我们要感谢 Wladislaw Maier (BMG Labtech 有限公司) 为他的支持, 为板阅读器建立自写脚本。这项工作由奥地利 "全宗 zur Förderung wissenschaftlichen 研究" (FWF) 到 pm (P24189) 和博士项目 "DK 分子酶学" (W901) 支持 pm。

1.大肠杆菌中莱克丝操纵的设计、制备和表达
注: 见资料表, 以了解本节使用的商业套件的信息。
<ol><li>为了将莱克丝操纵转移到大肠杆菌中, 选择一个标准的 pET 载体, 具有适当的限制部位和感兴趣的耐药性基因 (如pET28a;NcoI, XhoI, 卡那霉素)。</li>
	<li>根据发光 mandapamensis 27561 (基因库: DQ988878.2) 的 DNA 序列设计了吉布森组件的碎片和重叠底漆。</li>
	<li>建立一个标准的 PCR 反应与设计的引物和分离的基因组 DNA 的发光 mandapamensis 27561 作为模板 (见补充材料的底漆和条件)。 注: 分离细菌菌株基因组 DNA 提高 PCR 效果。</li>
	<li>通过自旋柱提纯纯化 PCR 产物。</li>
	<li>对孤立的 pET28a 载体进行限制消化, NcoI 和 XhoI 在37摄氏度45分钟。</li>
	<li>通过琼脂糖凝胶电泳和随后的自旋柱提纯纯化线性化载体和 PCR 片段。</li>
	<li>确定每个片段和线性化向量的 DNA 浓度, 并根据20、21号协议计算装配的最佳数量。 注: 装配效果取决于碎片大小和数量, 必须根据制造商的20、21号协议进行调整。</li>
	<li>结合 pcr 管中的所有片段和缓冲器后, 在50摄氏度的 pcr 机中孵育组装混合物1小时。</li>
	<li>按照标准转换协议将已装配的矢量产品转化为大肠杆菌细菌质粒转化为一种适宜的大肠杆菌系统高产质粒复制 (如大肠杆菌TOP10或 XL-1)。</li>
	<li>从变换板上选取菌落, 在新的板块上进行 DNA 分离。</li>
	<li>根据标准协议隔离质粒 DNA。</li>
	<li>为了验证质粒的正确组装, 包括所有片段, 首先根据标准协议执行菌落 PCR, 使用每个组装片段的特定底漆。</li>
	<li>此外, 菌落 PCR 和随后的琼脂糖凝胶电泳, 准备所有独立的组装载体 DNA 测序, 以验证正确的组装和正确的 DNA 序列。</li>
	<li>将经验证的质粒转化为大肠杆菌细菌质粒的标准转化协议, 转变为高产蛋白生产 (如大肠杆菌BL21) 的适宜大肠杆菌体系。 注意: 直接使用下面的表达式协议继续。为更长的储藏, 推荐甘油库存的准备。</li>
</ol>2. 改良大肠杆菌菌株的表达
<ol><li>准备一夜文化 (很久以前) 为表达通过接种适当的磅培养基 (例如, 100 毫升) 与早先准备好的甘油存货的大肠杆菌BL21 细胞转换与组装质粒或直接地从转换板。添加100µL 的卡那霉素 (50 毫克/毫升; pET28a 的抗生素耐药性基因), 并孵化很久以前在37摄氏度和 120 rpm 在一个孵化器的摇床过夜。</li>
	<li>接种主要表达培养 (例如, 800 毫升的 LB 培养基) 与8毫升的很久以前和添加800µL 卡那霉素 (50 毫克/毫升)。</li>
	<li>孵化的主要文化在37°c 和 120 rpm 的孵化器, 直到细胞密度达到 OD600 0.6-0.8 (约2.5 小时)。</li>
	<li>将孵化温度降低到28摄氏度。</li>
	<li>通过添加 IPTG 到最终浓度为0.1 毫米诱导蛋白表达。 注: 经验测试显示, 温度降低到28摄氏度时, 光照强度最高。</li>
	<li>观察细胞, 直到它们开始发光 (大约1小时)。 注意: 根据表达的目的, 细胞生长到第二天, 然后收获, 或细胞可以保持晃动, 只要他们是闪耀 (最大48小时)。采集细胞和纯化任何蛋白质都可以按照标准程序进行。</li>
</ol>3. 细菌生物发光菌株的表达
注: 细菌生物发光菌株需要特定的生长培养基/人工海水培养基进行生长和光生产。
