Name: bacterial cell culture at the single-cell level inside giant vesicles
Uploaded: 2019-04-30T14:30:00
Description: Watch this Scientific Journal Video about Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles at JoVE.com

这项工作得到了日本教育、文化、体育、科学和技术部(MEXT)的"优秀青年研究人员领导倡议"(LEADER,第16812285号)的支持,这是青年科学家研究资助(第18K18157,16K21034号)从日本科学促进会(JSPS)到M.M.,从MEXT到K.K.(第17H06417号,17H06413号)提供助学金。

1. 通过滴转移方法制备含有细菌细胞的GV
<ol><li>制备1-棕榈基-2-醇-sn-甘油-3磷胆碱(POPC, 10 mM, 1 mL) 和 1,2-distearoyl-sn-甘油-3-磷乙醇胺-N-生物素(聚乙烯乙二醇)-2000] (生物素-PEG-DSPE,0.1 mM,1 mL)氯仿/甲醇溶液(2/1,v/v),并将库存储存在-20°C。</li>
	<li>制备含脂油溶液
	<ol>
<li>将 20 μL 的 POPC 溶液和 4 μL 的生物素-PEG-DSPE 溶液倒入玻璃管中(图 1b (i))。</li>
		<li>通过气流蒸发有机?...

<ol>
	<li>Ozbudak, E. M., Thattai, M., Kurtser, I., Grossman, A. D., van Oudenaarden, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+noise+in+the+expression+of+a+single+gene.">Regulation of noise in the expression of a single gene.</a> Nature Genetics. 31, 69-73 (2002).</li><li>Rosenfeld, N., Young, J. W., Alon, U., Swain, P. S., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulation+at+the+single-cell+level.">Gene regulation at the single-cell level.</a> Science. 307, 1962-1965 (2005).</li><li>Eldar, A., Elowitz, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+for+noise+in+genetic+circuits.">Functional roles for noise in genetic circuits.</a> Nature. 467, 167-173 (2010).</li><li>Hashimoto, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noise-driven+growth+rate+gain+in+clonal+cellular+populations.">Noise-driven growth rate gain in clonal cellular populations.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (12), 3251-3256 (2016).</li><li>Boedicker, J. Q., Vincent, M. E., Ismagilov, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+confinement+of+single+cells+of+bacteria+in+small+volumes+initiates+high-density+behavior+of+quorum+sensing+and+growth+and+reveals+its+variability.">Microfluidic confinement of single cells of bacteria in small volumes initiates high-density behavior of quorum sensing and growth and reveals its variability.</a> Angewandte Chemie International Edition. 48, 5908-5911 (2009).</li><li>Christopher, M., Waters, B. L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quorum+sensing:+cell-to-cell+communication+in+bacteria.">Quorum sensing: cell-to-cell communication in bacteria.</a> Annual Review of Cell and Developmental Biology. 21, 319-346 (2005).</li><li>Inoue, I., Wakamoto, Y., Moriguchi, H., Okano, K., Yasuda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-chip+culture+system+for+observation+of+isolated+individual+cells.">On-chip culture system for observation of isolated individual cells.</a> Lab on a Chip. 1, 50-55 (2001).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+growth+of+Escherichia+coli.">Robust growth of Escherichia coli.</a> Current Biology. 20, 1099-1103 (2010).</li><li>Reshes, G., Vanounou, S., Fishov, I., Feingold, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+shape+dynamics+in+Escherichia+coli.">Cell shape dynamics in Escherichia coli.</a> Biophysical Journal. 94, 251-264 (2008).</li><li>Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L., Leibler, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+Persistence+as+a+Phenotypic+Switch.">Bacterial Persistence as a Phenotypic Switch.</a> Science. 305, 1622-1625 (2004).</li><li>Brouzes, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Droplet+microfluidic+technology+for+single-cell+high-throughput+screening.">Droplet microfluidic technology for single-cell high-throughput screening.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (34), 14195-14200 (2009).</li><li>Zengler, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivating+the+uncultured.">Cultivating the uncultured.</a> Proceedings of the National Academy of Sciences of the United States of America. 99 (24), 15681-15686 (2002).</li><li>Eun, Y., Utada, A. S., Copeland, M. F., Takeuchi, S., Weibel, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulating+bacteria+in+agarose+microparticles+using+microfluidics+for+high-throughput+cell+analysis+and+isolation.">Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation.</a> ACS Chemical Biology. 6, 260-266 (2011).</li><li>Allen, T. M., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposomal+drug+delivery+systems:+From+concept+to+clinical+applications.">Liposomal drug delivery systems: From concept to clinical applications.</a> Advanced Drug Delivery Reviews. 65, 36-48 (2013).</li><li>Szostak, J. W., Bartel, D. P., Luisi, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesizing+life.">Synthesizing life.</a> Nature. 409, 387-390 (2001).