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 border="0" cellpadding="0" cellspacing="0" style="width:896px;" width="895">
	<colgroup>
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			<td height="63" style="height:63px;width:335px;">NEBuilder High-Fidelity DNA Assembly Cloning Kit</td>
			<td style="width:171px;">New England Biolabs Inc.</td>
			<td style="width:115px;">E2621S</td>
			<td style="width:276px;">for 10 rxn 
			dx.doi.org/10.17504/protocols.io.cwaxad</td>
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			<td height="21" style="height:21px;">Phusion Polymerase&#160;</td>
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			<td height="42" style="height:42px;width:335px;">Q5 High Fidelity DNA Polymerase</td>
			<td style="width:171px;">New England Biolabs Inc.</td>
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			<td height="21" style="height:21px;">GeneJet Genomic DNA Purififcation Kit</td>
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			<td>for 50 rxn</td>
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			<td height="42" style="height:42px;width:335px;">Restriction enzymes and buffer (NcoI, XhoI)</td>
			<td style="width:171px;">New England Biolabs Inc.</td>
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			<td height="21" style="height:21px;width:335px;">OPTIMA/Mars Analysis Software</td>
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			<td height="21" style="height:21px;width:335px;">Microsoft Excel 2010</td>
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			<td height="21" style="height:21px;width:335px;">Multitron Standard Incubator Shaker</td>
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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 ...

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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

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation is a popular technique to detect protein-protein interactions. Subsequent Western blot analysis is the most common method to visualize co-immunoprecipitated proteins. Recently, the proximity ligation assay has become a powerful tool to visualize protein-protein interactions in situ and provides the possibility to quantify protein-protein interactions by this method. Similar to conventional immunocytochemistry, the proximity ligation assay technique is also based on the accessibility of primary antibodies to the antigens, but in contrast, proximity ligation assay detects protein-protein interactions with a unique technique involving rolling-circle PCR, while conventional immunocytochemistry only shows co-localization of proteins.
Nuclear factor 90 (NF90) and RNA-binding motif protein 3 (RBM3) have been previously demonstrated as interacting partners. They are predominantly localized in the nucleus, but also migrate into the cytoplasm and regulate signaling pathways in the cytoplasmic compartment. Here, we compared NF90-RBM3 interaction in both the nucleus and the cytoplasm by co-immunoprecipitation and proximity ligation assay. In addition, we discussed the advantages and limitations of these two techniques in visualizing protein-protein interactions in respect to spatial distribution and the properties of protein-protein interactions.

Visualization of Protein-protein Interaction in Nuclear and Cytoplasmic Fractions by Co-immunoprecipitation and In Situ Proximity Ligation Assay

Protein-protein interactions can occur in both the nucleus and the cytoplasm of a cell. To investigate these interactions, traditional co-immunoprecipitation and modern proximity ligation assay are applied. In this study, we compare these two methods to visualize the distribution of NF90-RBM3 interactions in the nucleus and the cytoplasm.

通过免疫共沉淀在核质馏分蛋白质相互作用的可视化和<em&gt;原位</em&gt;接近结扎分析

Protein-protein interactions are involved in thousands of cellular processes and occur in distinct spatial context. Traditionally, co-immunoprecipitation ...

科研

生物化学

Measurement of Basal and Forskolin-stimulated Lipolysis in Inguinal Adipose Fat Pads

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes the method for the measurement of basal and forskolin (FSK)-stimulated lipolysis in the inguinal fat pads isolated from wild type mice fed either normal chow diet (NCD), high fat diet (HFD) or a high fat diet containing 0.01% of capsaicin (CAP; transient receptor potential vanilloid subfamily 1 (TRPV1) agonist) for 32 weeks. The method described here for performing ex vivo lipolysis is adopted from Schweiger et al.1 We present a detailed protocol for measuring glycerol levels by UV-Visible (UV/VIS) spectrophotometry. The method described here can be used to successfully isolate inguinal fat pads for lipolysis measurements to obtain consistent results. The protocol described for inguinal fat pads can readily be extended to measure lipolysis in other tissues.

This protocol describes the method of determining basal and forskolin-stimulated lipolysis in inguinal fat pads obtained from normal chow diet (NCD) or high fat diet (HFD) &plusmn; capsaicin fed wild type mice. As an index for lipolysis, glycerol release was measured from inguinal adipose fat pads.