<ol><li>制备人工海水介质, 由两个单独制备的介质组成。 注: 人工海水介质的制备是根据原有的22号议定书改编的。以下数额为1升液体培养基或1升琼脂培养基。<ol>
<li>为人为海水媒介, 称在以下盐: 28.13 g 氯化钠, 0.77 g 氯化钾, 1.60 g CaCl2 ·2H2O, 4.80 g 氯化镁2 ·6H2O, 0.11 克 NaHCO3, 和 3.50 g MgSO4 ·7H2O。</li>
		<li>加1升蒸馏水, 溶解所有成分。</li>
		<li>对于 LB 培养基, 重量在以下成分:10 克酵母萃取物, 10 克蛋白胨, 并为琼脂板块, 另外20克琼脂。</li>
		<li>添加250毫升的自来水和溶解成分。</li>
		<li>高压釜两种制备介质分别在121°c 为20分钟。</li>
		<li>对于琼脂板材, 热处理后直接将250毫升的 LB 介质与750毫升人工海水介质结合在一起, 并制备板材。</li>
		<li>对于液体介质, 在热处理或冷却后直接将250毫升的 LB 介质与750毫升人工海水介质结合在一起。 注: 人工海水介质在盐沉淀下可能会混浊。</li>
	</ol>
</li>
	<li>对人工海水培养基琼脂板上的细菌生物发光菌株进行条纹, 并在 24-30 °c 隔夜孵化。 注: 细菌菌株的长期贮存通常是通过冷冻甘油储存的细菌培养来实现的。菌株应始终在琼脂板块上的条纹, 以确保所有菌株的统一启动条件, 在使用之前, 液体培养, 由于一个滞后阶段的增长后, 解冻。</li>
	<li>很久以前通过接种100毫升人工海水培养基, 用一个单一的菌落从板块中制备。孵化的很久以前在 24-30 °c 和 120 rpm 在一个孵化器的摇床过夜。</li>
	<li>接种800毫升人工海水培养基, 8 毫升的很久以前。</li>
	<li>孵化的细菌细胞在 24-30 °c 和 120 rpm 的孵化器振动筛。 注意: 发光细菌的光强分布与温度呈强烈的变化。根据各自细菌应变的光生产的调控机制, 光发射可能在大约 1-6 h 以后开始。</li>
	<li>观察细菌细胞培养, 直到它们开始发光 (大约 1-6 小时)。 注意: 根据表达的目的, 细胞生长到第二天, 然后收获, 或细胞可以保持晃动, 只要他们是闪亮的。采集细胞和纯化任何蛋白质都可以按照标准程序进行。</li>
</ol>4. 细菌生物发光菌株和改良大肠杆菌菌株体内活性测定
注: 长时间贮存菌株通常 acieved 通过冷冻甘油储存的细菌培养。菌株应始终在琼脂板块上的条纹, 以确保所有菌株的统一启动条件, 在使用之前, 液体培养, 由于一个滞后阶段的增长后, 解冻。
<ol><li>在琼脂盘上条纹所需的生物发光细菌菌株或修饰大肠杆菌菌株, 并在一夜之间孵化28摄氏度。 注: 孵化温度可能因应变而异, 必须进行经验评估。为了能够比较生物发光细菌菌株和改良大肠杆菌菌株, 生长条件必须相同。</li>
	<li>接种3毫升培养基以各自菌株与一个单一殖民地从琼脂板材和孵化细胞在28°c 和 180 rpm 在孵化器振动筛大约 1-2 h。</li>
	<li>测量液体培养的1:10 稀释的细胞密度在650毫微米。计算1毫升养殖的比例和体积, 外径650为0.05。 注: 随后的板读取器化验将确定细胞密度在 650 nm, 以避免干扰的光发射的菌株。</li>
	<li>将所计算的培养基和培养基的体积注入24井的黑壁板, 玻璃底部。对改良的大肠杆菌菌株, 添加1µL 卡那霉素 (pET28a 载体耐药性) 和1µL IPTG (诱导基因表达) 的样品。将盖子放在盘子上, 以避免在测量过程中蒸发。 注: 为了确保含有整个莱克丝操纵的 pET28a 向量不会被大肠杆菌培养, 卡那霉素必须添加到每一个大肠杆菌样本中, 以保证大肠杆菌的光生产细胞可以测量, 基因表达必须由 IPTG 诱导。为避免串扰和测量干扰, 具有玻璃底和透明盖子的黑壁井板显示出最佳效果。但是, 可以观察到串扰, 并且必须仔细选择良好的位置。</li>
	<li>在板读取器中开始测量。 注意: 车牌阅读器协议是基于专门开发的用于此检测的脚本 (参见补充材料), 它结合了两种测量、吸光度和生物发光。数据点被收集每10分钟与永久震动在测量和恒定的温度之间28°c。</li>
</ol>

