Name: super-resolution imaging of bacterial secreted proteins using genetic code expansion
Uploaded: 2023-02-10T13:23:00
Description: Watch this Scientific Journal Video about Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion at JoVE.com

这项工作得到了德克萨斯州加尔维斯顿德克萨斯大学医学分院的启动资金的支持，以及L.J.K.的德克萨斯州之星奖。我们感谢Edward Lemke教授（德国海德堡欧洲分子生物学实验室）提供的质粒pEVOL-PylRS-AF。 图1 中的图像是使用BioRender创建的。

1. 质粒构建
<ol><li>将表达POI的基因克隆到表达大肠杆菌BL21（DE3）中表达POI的表达质粒（例如pET28a-sseJ 10TAG）。此步骤有助于确定突变体是否具有功能。</li>
	<li>为了可视化宿主细胞中分泌的沙门氏菌效应子，构建一个表达质粒（pWSK29-sseJ-HA），该质粒在其天然启动子的控制下表达靶POI SseJ，如先前的报告7，12，<sup...

<ol>
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本文描述的方法用于跟踪感染后由细菌T3SS注射到宿主细胞中的效应蛋白的精确位置。T3SS被 沙门氏菌、 志贺氏菌和 耶尔森氏菌 等细胞内病原体用于将毒力成分转运到宿主中。超分辨率成像技术的发展使得以以前无法想象的分辨率12，13，24可视化毒力因子成为可能。然而，某些标记限制排除了更深入的研究...