测量基底和福斯柯林刺激的脂肪分解在腹股沟脂肪脂肪垫

Lipolysis is a process by which the lipid stored as triglycerides in adipose tissues are hydrolyzed into glycerol and fatty acids. This article describes ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to precisely maneuver the nucleation and crystal growth towards desired sizes of protein crystals. The controlled crystallization is based on sample investigation with in situ Dynamic Light Scattering (DLS) while all visual changes in the droplet are monitored online with the help of a microscope coupled to a CCD camera, thus enabling a full investigation of the protein droplet during all stages of crystallization. The use of in situ DLS measurements throughout the entire experiment allows a precise identification of the highly supersaturated protein solution transitioning to a new phase &#8211; the formation of crystal nuclei. By identifying the protein nucleation stage, the crystallization can be optimized from large protein crystals to the production of protein microcrystals. The experimental protocol shows an interactive crystallization approach based on precise automated steps such as precipitant addition, water evaporation for inducing high supersaturation, and sample dilution for slowing induced homogeneous nucleation or reversing phase transitions.

Growing Protein Crystals with Distinct Dimensions Using Automated Crystallization Coupled with In Situ Dynamic Light Scattering

Here we present a protocol for controlled production of protein microcrystals. The process uses an automated device allowing controlled manipulation of several crystallization parameters. The protein crystallization is carried-out by controlled and automated addition of crystallization solutions while monitoring and investigating the radius distribution of particles in the crystallization droplet.

采用自动结晶耦合原位动态光散射的不同尺寸生长蛋白晶体

The automated crystallization device is a patented technique1 especially developed for monitoring protein crystallization experiments with the aim to ...

Analysis of SEC-SAXS data via EFA deconvolution and Scatter

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ratio and conformational changes of macromolecules and macromolecular complexes. A high quality SAXS dataset for structural modeling must be from monodisperse, homogeneous samples and this is often only reached by a combination of inline chromatography and immediate SAXS measurement. Most commonly, size-exclusion chromatography is used to separate samples and exclude contaminants and aggregations from the particle of interest allowing SAXS measurements to be made from a well-resolved chromatographic peak of a single protein species. Still, in some cases, even inline purification is not a guarantee of monodisperse samples, either because multiple components are too close to each other in size or changes in shape induced through binding alter perceived elution time. In these cases, it may be possible to deconvolute the SAXS data of a mixture to obtain the idealized SAXS curves of individual components. Here, we show how this is achieved and the practical analysis of SEC-SAXS data is performed on ideal and difficult samples. Specifically, we show the SEC-SAXS analysis of the vaccinia E9 DNA polymerase exonuclease minus mutant.

SEC-BioSAXS measurements of biological macromolecules are a standard approach for determining solution structure of macromolecules and their complexes. Here, we analyze SEC-BioSAXS data from two types of commonly encountered SEC traces&#8212;chromatograms with fully resolved and partially resolved peaks. We demonstrate the analysis and deconvolution using scatter and BioXTAS RAW.

通过 EFA 去卷和散射分析 SEC-SAXS 数据

BioSAXS is a popular technique used in molecular and structural biology to determine the solution structure, particle size and shape, surface-to-volume ...

Optimizing the Growth of Endothiapepsin Crystals for Serial Crystallography Experiments

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial crystallography experiments at both synchrotrons and XFELs. The method is based upon an understanding of the protein crystal phase diagram, and how that knowledge can be utilized. The method is divided into three stages: (1) optimizing crystal morphology, (2) transitioning to batch, and (3) scaling. Stage 1 involves finding well diffracting, single crystals, hopefully but not necessarily, presenting in a cube-like morphology. In Stage 2, the Stage 1 condition is optimized by crystal growth time. This strategy can transform crystals grown by vapor diffusion to batch. Once crystal growth can occur within approximately 24 h, a morphogram of the protein and precipitant mixture can be plotted and used as the basis for a scaling strategy (Stage 3). When crystals can be grown in batch, scaling can be attempted, and the crystal size and concentration optimized as the volume is increased. Endothiapepsin has been used as a demonstration protein for this protocol. Some of the decisions presented are specific to endothiapepsin. However, it is hoped that the way they have been applied will inspire a way of thinking about this procedure that others can adapt to their own projects.

The aim of this article is to give the viewer a solid understanding of how to transform their small-volume, vapor-diffusion protocol, for growing large, single protein crystals, into a large-volume batch micro-crystallization method for serial crystallography.

优化用于连续晶体学实验的内硫肽晶体的生长

Here, a protocol is presented to facilitate the creation of large volumes (&#62; 100 &#181;L) of micro-crystalline slurries suitable for serial ...

Cell Fractionation of U937 Cells by Isopycnic Density Gradient Purification

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and isopycnic density gradient centrifugation. The major advantage of this procedure is that it does not rely on the sole use of solubilizing detergents to obtain subcellular fractions. This makes it possible to separate the plasma membrane from other membrane-bound organelles of the cell. This procedure will facilitate the determination of protein localization in cells with a reproducible, scalable, and selective method. This method has been successfully used to isolate cytosolic, nuclear, mitochondrial, and plasma membrane proteins from the human monocyte cell line, U937. Although optimized for this cell line, this procedure may serve as a suitable starting point for the subcellular fractionation of other cell lines. Potential pitfalls of the procedure and how to avoid them are discussed as are alterations that may need to be considered for other cell lines.