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作者没有什么可透露的。

通过吉布森克隆, 构建了一个含有四个片段组成 mandapamensis 27561 的操纵的表达质粒。这种基于大肠杆菌的莱克丝基因表达使大肠杆菌细胞发出光。避免仲裁传感调节和非标准生长条件是该系统的重要优点。
使用吉布森克隆策略的优点是易于组装多线 DNA 片段, 灵活性高, 不需要特定的限制站点18,19,20。这种方法可以很容易地改变一个莱克丝操纵, 不包括单一基因或基因簇, 或引入新的基因或交换基因从一个菌株与另一个。应用此方法的先决条件是各自的莱克丝操纵基因序列的可用性。仅为少量发光的细菌是完全脱氧核糖核酸序列各自莱克丝操纵知道并且/或者可利用。这些序列中的许多都是碎片, 因为它们是使用猎枪测序生成的。在被调查的P. mandapamensis 27561 的情况下,莱克丝操纵的完整 DNA 序列是已知的, 可从 NCBI 基因数据库 (基因库: DQ988878.2) 获得。
吉布森装配协议的一个关键步骤是计算单个碎片的 DNA 浓度。在制造商的装配协议, 建议使用 0.2-0.5 pmols 和总容量20µL 为 4-6 片断20,21。在本例中, luxCDABFEG-ribEBH由4个片段组成, 其浓度和总体积分别为 0.1 pmol 和45µL, 这取决于 PCR 产物的产量。一方面, 集中度必须减少, 另一方面, 必须增加体积。然而, 大会工作非常顺利, DNA 测序确认正确插入的第一次尝试。这一发现证实吉布森大会是一个稳健和适当的方法, 我们的调查, 可以很容易地修改18,19,20。
新建立的板读法是一种易于处理的方法。它可以对新的或 (非) 已知的生物发光细菌菌株进行简单的初步分析, 并给出了关于光生产调控机制的第一个提示 (例如,低细胞密度下的发光滞后)。此外, 通过简单地改变生长介质或温度, 可以很容易地评价生长条件与光生产的结合。
测量与板材读者被执行了在28°c。这种不寻常设置的原因是生物发光细菌菌株的温度敏感性, 温度高于30摄氏度会导致更少甚至没有生长和/或光发射。注意, 板读器能够保持一个定义的温度随着时间的限制, 一个缺少的有源冷却系统。因此, 环境温度必须低于测量的温度。
作为概念的证明, 对大肠杆菌结构与生物发光应变P. mandapamensis (图 5) 进行了比较, 同时进行了参考测量 (图 4), 并确认了这个新建立的系统的可靠性。此外, 长期测量还保证了光发射的寿命 (图 6)。但人们必须考虑到, OD 值在一定的光学密度之上是不可靠的, 这取决于使用的测量装置的特点 (在本例中约15小时), 在这种情况下需要适当的稀释来测量探测器的线性范围。这些高方差的 OD 值使得这些长时间的测量不可靠。因此, 建议使用此处报告的实验装置, 以缩短10小时的测量量。
在图 7中可以看到已建立的平板阅读器检测的局限性。难点是要找到适当的测量设置。"增益值" 调整光电倍增管 (PMT) 的灵敏度。增益是 PMT 中信号的放大, 这意味着较高的增益因数会增加信号。如果增益设置得过低, 信噪比越大, "低" 发光细菌的光强度就不能再测量 (接近零的信号, 没有显示的数据)。生物发光检测的挑战是设置增益, 使所有细菌菌株的测量结果停留在仪器的范围内。另外, 发光的测量范围取决于测量间隔时间 (例如,最大值为 200万, 1 秒)。
该增益设置为 2800, 经经验检验, 并选择用于建立此方法的特定板读取器。使用增益设置允许记录的最大发射光的生物发光大肠杆菌系统, p. mandapamensis 27561 和mandapamensis S1 没有溢出, 但为菌株P. mandapamensis TH1 和五. 弧菌14126 的增益太高。因此, 这些后应变超过检测极限, 真正的最大光强度是无法测量的。这种技术限制可能会阻止生物发光细菌的比较, 因为它们在最大光发射中表现出很高的变化, 尽管生长条件和细胞密度可能是可比的。
在用过的井板中分析的细菌菌株的定位必须进行实证评估。虽然使用了玻璃底的黑色井板, 样品之间的串扰被观察了。特定菌株的光强程度如此之高, 所有邻近的油井都将显示假的正光排放 (如空白)。因此, 对两种不同的菌株进行单独的测量或与某种空间的分离是很重要的。
已经有许多被修改过的大肠杆菌菌株, 其中含有莱克丝操纵的部分, 主要以应用为导向的15,16,17。这里描述的方法, 瞄准基础研究, 例如可能单独地分析每莱克丝基因。虽然生物发光研究有很长的历史, 但仍存在许多开放性问题。通过排除或引入莱克丝操纵的基因, 将luxAB基因与另一种菌株的基因交换, 或分析蛋白质复合物, 嵌入在易于处理的大肠杆菌系统中, 并进一步应用于板材读者分析, 也许可以获得更多的信息, 管理过程和功能的莱克丝基因。