</li><li>Noireaux, V., Libchaber, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vesicle+bioreactor+as+a+step+toward+an+artificial+cell+assembly.">A vesicle bioreactor as a step toward an artificial cell assembly.</a> Proceedings of the National Academy of Sciences of the United States of America. 101 (51), 17669-17674 (2004).</li><li>Tan, Y. C., Hettiarachchi, K., Siu, M., Pan, Y. R., Lee, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+microfluidic+encapsulation+of+cells,+proteins,+and+microbeads+in+lipid+vesicles.">Controlled microfluidic encapsulation of cells, proteins, and microbeads in lipid vesicles.</a> Journal of the American Chemical Society. 128 (17), 5656-5658 (2006).</li><li>Chowdhuri, S., Cole, C. M., Devaraj, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+Living+Cells+within+Giant+Phospholipid+Liposomes+Formed+by+the+Inverse-Emulsion+Technique.">Encapsulation of Living Cells within Giant Phospholipid Liposomes Formed by the Inverse-Emulsion Technique.</a> ChemBioChem. 17, 886-889 (2016).</li><li>Morita, M., Katoh, K., Noda, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+bacterial+growth+in+giant+unilamellar+vesicles:+a+novel+tool+for+bacterial+cultures.">Direct observation of bacterial growth in giant unilamellar vesicles: a novel tool for bacterial cultures.</a> ChemistryOpen. 7, 845-849 (2018).</li><li>Pautot, S., Frisken, B. J., Weitz, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+Unilamellar+Vesicles+Using+an+Inverted+Emulsion.">Production of Unilamellar Vesicles Using an Inverted Emulsion.</a> Langmuir. 19 (7), 2870-2879 (2003).</li><li>Hope, M. J., Bally, M. B., Webb, G., Cullis, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+large+unilamellar+vesicles+by+a+rapid+extrusion+procedure.+Characterization+of+size+distribution,+trapped+volume+and+ability+to+maintain+a+membrane+potential.">Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential.</a> Biochimica et Biophysica Acta (BBA) - Biomembranes. 812, 55-65 (1985).</li><li>Tsumoto, K., Matsuo, H., Tomita, M., Yoshimura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+formation+of+giant+liposomes+through+the+gentle+hydration+of+phosphatidylcholine+films+doped+with+sugar.">Efficient formation of giant liposomes through the gentle hydration of phosphatidylcholine films doped with sugar.</a> Colloids and Surfaces B: Biointerfaces. 68, 98-105 (2009).</li><li>Li, A., Pazzi, J., Xu, M., Subramaniam, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellulose+abetted+assembly+and+temporally+decoupled+loading+of+cargo+into+vesicles+synthesized+from+functionally+diverse+lamellar+phase+forming+amphiphiles.">Cellulose abetted assembly and temporally decoupled loading of cargo into vesicles synthesized from functionally diverse lamellar phase forming amphiphiles.</a> Biomacromolecules. 19, 849-859 (2018).</li><li>Weinberger, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gel-assisted+formation+of+giant+unilamellar+vesicles.">Gel-assisted formation of giant unilamellar vesicles.</a> Biophysical Journal. 105, 154-164 (2013).</li><li>Kurokawa, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+cytoskeleton+for+stabilizing+artificial+cells.">DNA cytoskeleton for stabilizing artificial cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (28), 7228-7233 (2017).</li><li>Nourian, Z., Roelofsen, W., Danelon, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Triggered+gene+expression+in+fed-vesicle+microreactors+with+a+multifunctional+membrane.">Triggered gene expression in fed-vesicle microreactors with a multifunctional membrane.</a> Angewandte Chemie International Edition. 51, 3114-3118 (2012).</li><li>Dezi, M., Di Cicco, A., Bassereau, P., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergent-mediated+incorporation+of+transmembrane+proteins+in+giant+unilamellar+vesicles+with+controlled+physiological+contents.">Detergent-mediated incorporation of transmembrane proteins in giant unilamellar vesicles with controlled physiological contents.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (18), 7276-7281 (2013).</li><li>Trantidou, T., Dekker, L., Polizzi, K., Ces, O., Elani, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functionalizing+cell-mimetic+giant+vesicles+with+encapsulated+bacterial+biosensors.">Functionalizing cell-mimetic giant vesicles with encapsulated bacterial biosensors.</a> Interface Focus. 8, 20180024 (2018).</li><li>Elani, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constructing+vesicle-based+artificial+cells+with+embedded+living+cells+as+organelle-like+modules.">Constructing vesicle-based artificial cells with embedded living cells as organelle-like modules.</a> Scientific Reports. 8, 4564 (2018).</li></ol>

