The authors thank members of the Shapiro lab and Erin Schrader for comments on the manuscript. The authors acknowledge financial support from: NIH postdoctoral fellowship F32 GM100732 to JMS and NIH grants R01 GM51426 and R01 GM32506 to LS.

1.大型同步性 - 最佳西方印迹，芯片/ RNA测序，和其他物资集约化化验<ol><li>从冰柜股票或板块，通过在28°C在PYE中颤抖成长应变NA1000的5毫升O / N培养。 </li><li>接种0.5ml所细胞从步骤1在25毫升M2G的（表1-2）和摇动在28℃，直到培养物达到0.5和0.6之间的OD 600。 </li><li>接种细胞到1升M2G和动摇，在28°C。 </li><li>一旦OD 600达到0.5至0.6，确认swarmer使用液相安装相位显微镜细胞的存在。点1微升的细胞在载玻片上的，盖上盖玻片，并通过图像相显微镜。通过在人口可视迅速游动细胞确认swarmer细胞的存在。 </li><li>在7 KXG在JA-10转子旋转细胞进行15分钟，在4℃。 </li><li>弃去上清液，加入冷M2的180毫升（表1-2），轻轻水库uspend所有使用血清吸管的细胞。放弃松散沉淀的细胞;他们是predivisional大步细胞。 </li><li>增加60毫升冷的胶体二氧化硅溶液（前务必同时增加了细胞混合胶体二氧化硅悬浮液）和混合细胞悬液好。 </li><li>倾细胞悬液分为八个30毫升管和在JA-20转子旋转30分钟，在6.4 KXG在4℃。 注：每个人都应该看到两个不同的频段;所述swarmer频带是低频带，而柄/ predivisional细胞是在顶带（ 图1B）。 </li><li>小心吸顶带，并取出液体约1厘米以上的swarmer频段（低频带）。 </li><li> （严重）用巴斯德吸管，小心地取出swarmer带，并放入一个干净的试管。洗去胶体二氧化硅，顶管断用冷M2和自旋10分钟，在6.4 KXG在4℃下在JA-20转子。 </li><li>小心弃去上清液，重悬细胞在20毫升冷M2的和自旋为在6.4 KXG在JA-20转子10分钟，在4℃。 </li><li>重悬的所有粒料于30毫升冷的M2的和使用分光光度计测量的OD 600和空白使用冷M2培养基。保存1微升相成像检查swarmer细胞;细胞的90-95％应是游动。 </li><li>在JA-20转子降速细胞5分钟，在6.4 KXG在4℃。悬浮细胞在28°C M2G中，这样的A 600为0.3-0.4〜并开始晃动，在28°C。 注：典型的产量是30和60毫升swarmer细胞培养，从非同步的细胞1L之间。 </li><li>开始服用的时间点（野生型文化将需要大约135-140分钟分）8,9。在每个时间点，测量OD 600。检查分割后的OD 600大约是2倍的初始OD 600。 </li><li>对于免疫印迹或基因表达分析，除去铜1毫升等分lture在所需的时间点，在台式离心机降速在最大速度为30秒，迅速倒出或者吸出培养基，并快速冷冻在液氮中的细胞沉淀。存储单元在-80°C，直到下游分析。 </li></ol> 2.小​​规模的同步性 - 最佳显微镜<ol><li>从冰柜股票或板块，成长5ml的O / N培养摇晃在28℃，M2G。 </li><li>稀释在15毫升M2G的（表1-2）和生长，直到数中期（OD 600 = 0.5-0.6）。 </li><li>旋在6.4 KXG为10分钟，在4℃下，在JA-20转子，和重悬在1ml冷的M2（表1-2），并转移到2ml微量离心管中。 </li><li>旋转15 KXG在离心管沉淀细胞，吸出上清液3分钟，放小球上的冰和重悬在900微升冷M2的。 </li><li>添加900微升冷的PVP在15 KXG涂覆的胶体二氧化硅和自旋20分钟，在4℃下在麦克风rocentrifuge管。 </li><li> （严重）吸或吸管从顶部大步/ predivisional细胞乐队和收集swarmer波段底部到一个新的离心管。 </li><li>在1ml冷的M2在15 KXG而离心3分钟洗swarmer细胞两次。 </li><li>在最后的旋转前，将细胞注入一预冷1毫升玻璃试管并测量细胞的OD 600相比M2的空白。 </li><li>重悬最终的细胞沉淀到28°C M2G 0.3之间的OD - 0.4摇/滚细胞在28°C。 注意：典型的产率是毫升swarmer细胞培养物2和4之间。 </li><li>显微镜的实验，在所希望的时间点处1微升的细胞到一个M2G琼脂糖垫用于成像。 </li></ol>