生物体的光的发射 (生物发光) 是一个迷人的过程中发现细菌, 真菌, 昆虫, 线虫, 鱼类和鱿鱼1。生物发光光的发射来自于化学能量 (部分地) 转化为光能 ("冷光") 的化学反应。在生物发光细菌中, heterodimeric 酶荧光素催化 monooxygenation 长链, 脂肪醛, 如 tetradecanal, 到相应的酸伴随着光发射最大值在 490 nm2,3。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57881/57881fig1v2.jpg"> 图 1: 细菌荧光素酶的一般反应方案.细菌荧光素酶 (LuxAB) 通过利用黄素单核苷酸 (FMNH2) 和分子氧 (O2) 催化 monooxygenation 长链醛 (ch3(ch2)), 产生产物长链酸 (ch3(ch2)nCOOH), 黄素单核苷酸 (FMN), 水 (H2O) 和光的发射以 490 nm 为中心。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig1v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在这种氧化过程中释放的能量会引起兴奋状态 FMN-4a-hydroxide, 它作为发光荧光素4。细菌生物发光所涉及的蛋白质, 即 LuxCDABEG,是由莱克丝操纵编码的, 并且高度保守于各种细菌菌株2,5。heterodimeric 荧光素酶的基因luxA和luxB编码;luxC、 luxD和奢侈品的基因产品是脂肪酸还原酶复合物的组成部分;luxG编码为黄素还原酶6。一些生物发光发光细菌(例如, 发光 mandapamensis 27561) 携带额外的luxF基因。据报道, LuxF 是一种 homodimeric 蛋白, 它结合了不寻常的黄素衍生物 6-(3 '-(R)-肉豆蔻)-FMN (myrFMN)7,8,9,10,11 ,12。还发现了对核黄素合成负责的其他基因 (例如, ribEBH), 而且还报告了调控基因, 这在生物发光的仲裁传感调控中起到了作用, 特别是对弧菌鲵和弧菌弧菌6,13。尽管高度保守的基因秩序, 生物发光细菌表现出高变化的特点, 如生长行为, 光强辐射, 或调节生物发光2,5,14.
一些含有部分或整个莱克丝操纵子的改良菌株或质粒是众所周知的, 利用生物发光作为记者系统。各种应用, 如确定启动子活动, 监测环境或食品样品中的细菌污染, 生物荧光共振能量转移 (),在体内成像的真核生物感染,pyrosequencing, 等等建立了15,16,17。有趣的是, 三最常用的生物发光报告系统来自北美萤火虫 (Photinus pyralis), 线虫的肠道病原体 (Photorhabdus luminescens) 和海的三色堇 (Renillareniformis)。这些系统都没有细菌起源, 但是使用莱克丝基因和操纵子从细菌起源获得更多的兴趣应用研究16。生物发光蛋白在细菌来源中的应用较少, 主要是由于细菌衍生的发光蛋白的稳定性和寿命较短, 与海洋生境有关。在标准实验室条件下, 海洋生境的生物发光细菌是不可耕种的。这些细菌需要特定的生长介质和条件, 如人工海水介质和较低的生长/孵化温度 (例如, 28 摄氏度)。
为了简化对不同生物发光细菌株的莱克丝操纵特征或单一莱克丝基因的比较, 一种规范莱克丝操纵表达和分析的方法是先决条件。因此, 将整个莱克丝肋操纵纳入标准研究细菌大肠杆菌 (大肠杆菌) 的想法应运而生 .为此, 吉布森装配被证明是一个有用的工具, 将多个线性, 重叠片段集成到一个表达式向量中, 而不需要特定的限制站点。当 DNA 插入量过大 (例如, mandapamensis 27561 luxCDABFEG-ribEBH; ~ 9 kb 操纵大小) 时, 该方法也适用于通过 PCR 扩增。莱克丝操纵可以分离成多个重叠片段, 然后组装成一个表达质粒, 最后序列验证的组装产品可以直接转化为适当的大肠杆菌系统高产蛋白质表达18,19,20。除了易于处理大肠杆菌的莱克丝基因表达, 一个简单的方法, 结合记录的细胞生长和生物发光光发射仍然有待建立。这里描述的方法允许原位测量和相关的细胞密度和光发射的生物发光细菌。
对莱克丝基因和莱克丝操纵的分析, 并对各种生物发光细菌进行了调控, 一方面是含有操纵的人工生物发光大肠杆菌系统 。mandapamensis 27561, 另一方面, 新开发的平板阅读器检测结合原位记录细胞密度和光发射, 有助于获得更多的信息, 各种细菌莱克丝系统。这种基本的特征和比较 luciferases 和相关的酶可能会导致替代已建立的记者系统, 增强稳定性和活性。