长期以来，细菌感染一直被视为对人体健康的严重危害。病原体使用高度进化、极其强大和复杂的防御系统，以及各种细菌毒力因子（称为效应蛋白）来逃避宿主免疫反应并建立感染1，2.然而，由于缺乏在发病过程中直接跟踪宿主细胞中关键蛋白质成分和效应子的合适方法，这些系统背后的分子机制和单个效应蛋白的作用仍然在很大程度上是未知的。
一个典型的例子是鼠伤寒沙门氏菌，它会导致急性胃肠炎。沙门氏菌鼠伤寒使用三型分泌系统（T3SS）将各种效应蛋白直接注射到宿主细胞中。一旦沙门氏菌进入宿主细胞，它就会驻留在酸性膜结合的隔室中，称为含沙门氏菌的液泡（SCV）3，4。SCV 的酸性 pH 值激活沙门氏菌致病性岛 2 （SPI-2） 编码的 T3SS，并将 20 种或更多效应蛋白的凌空翻位穿过液泡膜进入宿主细胞质5，6，7，8。在宿主内部，这些复杂的效应蛋白混合物协调操纵宿主细胞信号通路，导致形成高度动态、复杂的管状膜结构，从 SCV 沿微管延伸，称为沙门氏菌诱导的细丝 （SIF），使沙门氏菌能够在宿主细胞内存活和复制9，10，11。
可视化、跟踪和监测细菌效应子定位以及检查它们在宿主细胞内的运输和相互作用的方法，为支撑细菌发病机制提供了重要的见解。宿主细胞内沙 门氏菌 分泌的T3SS效应蛋白的标记和定位已被证明是一项技术挑战12，13;尽管如此，基因编码荧光蛋白的发展已经改变了我们研究和可视化生命系统中蛋白质的能力。然而，荧光蛋白的大小（~25-30 kDa）15通常与目标蛋白的大小相当甚至更大（POI;例如，SsaP为13.65 kDa，SifA为37.4 kDa）。事实上，效应器的荧光蛋白标记通常会阻断标记效应子的分泌并堵塞T3SS14。
此外，荧光蛋白稳定性较差，在光漂白前发射的光子数量很少，限制了它们在超分辨率显微技术中的使用16，17，18，特别是在光活化定位显微镜（PALM），STORM和受激发射耗尽（STED）显微镜中。虽然有机荧光染料的光物理性质优于荧光蛋白，但诸如CLIP/SNAP19，20，Split-GFP 21，ReAsH / FlAsH22，23和HA-Tags 24，25等方法/技术需要额外的蛋白质或肽附属物，这些附件可能通过干扰翻译后修饰或运输来损害目标效应蛋白的结构功能一种最大限度地减少必要蛋白质修饰的替代方法涉及在通过GCE翻译期间将ncAA掺入POI中。ncAA要么是荧光的，要么可以通过点击化学12，13，26，27，28制成荧光。
使用GCE，具有微小的、功能性的生物正交基团（如叠氮化物、环丙烯或环辛炔基团）的ncAA几乎可以在靶蛋白的任何位置引入。在该策略中，在POI基因的指定位置将天然密码子与稀有密码子（例如琥珀色（TAG）终止密码子）交换。修饰的蛋白质随后与正交氨酰基-tRNA合成酶/tRNA对一起在细胞中表达。tRNA合成酶活性位点被设计为仅接收一个特定的ncAA，然后将其共价连接到识别琥珀色密码子的tRNA的3'末端。ncAA被简单地引入生长培养基中，但它必须被细胞吸收并到达细胞质，其中氨酰基-tRNA合成酶（aaRS）可以将其连接到正交tRNA;然后将其合并到指定位置的POI中（见图1）12。因此，GCE能够将大量的生物正交反应基团（如酮，叠氮化物，炔烃，环辛炔，转环辛烯，四嗪，去甲骨烯，α，β-不饱和酰胺和双环[6.1.0]-壬炔）掺入POI中，可能克服传统蛋白质标记方法的局限性12，26，27，28。
超分辨率成像技术的最新新兴趋势为在分子水平上研究生物结构开辟了新的途径。特别是，STORM是一种基于定位的单分子超分辨率技术，已成为可视化低至~20-30纳米的细胞结构的宝贵工具，并且能够一次研究一个分子的生物过程，从而发现细胞内分子的作用，这些作用在传统的集合平均研究中尚不为人知13.单分子和超分辨率技术需要带有明亮、光稳定的有机荧光团的小标签，以获得最佳分辨率。我们最近证明GCE可用于结合合适的探针进行超分辨率成像12。
细胞中蛋白质标记的两个最佳选择是双环[6.1.0]壬炔赖氨酸（BCN）和反式环辛烯赖氨酸（TCO;如图1所示），它们可以使用tRNA/合成酶对的变体（此处称为tRNA Pyl/PylRS AF）进行遗传编码，其中Pyl代表吡咯赖氨酸，AF代表合理设计的双突变体（Y306A，Y384F），源自天然编码吡咯赖氨酸12的甲烷肉瘤，29，30，31.通过应变促进的逆电子需求Diels-Alder环加成（SPIEDAC）反应，这些氨基酸与四嗪偶联物发生化学选择性反应（图1）12，30，31。这种环加成反应非常快，并且与活细胞相容;如果适当的荧光团与四嗪部分12，26，32官能化，它们也可能是荧光的。本文提出了一种优化的方案，用于使用GCE监测传递到宿主细胞中的细菌效应子的动力学，然后使用dSTORM监测HeLa细胞中分泌蛋白的亚细胞定位。结果表明，通过GCE掺入ncAA，然后与含荧光四嗪的染料进行点击反应，代表了一种选择性标记，分泌蛋白可视化以及随后在宿主中进行亚细胞定位的通用方法。然而，这里详述的所有组件和程序都可以调整或替换，以便GCE系统可以适应其他生物学问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris/Glycine SDS running buffer</td>
			<td>BIO-RAD</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Fischer Scientific</td>
			<td>A661-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulphate</td>
			<td>Fischer Scientific</td>
			<td>BP212R-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>ThermiFischer Scientific</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Avanti J-26XP (High-Performance Centrifuge)</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Agar</td>
			<td>BD Diagnostics, Franklin Lakes, USA</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>BDP-FL-tetrazine</td>
			<td>Lumiprobe (USA)</td>
			<td>2.14E+02</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Millipore</td>
			<td>444203</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B8026</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C8654</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C1919</td>
			<td></td>
		</tr>
		<tr>
			<td>Click Amino Acid / trans-Cyclooct-2-en &#8211; L - Lysine (TCO*A)</td>
			<td>SiChem GmbH</td>
			<td>SC-8008</td>
			<td>Size: 500 mg</td>
		</tr>
		<tr>
			<td>DAPI (Hoechst33342)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>DeNovix DS-11+ Spectrophotometer</td>
			<td>DeNovix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM*</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>* Used for maintaining HeLa Cell</td>
		</tr>
		<tr>
			<td>DMEM**</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>**Used for bacterial infection in presence of ncAA, see section 5.4.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>250 g</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa fluoro555 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A-31572</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli strain BL21 (DE3)</td>
			<td>Novagen (Madison, WI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD Camera</td>
			<td>Andor</td>
			<td>iXon Ultra 897-BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tube 1.5 mL (PCR clean)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30123.328</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fischer</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Syringe Filters - Sterile (PVDF 0.22 &#181;m)</td>