This fractionation protocol will allow researchers to isolate cytoplasmic, nuclear, mitochondrial, and membrane proteins from mammalian cells. The latter two subcellular fractions are further purified via isopycnic density gradient.

通过等密度梯度纯化对U937细胞进行细胞分级分离

This protocol describes a method to obtain subcellular protein fractions from mammalian cells using a combination of detergents, mechanical lysis, and ...

Sample Preparation and Transfer Protocol for In-Vacuum Long-Wavelength Crystallography on Beamline I23 at Diamond Light Source

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, chlorine, or calcium, that are often natively present in macromolecules. This enables the direct structure solution of proteins and nucleic acids via experimental phasing without the need of additional labelling. To eliminate the significant air absorption of X-rays in this wavelength regime, these experiments are performed in a vacuum environment. Beamline I23 at Diamond Light Source, UK, is the first synchrotron instrument of its kind, designed and optimized for MX experiments in the long wavelength range towards 5 &#197;. 
To make this possible, a large vacuum vessel encloses all endstation components of the sample environment. The necessity to maintain samples at cryogenic temperatures during storage and data collection in vacuum requires the use of thermally conductive sample holders. This facilitates efficient heat removal to ensure sample cooling to approximately 50 K. The current protocol describes the procedures used for sample preparation and transfer of samples into vacuum on beamline I23. Ensuring uniformity in practices and methods already established within the macromolecular crystallography community, sample cooling to liquid nitrogen temperature can be performed in any laboratory setting equipped with standard MX tools. 
Cryogenic storage and transport of samples only require standard commercially available equipment. Specialized equipment is required for the transfer of cryogenically cooled crystals from liquid nitrogen into the vacuum endstation. Bespoke sample handling tools and a dedicated Cryogenic Transfer System (CTS) have been developed in house. Diffraction data collected on samples prepared using this protocol show excellent merging statistics, indicating that the quality of samples is unaltered during the procedure. This opens unique opportunities for in-vacuum MX in a wavelength range beyond standard synchrotron beamlines.

Here, we present a protocol for cryogenic sample preparation and transfer of crystals into the vacuum endstation on beamline I23 at Diamond Light Source, for long-wavelength macromolecular X-ray crystallography experiments.

金刚石光源下光束线I23真空长波长晶体学的样品制备和转移实验方案

Long-wavelength macromolecular crystallography (MX) exploits the anomalous scattering properties of elements, such as sulfur, phosphorus, potassium, ...

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

生物发光光遗传学2.0:利用生物发光在体外和体内激活光敏蛋白

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

An In-House-Built and Light-Emitting-Diode-Based Photodynamic Therapy Device for Enhancing Verteporfin Cytotoxicity in a 2D Cell Culture Model

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built using a set of conventional programmable light-emitting diodes (LEDs), a liquid crystal display (LCD) module, and a light sensor connected to a commercial microcontroller board. The box-based structure of the prototype was made with medium-density fiberboards (MDFs). The internal compartment can simultaneously allocate four cell culture multiwell microplates.
As a proof of concept, we studied the cytotoxic effect of the photosensitizer (PS) verteporfin against the HeLa cell line in two-dimensional (2D) culture. HeLa cells were treated with increasing concentrations of verteporfin for 24 h. The drug-containing supernatant medium was discarded, the adherent cells were washed with phosphate-buffered saline (PBS), and drug-free medium was added. In this study, the effect of verteporfin on cells was examined either without light exposure or after exposure for 1 h to light using red-green-blue (RGB) values of 255, 255, and 255 (average fluence of 49.1 &#177; 0.6 J/cm2). After 24 h, the cell viability was assessed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) assay.
Experimental results showed that exposure of cells treated with verteporfin to the light from the device enhances the drug&#39;s cytotoxic effect via a mechanism mediated by reactive oxygen species (ROS). In addition, the use of the prototype described in this work was validated by comparing the results with a commercial PDT device. Thus, this LED-based photodynamic therapy prototype represents a good alternative for in vitro studies of PDT.

Here, we describe a novel, simple, and low-cost device to successfully perform in vitro photodynamic therapy (PDT) assays using two-dimensional HeLa cell culture and verteporfin as a photosensitizer.

一种内部构建的基于发光二极管的光动力治疗设备，用于增强维替泊芬在 2D 细胞培养模型中的细胞毒性

This paper describes a novel, simple, and low-cost device to perform in vitro photodynamic therapy (PDT) assays, named the PhotoACT. The device was built ...