<table><tbody><tr><td>NEBuilder High-Fidelity DNA Assembly Cloning Kit</td><td>New England Biolabs Inc.</td><td>E2621S</td><td>for 10 rxn&lt;br/&gt; dx.doi.org/10.17504/protocols.io.cwaxad</td></tr><tr><td>Phusion Polymerase&amp;nbsp;</td><td>Thermo Scientific</td><td>F530S</td><td></td></tr><tr><td>Q5 High Fidelity DNA Polymerase</td><td>New England Biolabs Inc.</td><td>M0491S</td><td></td></tr><tr><td>GeneJet Genomic DNA Purififcation Kit</td><td>Thermo Scientific</td><td>K0721</td><td>for 50 rxn</td></tr><tr><td>Restriction enzymes and buffer (NcoI, XhoI)</td><td>New England Biolabs Inc.</td><td>R3193S/R0146S</td><td></td></tr><tr><td>Wizard SV Gel and PCR Clean-Up kit&amp;nbsp;</td><td>Promega</td><td>A9282</td><td>for 250 rxn</td></tr><tr><td>Monarch DNA Gel Extraction Kit&amp;nbsp;</td><td>New England Biolabs Inc.</td><td>#T1020S</td><td>for 50 rxn</td></tr><tr><td>GeneJet Plasmid Miniprep Kit&amp;nbsp;</td><td>Thermo Scientific</td><td>K0503</td><td>for 250 rxn</td></tr><tr><td>GoTaq G2 DNA Polymerase</td><td>Promega</td><td>M7841</td><td></td></tr><tr><td>24-well black sensoplate with glass bottom</td><td>Greiner Bio One</td><td>662892</td><td></td></tr><tr><td>FLUOStar Omega plate reader&amp;nbsp;</td><td>BMG Labtech</td><td></td><td></td></tr><tr><td>OPTIMA/Mars Analysis Software</td><td>BMG Labtech</td><td></td><td>Version 2.20</td></tr><tr><td>Microsoft Excel 2010</td><td>Microsoft</td><td></td><td></td></tr><tr><td>Multitron Standard Incubator Shaker</td><td>Infors AG</td><td></td><td></td></tr></tbody></table>

in situ measurement and correlation of cell density and light emission of bioluminescent bacteria

有相当多的细菌物种能够发射光。他们都有相同的基因簇, 即莱克丝操纵。尽管有这种相似之处, 这些细菌在生长行为、光发射强度或生物发光的调控等特性上表现出极端的变化。这里提出的方法是一个新的研究, 结合记录的细胞生长和生物发光光发射强度随着时间的推移, 利用一个板块阅读器。由此产生的生长和光发射特性可与各自细菌株的重要特征 (如仲裁传感调节) 联系起来。培养一系列的生物发光细菌需要一个特定的介质 (例如,人工海水介质) 和定义的温度。另一方面, 易于处理的非生物发光标准研究细菌大肠杆菌 (大肠杆菌)可以在实验室规模内以较高的数量进行廉价栽培。通过引入含有整个莱克丝操纵的质粒来开发大肠杆菌可以简化实验条件, 另外还为将来的应用开辟了许多可能性。通过吉布森克隆和插入四块含有七莱克丝基因和三根肋骨基因的表达质粒, 实现了利用大肠杆菌表达菌株的所有莱克丝基因的表达。莱克丝肋操纵成 pET28a 向量。大肠杆菌基的莱克丝基因表达可以诱导和控制通过异丙基β-thiogalactopyranosid (IPTG) 添加导致生物发光大肠杆菌细胞。该系统的优点是避免了仲裁传感调节限制和复杂介质组成以及非标准生长条件, 如定义的温度。该系统能够分析莱克丝基因及其相互作用, 通过排斥操纵的相应基因, 甚至添加新的基因, 将luxAB基因从一个细菌菌株交换, 或分析蛋白质复合物, 如luxCDE。