在这里,我们描述了一种在GV内单细胞水平上培养细菌细胞的方法。这个简单的方法涉及使用液滴转移方法在单细胞水平上形成含有细菌细胞的GV。与获得含有细菌细胞的GV的其他方法相比,这种方法有两个优点:(i)易于开发,(ii)制备GV需要小体积(2μL)样品溶液。用于制备含有细菌细胞的GV的液滴转移方法20比传统的水化22和微流体方法17简单。例如,经...

单细胞培养受到越来越多的关注。在密闭空间内的单细胞水平上培养细菌细胞可以阐明细菌功能,如型皮变异性1,2,3,4,细胞行为5,6,7,8,9,和抗生素耐药性10,11。由于培养技术的最新进展,单细菌的培养可以在密闭的空间内实现,如在井芯片4,7,8,凝胶滴12,13,和油中水(W/O)滴5,11 。为了促进对单一细菌细胞的了解或利用,需要进一步的技术发展栽培技术。
模仿生物细胞膜的囊泡是由两栖分子组成的球形隔间,可以容纳各种材料。囊泡根据大小进行分类,包括小囊泡(SV,直径<100nm)、大囊泡(LV,<1 μm)和巨型囊泡(GV,>1 μm)。SV或LV通常用作药物载体,因为它们与生物细胞膜14的亲和力。GV还被用作用于建造原细胞15或人工细胞16的反应堆系统。据报道,生物细胞封装到GV中已有17、18种,因此,当与反应器系统结合时,GV显示出细胞培养系统的潜力。
在这里,连同实验程序的视频,我们描述了如何GV可以用作新型细胞培养容器19。含有细菌的GV由滴转移方法20制成,然后固定在盖玻璃上的支撑膜上。我们使用该系统实时观察GV内单细胞水平的细菌生长。