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The authors have nothing to disclose.

The bacterial cell cycle is a fundamental process in life and is important for the study of growth and as a target for next generation antibiotics. Here, we detailed the rapid synchronization procedures for C. crescentus NA1000, a model organism for the study of the bacterial cell cycle and asymmetric cell division. This method is amendable to western blot, gene expression profiling, and fluorescence microscopy assays to investigate the spatiotemporal regulation of the bacterial cell cycle. 
<p class='jove_...

细菌细胞周期控制的基因组中的两个复制和子细胞的分裂。重要的是，抗生素耐药性是一个日益严重的威胁到公众健康，细菌细胞周期呈现抗生素发展的尚未开发的目标。 在细菌柄杆菌新月 ，每个细胞周期导致一个不对称分裂，产生不同的命运（ 图1A）1,2的两个子细胞。一个子细胞继承了鞭毛是能动的，而另一个女儿继承柄和无柄是。一个集成的基因电路控制细胞周期进程和细胞命运的转录调控，磷酸化信号，和监管蛋白水解3。此外，染色体复制和并发分流率的子细胞包含4号染色体只有一个拷贝。重要的是，这两种类型的细胞可迅速地通过胶体分离在可同步NA1000菌株5-7允许swarmer细胞从具有高产率（ 图1B）的人口的其余部分隔离IDAL二氧化硅粒子密度离心。隔离swarmer细胞，然后通过不对称细胞分裂进行同步。在这里，我们详细介绍了协议用 ​​于同步柄杆菌菌株NA1000。我们提供的协议，并为大规模和小规模的同步常见故障排除技巧。这个实验的过程提供了一个功能强大的工具来询问柄杆菌细胞周期和细胞命运的时空控制。

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PVP Coated Colloidal Silica (Percoll)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4937</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colloidal Silica (Ludox AS-40)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">420840</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA10 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">369687</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">JA20 Rotor</td>
			<td style="width:71px;">Beckman-Coulter</td>
			<td style="width:119px;">334831</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ferrous Sulfate Chelate Solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>F0518</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">30 mL Centrifuge Tubes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">8445</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Na2HPO4</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0720-1</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">KH2PO4</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">BDH9268-500G</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">NH4Cl</td>
			<td style="width:71px;">Amresco</td>
			<td style="width:119px;">0621-500g</td>
			<td></td>
		</tr>
	</tbody>
</table>

synchronization of caulobacter crescentus for investigation of the bacterial cell cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

同步通常产生细胞的两条带（ 图1B）：对swarmer带，它具有较高的密度和较低的密度的一个柄/ predivisional细胞带。为了确保高效的同步常见的控制包括监测OD 600和测量CTRA蛋白水平通过免疫印迹在不同的细胞周期的时间点。的OD 600应在细胞周期的过程中（ 图2）中约2倍的增加。在免疫印迹的细胞周期主控调节CTRA是一个有用的控制，以验证一个好的同步（ <str...

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Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

同步<em&gt;新月柄杆菌</em&gt;对细菌细胞周期的研究

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

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Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

11.8K 次观看.  Stanford University School of Medicine. Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.