莱克丝操纵- luxCDABFEG的基因顺序是高度保守的在各种各样的菌株2,5,14。对质粒的设计, 从生物发光细菌株发光 mandapamensis 27561 中取序信息, 并保留其基因顺序, 并考虑了单基因间的非编码序列。图 2描述了应用的吉布森克隆策略的示意图。四个片段总共, luxCDAB, luxF, luxEG和ribEBH, 产生了 20-40 基对重叠序列。在执行吉布森组件20的所有步骤之后, DNA 测序确认了质粒的正确组装, 包括所有碎片。图 3描述了包含莱克丝肋操纵的最终装配产品 pET28a 的矢量图。这一改良的 pET28a 载体的一个显著优势是利用标准化大肠杆菌生长条件和控制诱导与 IPTG。
为了测量生物发光细菌的光发射和各自的细胞密度, 研制了一种基于平板阅读器的方法。将单测量脚本与光强和细胞密度相结合, 生成了板读写器的方法。这个新的剧本使测量的 OD650和光强度每10分钟为用户定义的时间框架, 必须调整到使用的细菌的世代时间为各自分析 (例如, 10 h)。光密度的测量是在650纳米上进行的, 以避免干扰光的发射。作为概念的证明, 并确保大肠杆菌细胞的健康和正确的生长行为, 进行了参考测量。在图 4中, 本文介绍了大肠杆菌BL21 细胞、大肠杆菌BL21 细胞、含有空 pET28a 载体的大肠杆菌 BL21 细胞 , 以及含 pET28a 向量的 e. 对于后应变, 由于 T7 启动子的泄漏, 没有添加 IPTG 来分析光的发射。所有三参考测量显示一个 sigmoidal 生长曲线与三个成长阶段 (滞后, 指数和固定相)。只有大肠杆菌BL21 细胞含有 pET28a 载体与莱克丝肋骨操纵插入开始发出光, 但与测量的对比, 表达是由添加 IPTG 和光在30分钟后发出, 非诱导细胞仅在大约5小时后开始发光, 并显示出比诱导系统更低的光发射 (约4倍)。
图 5给出了在大肠杆菌和生物发光细菌菌株 mandapamensis 27561 中, 无论是在 LB 介质中还是在人工海水介质中, 操纵的生长曲线和光强的比较,利用本发明建立的原位法。为了比较这些细菌, 测量结果是在28摄氏度的孵育温度下进行的。这种温度降低了 LB 介质中改性大肠杆菌的生长速率以及人工海水介质, 但对生物发光细菌菌株的温度较低是至关重要的。这种温度依赖性在图 5A中可见, 因为P. mandapamensis 27561 的细胞密度比大肠杆菌高得多。此外, 对于大肠杆菌菌株, LB 培养基允许产生较高的细胞密度, 对于天然的生物发光细菌菌株, 人工海水介质是首选和必要的生物发光。记录的细胞密度与各自的光强度相关, 如图 5B所示。值得注意的是, 荧光大肠杆菌细胞在 LB 介质和人工海水介质中均达到类似的光发射最大值, 尽管在不同的时间点记录了最高强度。与此不同的是, P. mandapamensis 27561 在 LB 培养基中的生长速率很低, 但细菌细胞根本不发光 (图 5B)。在人工海水介质中, P. mandapamensis 27561 显示, 每秒约 1 x 104计数的光发射量最高, 这几乎是一个比大肠杆菌低200的因素。图 5C代表光生物发光被 OD 规范的相对光线单位。这些结果证实, 不仅是插入含有莱克丝操纵的质粒在大肠杆菌中的成功和功能, 而且这种改良大肠杆菌菌株是一个有效的替代品, 甚至更高的光发射产量和无限制的细菌生物发光的滨海细菌, 如复杂的海水介质和较低的温度。
此外, 还对大肠杆菌基的24小时以上的大肠埃希氏肋基因表达进行了长期测量, 以分析光发射的寿命 (图 6)。光发射持续了19.5 小时, 比细菌菌株 (例如, mandapamensis 27561) 的时间长得多, 在那里, 观察到的结果是, 在10小时后, 光的发射非常低。
为了说明所研制的试验的局限性,图 7显示了三种生物荧光细菌菌株的测量结果, 即发光 mandapamensis S1、发光 mandapamensis TH1 和弧菌弧菌14126。对于第一应变 (S1), 该方法工作良好, 并显示了最大的生物发光强度近 2 x 106。对于另外两个菌株 (TH1 和 14126), 由于两者所产生的光强度超过了所用仪器设置的检测极限, 所以无法确定最大光强度。为这两种菌株开发的方法 (脚本) 定义的增益值太高。然而, 生物发光活动的开始可以互相比较。mandapamensis TH1 和mandapamensis S1 在大约1小时后开始发光, 外径650值分别为 0.1-0.2。与此相反, v. 弧菌14126 在大约5.5 小时后开始发出光, 其外径650值为1.0。观测到的光发射伴随着 OD 和生物发光指数的增加。众所周知,弧菌14126 的生物发光基础上的法定人数传感调节, 因此一个特定的细胞密度允许激活莱克丝操纵, 这可以清楚地看到在图 713。这一结果表明, 用这种新的原位板阅读器分析, 可以很容易地比较生物发光细菌, 并粗略地定义了这些菌株的调节机制, 通过确定仲裁传感调节是否可以观察或不。
图 8显示了大肠杆菌BL21 细胞的表达文化的一个例子, 它窝藏在 IPTG 诱导下的 pET28a 质粒, 含有莱克丝操纵。在诱导后大约一小时后,大肠杆菌细胞开始以蓝绿色的颜色发光。图 8A显示在光中拍摄的大肠杆菌表达文化,图 8B在黑暗中给予相同的文化。图 8C描述了一种人工海水培养基的琼脂板, mandapamensis S1 在黑暗中以相同的蓝绿色颜色特征进行细菌生物发光。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57881/57881fig2v3.jpg"> 图 2: 应用吉布森克隆策略的示意图表示法。步骤 (I):重叠引物 (彩色箭头; 约 20-40 基对重叠) 设计。重叠引物包含退火序列, 由一个片断的各自 5 ' 和 3 ' 区域和相邻段的各自 3 ' 和 5 ' 区域组成。步骤 (二):通过标准 PCR 反应生成装配的设计片段。步骤 (三):目标向量通过限制消化 (如NcoI、XhoI) 进行线性化。步骤 (IV):所有碎片和线性化向量的 DNA 浓度必须确定, 以调整适合吉布森装配 (根据制造商的协议) 的浓度。