<table>
	<tbody>
		<tr>
			<td>Bactotryptone</td>
			<td>BD Biosciences</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Wako Pure Chemicals</td>
			<td>032-21921</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (18 &#215; 18 mm)</td>
			<td>Matsunami Glass Ind.</td>
			<td>C018181</td>
			<td>thickness 0.13&#8211;0.17 mm</td>
		</tr>
		<tr>
			<td>Cover glass (30 &#215; 40 mm)</td>
			<td>Matsunami Glass Ind.</td>
			<td>custom-order</td>
			<td>thickness 0.25&#8211;0.35 mm</td>
		</tr>
		<tr>
			<td>Desktop centrifuge</td>
			<td>Hi-Tech Co.</td>
			<td>ATT101</td>
			<td>swing rotor type</td>
		</tr>
		<tr>
			<td>Double-faced seal (10 &#215; 10 &#215; 1 mm)</td>
			<td>Nitoms</td>
			<td>T4613</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vial</td>
			<td>AS ONE</td>
			<td>6-306-01</td>
			<td>Durham fermentation tube</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Wako Pure Chemicals</td>
			<td>049-31165</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted microscope</td>
			<td>Olympus</td>
			<td>IX-73</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Wako Pure Chemicals</td>
			<td>133-16771</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopic heating stage system</td>
			<td>TOKAI HIT</td>
			<td>TP-110R-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Nacalai Tesque</td>
			<td>23334-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-extruder</td>
			<td>Avanti Polar Lipids</td>
			<td>610000</td>
			<td></td>
		</tr>
		<tr>
			<td>Neutravidin</td>
			<td>Thermo Fisher Scientific</td>
			<td>31000</td>
			<td></td>
		</tr>
		<tr>
			<td>Objective lens</td>
			<td>Olympus</td>
			<td>LUCPLFLN 40&#215;/0.6 NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membranes</td>
			<td>Avanti Polar Lipids</td>
			<td>610005</td>
			<td>pore size 100 nm</td>
		</tr>
		<tr>
			<td>sCMOS camera</td>
			<td>Andor</td>
			<td>Zyla 4.2 plus</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Wako Pure Chemicals</td>
			<td>191-01665</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Wako Pure Chemicals</td>
			<td>196-00015</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrasonic bath</td>
			<td>AS ONE</td>
			<td>ASU-3D</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>BD Biosciences</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL lidded plastic tube</td>
			<td>Watson</td>
			<td>130-806C</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL lidded plastic tube</td>
			<td>Sumitomo Bakelite Co.</td>
			<td>MS4265-M</td>
			<td></td>
		</tr>
		<tr>
			<td>1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline</td>
			<td>Avanti Polar Lipids</td>
			<td>850457P</td>
			<td>POPC</td>
		</tr>
		<tr>
			<td>1,2-distearoyl-snglycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000]</td>
			<td>Avanti Polar Lipids</td>
			<td>880129P</td>
			<td>Biotin-PEG-DSPE</td>
		</tr>
	</tbody>
</table>

bacterial cell culture at the single-cell level inside giant vesicles

我们开发了一种在巨囊(GVs)内单细胞水平上培养细菌细胞的方法。细菌细胞培养对于了解细菌细胞在自然环境中的功能非常重要。由于技术的进步,各种细菌细胞功能可以在密闭空间内的单细胞水平上显现出来。GV是由两栖脂质分子组成的球形微尺寸隔间,可以容纳各种材料,包括细胞。在这项研究中,通过滴移送法将单个细菌细胞封装到10~30μm的GV中,含有细菌细胞的GV被固定在玻璃基板上的支撑膜上。我们的方法对于观察GV内部单细菌的实时生长非常有用。我们培养大肠杆菌(大肠杆菌) 细胞作为 GV 内的模型,但这种方法可以适应其他细胞类型。我们的方法可用于微生物学、生物学、生物技术和合成生物学的科学和工业领域。

我们提出了一种使用滴转移法生成包含单个细菌细胞的GV的简单方法(图1)。图1a显示了含有细菌的GV降水的示意图。含有细菌的W/O液滴通过离心在油水(脂单层)界面上传递,形成GV。蔗糖(内部水溶液)和葡萄糖(外水溶液)之间的密度差异也有助于跨越W/O液滴的油水界面。有必要监测内水溶液和外水溶液中的渗透压力,因为这些溶液之间...