			<td>Fischer Scientific</td>
			<td>97203</td>
			<td>Pack of 100</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporator</td>
			<td>BIO-RAD</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Gibco L-Glutamine (200 mM), 100x</td>
			<td>Fischer Scientific</td>
			<td>25-030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>G7141-50KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fischer Scientific</td>
			<td>BF229-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATTC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Corning</td>
			<td>25-060-C1</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Hydrocloric acid</td>
			<td>Fischer Scientific</td>
			<td>A144-212</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td>Image processing and analysis:&#160; http://rsbweb.nih.gov/ij</td>
		</tr>
		<tr>
			<td>IntantBlue</td>
			<td>Expedeon</td>
			<td>ISB1L</td>
			<td>Coomassie-based stain</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td></td>
		</tr>
		<tr>
			<td>Janelia Fluoro 646-tetrazine</td>
			<td>Tocris Bioscience</td>
			<td>7279</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;Manager (v. 1.4.2)</td>
			<td></td>
			<td></td>
			<td>https://micro-manager.org/Download_Micro-Manager_Latest_Release</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>250 g</td>
		</tr>
		<tr>
			<td>Micro pulser cuvette</td>
			<td>BIO-RAD</td>
			<td>165-2086</td>
			<td>0.2 cm electrode gap, pkg. of 50</td>
		</tr>
		<tr>
			<td>Nikon N-STORM</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>https://www.microscope.healthcare.nikon.com/products/super-resolution-microscopes/n-storm-super-resolution</td>
		</tr>
		<tr>
			<td>Nunc EasYFlask Cell Culture Flasks</td>
			<td>ThermiFischer Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnipur Casamino Acid</td>
			<td>Calbiochem</td>
			<td>2240</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Paraformaldehhyde (PFA)</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution (100x)</td>
			<td>GenDEPOT&#160;</td>
			<td>CA005-010</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>pEVOL-PylRS-AF</td>
			<td></td>
			<td></td>
			<td>For plasmid construction and map see following references 
			1. Angew Chem Int Ed Engl. 2011, 50(17), 3878-81. 
			2. Angew Chem Int Ed Engl., 2012, 51, 4166-70.</td>
		</tr>
		<tr>
			<td>Plasmid Mini-prep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pWSK29-sseJ10TAG-HA</td>
			<td></td>
			<td></td>
			<td>Ref.: elife. 2021, 10, e67789.</td>
		</tr>
		<tr>
			<td>plasmid pWSK29-sseJ-HA</td>
			<td></td>
			<td></td>
			<td>Ref.: elife. 2021, 10, e67789.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Vector map of PWSK29: https://www.addgene.org/172972/</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Millipore</td>
			<td>540025</td>
			<td>Protein grade, 10% Solution</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fischer Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium sulphate</td>
			<td>Acros Organic</td>
			<td>424220250</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Set I - Calbiochem</td>
			<td>Sigma-Aldrich</td>
			<td>539131</td>
			<td>100x Solution</td>
		</tr>
		<tr>
			<td>Rabbit anti-HA primary antibody</td>
			<td>Sigma-Aldrich</td>
			<td>H6908</td>
			<td></td>
		</tr>
		<tr>
			<td>S. enterica. serovar Typhimurium 14028s</td>
			<td></td>
			<td></td>
			<td>Ref.: PLoS Biol. 2015, 13, e1002116.</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036-50G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fischer Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM), 100x</td>
			<td>Corning</td>
			<td>25-060-C1</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 100</td>
			<td>Fischer Scientific</td>
			<td>24932</td>
			<td></td>
		</tr>
		<tr>
			<td>STED microsocpe (Leica TCS SP8 STED 3X system)</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td></td>
			<td>https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/</td>
		</tr>
		<tr>
			<td>ThunderSTORM</td>
			<td></td>
			<td></td>
			<td>https://zitmen.github.io/thunderstorm/</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (1x), 0.25%&#160;&#160;</td>
			<td>GenDEPOT&#160;</td>
			<td>CA014-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>X-well tissue culture chamber slides</td>
			<td>SARSTEDT</td>
			<td>94.6190.802</td>
			<td></td>
		</tr>
	</tbody>
</table>