步骤 (V):所有片段和优化的 DNA 浓度的线性化向量结合吉布森装配主组合 (T5 exonuclease, dna 聚合酶, 和 dna 连接酶), 并在50°c 孵化 1 h.步骤 (VI):装配产品被变换根据标准的协议, 进入一个适当的大肠杆菌菌株的高产质粒复制 (如大肠杆菌TOP10 或 XL-1)。步骤 (七):为了验证质粒的正确组装, 必须进行组装质粒的 DNA 测序。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig2v3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57881/57881fig3v2.jpg"> 图 3: 载有莱克丝肋操纵的 pET28a 的矢量图.mandapamensis 27561 的莱克丝肋操纵插入到 pET28a 的多个克隆站点中, 原始基因顺序 (luxCDABFEG ribEBH)。用于克隆的限制站点是 NcoI 和 XhoI。操纵的吉布森组装用的碎片是luxCDAB在橙色, luxF绿色, luxEG在蓝色和ribEBH的薰衣草;片段中的基因显示为一个单独的盒子。在应用的克隆策略中, 操纵的每个基因之间的非编码序列都包括在内。pET28a 的最终粒径操纵为14625基对。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig3v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57881/57881fig4v2.jpg"> 图 4:参考菌株生长曲线与光照强度的比较.每秒650毫微米和生物发光强度的 OD 测量每10分钟 10 h 在28°c。所有测量值都是三生物复制的平均值, 每四个技术复制。误差线表示标准偏差。 BL21 细胞 (灰色方块),大肠杆菌BL21 细胞含有一个空的 pET28a 向量 (灰色圆圈), 大肠杆菌 BL21 细胞包含 pET28a 向量与莱克丝肋骨操纵插入 (黑色钻石), 以确保我们的大肠杆菌细胞的生长行为正确。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig4v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a> 
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57881/57881fig5v2.jpg"> 图 5: 在大肠杆菌(正方形) 和mandapamensis 27561 ( 圆圈) 中以 LB 介质 (开放符号) 或人工海水介质 (填充符号) 表示的操纵的生长曲线和光强度的比较.所有测量值都是三生物复制的平均值, 每四个技术复制。误差线表示标准偏差。所有实验都是在28摄氏度的孵化温度下进行的。(A) 650 nm 的光学密度 (OD) 测量每10分钟进行10小时大肠杆菌莱克丝操纵表达 (左面板) 的比较与mandapamensis 27561 (右面板) 的 LB 介质和人工海水培养基。细胞密度确定在 650 nm, 以避免生物发光干扰。(B)对10小时大肠杆菌操纵表达 (左面板) 的光强度 (生物荧光 [计数/s]) 进行了每10分钟的测量, 并与 mandapamensis 27561 (右面板) 进行比较.水介质。(C)在大肠杆菌(左面板) 和P. mandapamensis 27561 (右面板) 中表达的莱克丝操纵的相对光照强度 (RLU/OD) 是通过将生物发光正常化为细胞密度来确定的。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig5v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57881/57881fig6v2.jpg"> 图 6: 对 24 h 型大肠杆菌的生长曲线和光强度的比较.每秒650毫微米和生物发光强度的 OD 测量每10分钟 24 h 在28°c。所有测量值都是三生物复制的平均值, 每四个技术复制。误差线表示标准偏差。此外, 相对光强度 (RLU/OD) 的生物发光是标准化的细胞密度表示。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig6v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57881/57881fig7v2.jpg"> 图 7: 对生物发光细菌进行比较, 评估潜在的仲裁传感规则.光发射和细胞密度每10分钟测量10小时, 并代表三生物复制的平均值, 每四个技术复制。误差线表示标准偏差。比较了发光 mandapamensis TH1 (黑方块)、弧菌弧菌14126 (灰色圆圈) 和发光mandapamensis S1 (灰钻石) 的测量结果;(A)描述光学密度 (OD) 在650毫微米, (B)光强度 (生物发光 [计数/秒]) 和(C)相对光强度 (RLU/OD)。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig7v2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57881/57881fig8.jpg"> 图 8: 在液态培养基和琼脂板上的生物发光。(A) 5 l 烧瓶, 2 升磅培养基接种大肠杆菌BL21 细胞, 表达 pET28a 操纵质粒 , 光照。( B)与 (A) 在黑暗中拍照的文化相同。图片 A 和 B 被采取了大约2小时在表示的归纳以后。(C) mandapamensis S1 的人工海水培养基琼脂板, 在黑暗中拍照。<a href="https://www.jove.com/files/ftp_upload/57881/57881fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria at JoVE.com