Watch this Scientific Journal Video about Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles at JoVE.com

Bacterial Cell Culture at the Single-cell Level Inside Giant Vesicles

巨型囊泡内单细胞层的细菌细胞培养

我们演示了巨型囊泡 (GV) 内的细菌的单细胞培养。含有细菌细胞的GV通过滴转移方法制备,固定在玻璃基板上的支撑膜上,直接观察细菌生长。这种方法也可以适应其他细胞。

We developed a method for culturing bacterial cells at the single-cell level inside giant vesicles (GVs). Bacterial cell culture is important for ...

该协议为在巨型囊泡内的单细胞水平上培养细菌细胞提供了一种手段。开发巨型囊泡很容易，只需非常小的样品溶液来制备囊泡。演示该程序将是尤里大田，一个博士候选人从我们的实验室。首先，在玻璃瓶中加入20微升新鲜准备的 POPC 溶液和 4 微升新鲜准备的生物素 PEG-DSPE 溶液。通过气流蒸发有机溶剂，直到形成脂质膜。将薄膜放在干燥器中一小时，以完全蒸发有机溶剂。然后在小瓶中加入200微升矿物油。用塑料石蜡膜盖住小瓶的开口，在120瓦的超声波浴中对小瓶内的内容物进行声波化，至少一小时。对于小囊泡制备，创建脂质膜，正如刚刚演示的，并在 LB 介质中加入 200 微升 200 毫米葡萄糖。然后用120瓦对薄膜进行声波化至少一小时，然后用迷你挤出机和100纳米孔径的聚碳酸酯膜将产生的巨型囊泡挤出到小囊泡中。对于细菌细胞预培养，将大肠杆菌从LB板接种到LB培养基的毫升中，在37摄氏度下进行过夜培养。第二天早上，将20微升的培养物的上一代转移到1.98毫升的新鲜LB培养基上，再进行两个小时的培养。在孵化结束时，检查分光光度计上培养前溶液的光学密度为 600 纳米（OD 600 值）。应使用 OD 600 的 1 到 1.5 的培养前解决方案。然后根据表一将培养前溶液与新鲜准备的蔗糖溶液和新鲜的 LB 介质混合。对于巨型囊泡的形成，在1.5毫升盖塑料管中加入50微升新鲜准备好的巨型囊泡外水溶液。在巨型囊泡的外水溶液上轻轻层150微升含脂油溶液。在室温下孵育所得溶液 10 至 15 分钟，然后检查以确保油和水溶液的界面是平的。对于含有液滴形成的水和油细菌细胞，在0.6毫升盖盖塑料管中，将新鲜准备的巨型囊泡内水溶液的两微升添加到含有脂质的声波含脂油溶液的50微升中。然后，点按管子对两个组件进行乳化。接下来，使用移液器将50微升水和油滴溶液加入油和水溶液的界面，通过离心沉淀含有细菌细胞的巨型囊泡。然后吸气顶部油层，并收集巨大的囊泡。要准备一个手工制作的腔室，在 10 比 10 的双面密封上钻一个 7 毫米的孔，然后将双面密封粘贴到 30 到 40 毫米盖玻璃上。要准备一个支持的双层膜，在手工腔室的孔中加入30微升的小囊泡溶液。在室温下孵育囊泡30分钟。在孵育结束时，用20微升LB介质轻轻洗孔两次，辅以200毫米勒葡萄糖。为了固定支架双层膜上的巨型囊泡，在外水巨型囊泡溶液中引入10微升的中性铁丹溶液，在室温下孵育15分钟。在孵育结束时，用20微升的LB介质轻轻洗孔两次，辅以200毫米勒葡萄糖，并将整个体积的巨型囊泡溶液加入到腔室的孔中。然后用 18 由 18 毫米盖玻璃密封孔。要监测巨型囊泡内的细菌生长，请在倒置显微镜上选择 40 倍的镜面，然后将腔室放在显微镜加热阶段系统上。然后在37摄氏度的静电条件下孵育室内含有细菌细胞的巨型囊泡6小时，每30分钟拍摄一次细菌细胞生长的图像，并记录一次，使用科学互补的金属氧化物半导体摄像机。含有细菌细胞的巨型囊泡通常大小在10至30微米之间。对于两种大小的大囊泡，大肠杆菌细胞都经历了伸长和分裂过程，一些大肠杆菌细胞在6小时的观察期内长到大量的细胞。包含单个细胞水平的巨型囊泡的相对频率约为获得的巨型囊泡的10%，封装在单个细胞水平的巨型囊泡的大约50%是含有细菌细胞的巨型囊泡。油水界面的稳定性对于获得含有细菌细胞的巨型囊泡非常重要。在巨大的囊泡矫正前，小心吸油。看完这段视频后，你应该对如何在单个巨型囊泡中的单个细胞水平上培养细菌细胞有一个很好的理解。我们的细菌培养方法是微生物学中培养未知环境细菌获取或分析其代谢产物的新工具。