super-resolution imaging of bacterial secreted proteins using genetic code expansion

三型分泌系统（T3SS）使革兰氏阴性菌能够将一系列效应蛋白直接注射到真核宿主细胞的细胞质中。进入后，注射的效应蛋白协同调节真核信号通路并重新编程细胞功能，使细菌进入并存活。在感染背景下监测和定位这些分泌的效应蛋白为定义宿主-病原体相互作用的动态界面提供了足迹。然而，在不破坏宿主细胞结构/功能的情况下标记和成像宿主细胞中的细菌蛋白在技术上具有挑战性。 
构建荧光融合蛋白并不能解决这个问题，因为融合蛋白会堵塞分泌装置，因此不会被分泌。为了克服这些障碍，我们最近采用了一种使用遗传密码扩展（GCE）对细菌分泌的效应子以及其他难以标记的蛋白质进行位点特异性荧光标记的方法。本文提供了一个完整的分步方案，使用GCE位点特异性标记 沙门氏菌 分泌效应子，然后使用直接随机光学重建显微镜（dSTORM）对HeLa细胞中分泌蛋白的亚细胞定位进行成像 
最近的研究结果表明，通过GCE 掺 入非规范氨基酸（ncAAs），然后用含四嗪的染料进行生物正交标记，是一种可行的技术，用于选择性标记和可视化细菌分泌蛋白以及随后在宿主中进行图像分析。本文的目的是提供一个简单明了的方案，可供有兴趣使用GCE进行超分辨率成像的研究人员使用，以研究细菌和病毒中的各种生物过程，以及宿主 - 病原体相互作用。

该协议论文描述了一种基于GCE的方法，用于沙门氏菌分泌效应子的位点特异性荧光标记和可视化，如图1所示。含有ncAA的反式环辛烯生物正交基团（TCO）和荧光染料的化学结构如图1A所示。SseJ标记是通过GCE技术使用正交氨基酰基-tRNA合成酶/tRNA对在琥珀色终止密码子（见图1B）上遗传掺入生物?...

Watch this Scientific Journal Video about Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion at JoVE.com

Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

本文提供了一种简单明了的方案，使用遗传密码扩展 （GCE） 位点特异性标记 沙门氏菌 分泌效应子，并使用直接随机光学重建显微镜 （dSTORM） 对 HeLa 细胞中分泌蛋白的亚细胞定位进行成像

使用遗传密码扩增对细菌分泌蛋白进行超分辨率成像

Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host ...

