	Single-cell isolation and cultivation play an important role in studying physiology, gene expression, and functions of microorganisms. A series of single-cell isolation technologies have been developed, among which single-cell ejection technology is one of the most promising. Single-cell ejection technology has applied laser-induced forward transfer (LIFT) techniques to isolate bacteria, but the viability (or recovery rate) of cells after sorting has not been clarified in current research. In this work, to keep the cells alive as long as possible, we propose a three-layer LIFT system (top layer, 25-nm aluminum film; second layer, 3 mu m agar media; third layer, liquid containing bacteria) for the isolation and cultivation of single Gram-negative (Escherichia colt), Gram-positive (Lactobacillus rhamnosus GG (LGG)), and eukaryotic (Saccharomyces cerevisiae) microorganisms. The experiment results showed that the average survival rates for ejected pure single cells were 63% for Saccharomyces cerevisiae, 22% for E. colt DH5 alpha, and 74% for LGG. In addition, we successfully isolated and cultured the green fluorescent protein (GFP)-expressing E. coli JM109 from a mixture containing complex communities of soil bacteria by fluorescence signal. The average survival rate of E. coli JM109 was demonstrated to be 25.3%. In this study, the isolated and cultured single colonies were further confirmed by colony PCR and sequencing. Such precise sorting and cultivation techniques of live single microbial cells could be coupled with other microscopic approaches to isolate single microorganisms with specific functions, revealing their roles in the natural community. IMPORTANCE We developed a laser-induced forward transfer (LIFT) technology to accurately isolate single live microbial cells. The cultivation recovery rates of the ejected single cells were 63% for Saccharomyces cerevisiae, 22% for E. coli DH5 alpha, and 74% for Lactobacillus rhamnosus GG (LGG). With coupled LIFT with a fluorescence microscope, we demonstrated that single cells of GFP-expressing E. coli JM109 were sorted according to fluorescence signal from a complex community of soil bacteria and subsequently cultured with 25% cultivation recovery rate. This single-cell live sorting technology could isolate single microbes with specific functions, revealing their roles in the natural community.
DOI	10.1128/aem.01165-21
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收录类别	sci ; ei
语种	英语
引用统计
文献类型	期刊论文
条目标识符	http://ir.ciomp.ac.cn/handle/181722/66781
专题	中国科学院长春光学精密机械与物理研究所
推荐引用方式 GB/T 7714	P. Liang,B. Liu,Y. Wang,et al. Isolation and Culture of Single Microbial Cells by Laser Ejection Sorting Technology[J]. Applied and Environmental Microbiology,2022,88(3):12.
APA	P. Liang,B. Liu,Y. Wang,K. X. Liu,Y. P. Zhao,&W. E. Huang and B. Li.(2022).Isolation and Culture of Single Microbial Cells by Laser Ejection Sorting Technology.Applied and Environmental Microbiology,88(3),12.
MLA	P. Liang,et al."Isolation and Culture of Single Microbial Cells by Laser Ejection Sorting Technology".Applied and Environmental Microbiology 88.3(2022):12.

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