Research

JoVE Journal

Immunology and Infection

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture

传播<em&gt; Homalodisca coagulata病毒01</em&gt;通过<em&gt; Homalodisca vitripennis</em&gt;细胞培养

The glassy-winged sharpshooter (Homalodisca vitripennis) is a highly vagile and polyphagous insect found throughout the southwestern United States. These ...

科研

免疫与感染

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

Assessing Bacterial Invasion of Cardiac Cells in Culture and Heart Colonization in Infected Mice Using Listeria monocytogenes

使用李斯特菌单细胞基因评估受感染小鼠培养和心脏殖民化中心脏细胞的细菌入侵

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is capable of causing serious invasive infections in immunocompromised ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

生成和多表型高内涵筛选<em&gt;贝氏柯克斯体</em&gt;转座子突变体

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

Development and Identification of a Novel Subpopulation of Human Neutrophil-derived Giant Phagocytes In Vitro

开发和鉴定人的中性粒细胞源性巨噬细胞新型亚群<em&gt;体外</em

Neutrophils (PMN) are best known for their phagocytic functions against invading pathogens and microorganisms. They have the shortest half-life amongst ...

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

Quantification of Intracellular Growth Inside Macrophages is a Fast and Reliable Method for Assessing the Virulence of Leishmania Parasites

巨噬细胞内细胞内生长的定量化是评价利什曼原虫寄生虫毒力的快速可靠方法

The lifecycle of Leishmania, the causative agent of leishmaniasis, alternates between promastigote and amastigote stages inside the insect and vertebrate ...

Quantification of Efferocytosis by Single-cell Fluorescence Microscopy

单细胞荧光显微镜定量 Efferocytosis 的研究

Studying the regulation of efferocytosis requires methods that are able to accurately quantify the uptake of apoptotic cells and to probe the signaling ...

Using a Bacterial Pathogen to Probe for Cellular and Organismic-level Host Responses

细菌病原体在细胞和有机水平宿主反应探针中的应用

There are a variety of strategies bacterial pathogens employ to survive and proliferate once inside the eukaryotic cell. The so-called 'cytosolic' ...

Isolation of Single Intracellular Bacterial Communities Generated from a Murine Model of Urinary Tract Infection for Downstream Single-cell Analysis

尿道感染小鼠模型产生的单细胞内细菌群落的分离, 用于下游单细胞分析

In this article, we outline a procedure used to isolate individual intracellular bacterial communities from a mouse that has been experimentally infected ...

A High-throughput Shigella-specific Bactericidal Assay

A High-throughput Shigella-specific Bactericidal Assay

一种高通量志贺氏菌特异性杀菌试验

Serum bactericidal assays (SBAs) measure the functional activity of antibodies and have been used for many decades. SBAs directly measure antibody killing ...

Electrophysiological Recordings of Single-cell Ion Currents Under Well-defined Shear Stress

在定义良好的剪切应力下单细胞电电流的电生理记录

Fluid shear stress is well known to play a major role in endothelial function. In most vascular beds, elevated shear stress from acute increases in blood ...