遗传密码扩展（GCE）克服了其他标记策略的局限性。它使我们能够定位特异性标记的蛋白质，并且我们有明亮的有机荧光团可以使它们可视化，因此我们可以避免进行荧光蛋白融合的策略，这可能会破坏蛋白质功能。此方法可以应用于任何其他系统。例如，我们已经用它来标记寨卡一氧化碳蛋白，这使我们能够提供和生产标记的寨卡病毒，然后我们可以观察感染宿主细胞。首先，将100微升原代培养物转移到含有每毫升35微克氯霉素和每毫升100微克氨苄西林的5毫升LB培养基中。在37摄氏度下孵育，以250RPM振荡，直到600纳米处的光密度达到0.6。用补充有一毫摩尔TCO（一种非规范氨基酸）的改良N-最小培养基替换LB培养基。并将细菌在 34 摄氏度下培养 30 分钟。加入0.2%阿拉伯糖，每毫升25毫克氯霉素和每毫升氨苄西林100毫克，再培养细胞6小时，以250RPM振荡。六小时后，用不含非规范氨基酸或ncAA的新鲜MgM培养基在30分钟内洗涤细菌四次。离心细菌，重新悬浮在PBS缓冲液中，并在黑暗中以4摄氏度孵育一小时以去除多余的ncAA。一小时后，将细菌在4摄氏度下以3， 000克离心15分钟，并储存以备进一步使用。对于对照实验，在没有ncAA的情况下通过表达携带ncAA的蛋白质来重复相同的实验。在PBS中不存在或存在TCO的情况下，重新悬浮表达与TCO结合的SseJ的沙门氏菌细胞。将 600 纳米处的光密度调节至 PBS 中的 4，并在黑暗中用 20 微摩尔 Janelia Fluor 646 四烯或 20 微摩尔 BDPFL 四嗪在 37 摄氏度下孵育细胞，并以 250 RPM 摇动一至两个小时。沉淀细胞并用含有5%DMSO和0.2%Pluronic F-127的PBS洗涤三到四次。将沉淀重新悬浮在含有5%DMSO的PBS中。在黑暗中在四摄氏度下孵育过夜后，用PBS再次洗涤两次。立即使用共聚焦显微镜对细胞进行成像，或在室温下在黑暗中用PBS中的1.5%多聚甲醛固定30至45分钟。在高糖DMEM中培养并保持HeLa细胞在37摄氏度，5%二氧化碳和95%湿度下，除青霉素链霉素外还补充了10%FBS。在感染前一天培养细菌，并在含有正交tRNA合成酶对的pEVOL质粒和含有SseJ基因的pWSK29质粒中接种含有pEVOL质粒的野生型沙门氏菌的单个菌落，在含有标准LB肉汤的5毫米抗生素中，在37摄氏度下过夜，以250 RPM振荡。以每毫升 100， 000 个细胞修复稀释的细胞原液，并在含有 10%FBS 生长培养基的 500 微升 DMEM 中每孔接种 50， 000 个 HeLA 细胞。将腔室载玻片在培养箱中在 37 摄氏度、5% 二氧化碳和 95% 湿度下保持 24 小时。对于细菌感染，通过将100微升过夜细菌培养物稀释到含有35微克/毫升氯霉素和100微克/毫升氨苄西林的LB培养基中，将携带pEVOL质粒和含有SseJ基因的pWSK29质粒的野生型沙门氏菌传代培养。在 37 摄氏度下以 250 RPM 振荡孵育 5 到 7 小时。为了启动HeLa细胞感染，从培养箱中取出HeLa细胞后，用预热的DPBS洗涤细胞，并向每个孔中加入500微升含有10%FBS的新鲜DMEM。将腔室载玻片放回二氧化碳培养箱中，直到感染开始。孵育五至七个小时后，稀释沙门氏菌培养物，使600纳米处的光密度在一毫升DMEM生长培养基中为0.2，并向腔室载玻片的每个孔中加入必要量的沙门氏菌接种物，使感染的多重性为100。将感染的细胞在二氧化碳培养箱中孵育30分钟后，用预热的DPBS洗涤细胞三次以去除细胞外沙门氏菌。将此时间点设置为感染后零小时，并加入 500 微升每毫升庆大霉素含有 100 微克的完全培养基，持续 1 小时。孵育后，再次如前所示用DPBS洗涤细胞三次，并向腔室载玻片的每个孔中加入500微升补充有0.2%阿拉伯糖，每毫升庆大霉素10微克的完整培养基。在对照实验中，在没有TCO的情况下对HeLa细胞进行类似的感染。在二氧化碳培养箱中孵育室载玻片10小时后，用不含TCO的新鲜完全培养基替换完全培养基，并在30分钟的间隔内用预热的DPBS洗涤HeLa细胞四次。然后用不含TCO的新鲜完全培养基洗涤细胞。感染后12小时后，从细胞中吸出培养基，并用预热的DPBS洗涤细胞两次或三次。在一组孔中，加入500微升染料溶液混合物一个，在另一组孔的同一腔室载玻片中，加入500微升染料溶液混合物两个。将腔室载玻片放回二氧化碳培养箱中一小时和 1/2 至两个小时。感染后13.5至14小时后，用预热的DPBS冲洗HeLa细胞两次，并加入500微升补充FBS的新鲜DMEM。孵育30分钟后，如前所述，用预热的DPBS再次洗涤细胞，然后在30分钟的间隔内用新鲜DMEM洗涤四次。在感染后16小时，通过向每个孔中加入200微升4%多聚甲醛并在室温下在黑暗中孵育10分钟，用多聚甲醛固定HeLa细胞。吸去多聚甲醛。用PBS冲洗三次，并将细胞储存在PBS中，在黑暗中温度为四摄氏度。将细胞带到显微镜下，并将成像室放在样品架的显微镜载物台上。然后用0.4毫升新鲜制作的GLOX BME成像缓冲液更换孔中的培养基。将激光功率降低到大约一毫瓦以识别感兴趣的HeLa细胞并调整焦平面和激光束角度，同时用低647纳米激光密度照射样品。调整 HILO 照明角度。捕获目标结构的分数限制图像的参考，并通过将激光旋转到其最大功率将荧光团切换到暗状态。将前置放大器增益设置为3并激活帧传输。然后将 EM 增益设置为 200，以提高 dSTORM 测量的灵敏度。将激光强度调整到合适的水平，其中闪烁事件在空间和时间上分开。将曝光时间设置为30毫秒并开始采集。获取 10， 000 到 30， 000 帧。在不同的投资回报率下重复类似的采集。对于dSTORM的图像重建，请打开图像J并导入原始数据。打开雷暴插件，配置设备对应的相机设置。转到运行分析并设置适当的设置，例如图像过滤、定位方法和分子的亚像素定位。单击“确定”开始图像重建，开始后处理最终结果分析。表达与TCO结合的SseJ的沙门氏菌用珍妮莉亚福陆646四嗪处理，并在不存在或存在TCO的情况下用珍妮莉亚福陆646荧光成像。SseJ的荧光仅在存在TCO时检测。HeLa细胞感染含有psseJ-HA的沙门氏菌16小时，用抗HA抗体，LPS和DAPI固定和免疫染色。正如预期的那样，SseJ依赖性沙门氏菌诱导的细丝或SIF在感染细胞中形成。在没有TCO的情况下，琥珀色密码子不被抑制，并且在感染的HeLa细胞中不存在SseJ依赖性SIF。在存在TCO的情况下观察到SseJ依赖性SIF，这清楚地表明SseJ是通过使用遗传密码扩展或GCE表达SseJF10TCOHA来拯救的。此处显示了使用两种交替染料混合物一和染料混合物二通过SPIEDAC点击反应标记HeLa细胞中分泌的SseJF10TCOHA的特异性。点击染料Janelia Fluor 646 TZ进一步用于观察在TCO和Pvault质粒存在下感染携带SseJ HA的野生型Salmanella感染的HeLa细胞中是否存在任何背景标记。SseJ被释放到宿主细胞的细胞质中，没有细胞内或非特异性荧光信号，表明荧光信号对SseJ具有特异性。该图中显示了HeLa细胞中GCE标记的SseJ的超分辨率成像。此处显示了正在观察的HeLa细胞的明场图像。这里展示了用TCO和Janelia Fluor 646标记的分泌SseJ的衍射限制宽场图像以及SseJ装饰的SIF的相应dSTORM图像。插页显示了盒装区域中超分辨率 SseJ 的放大视图。NCAA储备溶液在NaOH中制备。不要忘记在添加到介质之前或之后中和它。标记感兴趣的蛋白质后，广泛洗涤细胞，使背景信号最小。按照我们的GCE程序，现在，我们可以标记细菌中的任何毒力因子，并使用我们的成像技术跟踪其活性。