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

生物发光细菌通过多种机制 (如聚类传感) 来调节光的产生。这种新方法允许对生物发光的原位研究和光发射与细胞密度的相关性。人工生物发光大肠杆菌系统允许对莱克丝操纵子、莱克丝蛋白及其相互作用的表征。

就地生物发光细菌细胞密度与光发射的测量与相关性研究

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

in-situ-measurement-correlation-cell-density-light-emission

Research

JoVE Journal

Biochemistry

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

11.5K 次观看.  Graz University of Technology. 生物发光细菌通过多种机制 (如聚类传感) 来调节光的产生。这种新方法允许对生物发光的原位研究和光发射与细胞密度的相关性。人工生物发光大肠杆菌系统允许对莱克丝操纵子、莱克丝蛋白及其相互作用的表征。

视频: In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

科研

免疫与感染

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

遗传学

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

"发光法"的分类检测细菌病原体的记者噬菌体

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

生物发光细菌成像<em在体内</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

生物学

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

优化人肠道类肠生物用于昼夜节律研究

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

作者聚焦：多细胞系统中昼夜节律的体外研究

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

使用类器官中的生物发光报告基因监测昼夜节律振荡

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...

就地生物发光细菌细胞密度与光发射的测量与相关性研究

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此视频中的章节

用荧光素酶在小鼠细菌性感染的形象

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

"发光法"的分类检测细菌病原体的记者噬菌体

生物发光细菌成像

监测细胞自主的生物钟节律基因表达，利用荧光素酶生物发光记者

自主的生物发光哺乳动物细胞连续和实时监测细胞毒性

研究蛋白质 - 蛋白质相互作用活细胞使用生物发光共振能量转移

利用自动纵向荧光成像气体和温度优化记录仪 (鳄鱼) 对生物发光记者进行灵活测量

使用类器官中的生物发光报告基因监测昼夜节律振荡

使用类器官中的生物发光报告基因监测昼夜节律振荡