Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips.
Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips.
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DOI：
10.1039/c4ib00109e
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发表时间：
2014-08
期刊：
影响因子：
--
通讯作者：
Ismagilov RF
中科院分区：
文献类型：
--
作者：
Ma L;Datta SS;Karymov MA;Pan Q;Begolo S;Ismagilov RF
从环境样品中分离携带感兴趣基因的微生物对于生物学和医学的应用是重要的。然而，这涉及使用通常需要裂解微生物细胞的遗传测定，这与获得活细胞用于分离和培养的目标不相容。本文介绍了设计，制造，生物学验证，和基本的物理微流体滑动芯片设备，解决这一挑战。该装置由两个含有1，000个微隔室的连接板组成，每个微隔室包括两个并列的威尔斯孔，每个相对板上有一个孔。单个微生物细胞被随机限制，随后在微区室中培养。然后，我们将每个微隔室分成两个复制液滴，两者都含有微生物培养物，然后可控地分离两个板，同时将每个液滴保留在每个孔内。我们实验描述的液滴保留作为毛细管压力，粘性压力，和水相的粘度的函数。在每对复制品中，一个可用于遗传分析，另一个保存活细胞用于生长。这种微流体方法提供了一种从复杂群落中培养厌氧菌的简便方法。我们通过从临床样本中靶向、分离和培养核心肠道厌氧菌普通拟杆菌来验证这种方法。到目前为止，这种方法已经能够分离一种新的微生物分类群，代表一个新的属。这种方法也可以扩展到其他微生物甚至哺乳动物系统的研究，并可以在包括数字PCR，测序，单细胞分析和蛋白质结晶在内的应用中实现解决方案的靶向检索。
Isolating microbes carrying genes of interest from environmental samples is important for applications in biology and medicine. However, this involves the use of genetic assays that often require lysis of microbial cells, which is not compatible with the goal of obtaining live cells for isolation and culture. This paper describes the design, fabrication, biological validation, and underlying physics of a microfluidic SlipChip device that addresses this challenge. The device is composed of two conjoined plates containing 1,000 microcompartments, each comprising two juxtaposed wells, one on each opposing plate. Single microbial cells are stochastically confined and subsequently cultured within the microcompartments. Then, we split each microcompartment into two replica droplets, both containing microbial culture, and then controllably separate the two plates while retaining each droplet within each well. We experimentally describe the droplet retention as a function of capillary pressure, viscous pressure, and viscosity of the aqueous phase. Within each pair of replicas, one can be used for genetic analysis, and the other preserves live cells for growth. This microfluidic approach provides a facile way to cultivate anaerobes from complex communities. We validate this method by targeting, isolating, and culturing Bacteroides vulgatus, a core gut anaerobe, from a clinical sample. To date, this methodology has enabled isolation of a novel microbial taxon, representing a new genus. This approach could also be extended to the study of other microorganisms and even mammalian systems, and may enable targeted retrieval of solutions in applications including digital PCR, sequencing, single cell analysis, and protein crystallization.
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影响因子：
15
作者：
Li, Liang;Du, Wenbin;Ismagilov, Rustem F.
通讯作者：
Ismagilov, Rustem F.
影响因子：
64.8
作者：
Arumugam, Manimozhiyan;Raes, Jeroen;Pelletier, Eric;Le Paslier, Denis;Yamada, Takuji;Mende, Daniel R.;Fernandes, Gabriel R.;Tap, Julien;Bruls, Thomas;Batto, Jean-Michel;Bertalan, Marcelo;Borruel, Natalia;Casellas, Francesc;Fernandez, Leyden;Gautier, Laurent;Hansen, Torben;Hattori, Masahira;Hayashi, Tetsuya;Kleerebezem, Michiel;Kurokawa, Ken;Leclerc, Marion;Levenez, Florence;Manichanh, Chaysavanh;Nielsen, H. Bjorn;Nielsen, Trine;Pons, Nicolas;Poulain, Julie;Qin, Junjie;Sicheritz-Ponten, Thomas;Tims, Sebastian;Torrents, David;Ugarte, Edgardo;Zoetendal, Erwin G.;Wang, Jun;Guarner, Francisco;Pedersen, Oluf;de Vos, Willem M.;Brunak, Soren;Dore, Joel;Weissenbach, Jean;Ehrlich, S. Dusko;Bork, Peer
通讯作者：
Bork, Peer
DOI：
10.1073/pnas.1106752109
发表时间：
2012-05-15
影响因子：
11.1
作者：
Leung, Kaston;Zahn, Hans;Hansen, Carl L.
通讯作者：
Hansen, Carl L.
影响因子：
6.1
作者：
Begolo S;Shen F;Ismagilov RF
通讯作者：
Ismagilov RF
影响因子：
6.1
作者：
Jakiela, Slawomir;Makulska, Sylwia;Garstecki, Piotr
通讯作者：
Garstecki, Piotr