Research

JoVE Journal

Biology

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

人类DNA修复蛋白的遗传研究利用酵母作为模型系统

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

科研

生物学

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays

测绘细菌功能的网络和途径<em&gt;大肠杆菌</em使用合成基因阵列

Phenotypes  are determined by a complex series of physical (e.g. protein-protein) and  functional (e.g. gene-gene or genetic) interactions (GI)1. While ...

Super-resolution Imaging of the Bacterial Division Machinery

超分辨率成像细菌科机械

Bacterial cell division requires the coordinated  assembly of more than ten essential proteins at midcell1,2. Central  to this process is the formation of ...

Test Samples for Optimizing STORM Super-Resolution Microscopy

试验样品为优化风暴超高分辨率显微镜

STORM is a recently developed super-resolution microscopy technique with up to 10 times better resolution than standard fluorescence microscopy ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

在Cytokineticž圈的活细菌超分辨率成像使用快速三维结构照明显微镜（F3D-SIM卡）

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Genetic Barcoding with Fluorescent Proteins for Multiplexed Applications

遗传条形码与荧光蛋白的复用的应用程序

Fluorescent proteins, fluorescent dyes and fluorophores in general have revolutionized the field of molecular cell biology. In particular, the discovery ...

DNA Electroporation, Isolation and Imaging of Myofibers

DNA电穿孔，肌纤维的分离和成像

Mature muscle has a unique structure that is amenable to live cell imaging. Herein, we describe the experimental protocol for expressing fluorescently ...

Open-source Single-particle Analysis for Super-resolution Microscopy with VirusMapper

与VirusMapper超分辨率显微镜开源单粒子分析

Super-resolution fluorescence microscopy is currently revolutionizing cell biology research. Its capacity to break the resolution limit of around 300 nm ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

哺乳动物细胞基态衰竭超分辨成像

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...