作者实验室的工作得到了德国福雄格梅因沙夫特（http://www.dfg.de） 的支持;CO 1139/1-1）、化学工业基金会（http://www.vci.de/fonds）和哥廷根分子生物学中心（GZMB）。作者要感谢约尔格·斯图尔克对手稿的有益评论和批判性解读。

1. 准备阿加板块、文化媒体、冷冻库和前文化
<ol><li>准备生长介质和所需的试剂（参见材料和试剂表）。</li>
	<li>条纹B.亚提利菌株（例如BP40 （罗克格+ 古德布CR amyE::P古德布- yfp）和 BP52 （罗克格+ 古德布+ 艾米: :P古德布- cfp）分别表示一个和两个活跃的 Gdhs）2，将用于 SP 中等 agar 板的竞争实验， 以获得单一菌落。在 37 °C 处连夜孵化板。</li>
	<li>以单个菌落和接种无菌培养管，含有4毫升LB液体介质。在 28 °C 和 220 rpm 的夜间生长细菌。</li>
	<li>在 28 °C 的夜间培养中生长，以避免细胞裂解或孢子。</li>
	<li>使用标准光谱仪测量波长为 600 nm （OD600）的培养物的光学密度。</li>
	<li>如果培养物已达到 OD600 的 2.0，将 0.75 毫升培养物与 1.5 毫升反应管中的无菌 50% 甘油溶液混合 0.75 毫升。最终的OD600 应该是1.0，以获得含有约108 个细胞/毫升的低温素。</li>
	<li>将管子存放在-80°C。</li>
	<li>对标有 cfp 和 yfp 编码氟化基因的每个菌株进行三种预培养。接种预培养（含有4毫升LB液体介质的无菌培养管）与1个μl细胞从-80°C低温库存。在 28 °C 和 220 rpm 的夜间孵化文化。</li>
</ol>2. 细菌共生、样品收集和样品储存
<ol><li>新鲜准备 100 毫升 C-Glc 和 CE-Glc 最小介质（参见试剂和材料表），并将每个介质的 20 毫升转移到无菌 100 毫升摇动烧瓶中。</li>
	<li>以一夜之间生长的 0.1 毫升预栽培为例，用 1.5 毫升的 1.5 毫升小面包中 0.9 毫升 LB 介质稀释它们，并确定 OD600。</li>
	<li>对于竞争实验，采取1.0-1.5 之间具有类似OD 600的不同菌株的预培养。</li>
	<li>为了获得混合细胞群，稀释具有适当OD600 的预培养细胞到20毫升C-Glc和CE-Glc中以100毫升摇瓶补充的0.05的OD600。 对于比赛实验，两个菌株应以1:1的比例混合。</li>
	<li>从烧瓶中取出 10 毫升样品，将其转移到 15 毫升塑料管中，在标准桌面离心机中通过离心机在 4，000 x g 中通过离心机采集细胞 10 分钟，然后丢弃超自然物。</li>
	<li>将细胞以0.5毫升新鲜的LB介质中补充，并将细胞转移到无菌的1.5毫升反应杯中。加入 0.5 毫升 50% 无菌甘油，通过严格的涡流混合悬浮，并将样品存放在 -80 °C 冰柜中，直到进一步治疗。</li>
	<li>在 37 °C 和 220 rpm 时，将留在摇动烧瓶中的细胞孵化长达 24 小时。将摇瓶放在黑暗中，以防止荧光片的照片漂白。</li>
	<li>在 7 小时和 24 小时的生长后，进一步采集 0.1 毫升的样本。测量并注意样品的1:10 稀释的 OD 600（C-Glc 或 CE-Glc 最小介质）。</li>
	<li>从每个培养物中取出适量的细胞，使低温素具有 OD600 的 1.0。将样品储存在-80°C，直到进一步治疗。</li>
</ol>3. 定量分析的样品处理、电镀和孵化
<ol><li>收集完所有样品后，解冻冷冻库，并在0.9%的盐溶液（见试剂和材料表）中稀释细胞，至多10-3。</li>
	<li>SP中等胶板上10-3 稀释的板0.1毫升，使用无菌玻璃移液器分配细胞。</li>
	<li>在黑暗中以 37 °C 的夜间孵化板，直到出现单个菌落。</li>
</ol>4. 通过立体荧光显微镜计算幸存者进行定量分析
<ol><li>用黑色笔将阿加板底部分成四部分，以便更好地定位，同时在立体荧光显微镜下计算幸存者。</li>
	<li>将板倒置在显微镜下，并使用冷光源将菌落聚焦。</li>
	<li>一旦菌落聚焦，切换到相应的过滤器集 CFP，以可视化 cfp标记菌株的幸存细胞。用笔给殖民地贴上标签并注明数字，来计算这种菌株的幸存者。</li>
	<li>用乙醇去除标签，切换到YFP过滤器集，以可视化 yfp标记菌株的幸存细胞。再次计算幸存者，用笔给殖民地贴上标签并注明数字。</li>
</ol>5. 半量子分析的样品处理和显微镜
<ol><li>对于说明性数字，在 SP agar 板上从步骤 3.1 中发现 10-4稀释（约 100 个细胞）中的 10 μl（见图 3）。</li>
	<li>在黑暗中以 37 °C 的夜间孵化板，直到出现单个菌落。</li>
	<li>将无盖的培养皿放在显微镜下，并使用冷光源将点聚焦。</li>
	<li>拍摄斑点的照片，以图示数字。选择适当的曝光时间，用冷光源拍摄殖民地的照片。</li>
	<li>无需移动板，请更改为 CFP 滤镜集，调整曝光时间并拍照。使用 YFP 过滤器设置进行相同的处理，并将图片保存以供进一步分析。</li>
</ol>6. 数据分析
<ol><li>使用 Excel 等软件进行定量数据分析。根据计算的殖民地，计算黄殖民地和蓝殖民地占整个殖民地数的百分比，该殖民地的百分比设定为100%。</li>
	<li>使用计算的数字创建堆叠条图（参见 图 4 和图 5）。使用 Adobe 照片商店等图像处理程序构建从不同地点拍摄的照片的合并图片。或者，可从 <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> 下载的免费软件 ImageJ 可用于图像处理。</li>
	<li>打开使用 CFP 过滤器集和 YFP 过滤器集拍摄的单个位置的图片。优化对比度和亮度，以减少来自媒体的任何背景荧光。</li>
	<li>转到其中一张图片并选择所有图片。复制图片并粘贴到另一张图片上。</li>
	<li>要么使用功能"颜色闪避"合并图片，要么使用通道选项卡覆盖荧光图片。来自不同媒体和不同时间点的殖民地合并图片代表了液体文化中不同菌株的生长。</li>
</ol>7. 具体提示:染料开关实验和同源菌株的培养，标有 cfp 和 yfp
B.亚提利中两个氟编码基因中任何一个的表达可能会影响细菌的生长速度。因此，建议进行以下实验，以排除在培养过程中从细胞群中消除一个竞争对手菌株只是由于氟磷的负面影响:
<ol><li>重复整个实验并共生反标记的菌株（例如，较早 的 cfp标记菌株现在标记为 yfp 基因， 反之亦然）。虽然相反，但获得的结果应与先前的观察结果相媲美，即一种菌株比另一株具有选择性生长优势。</li>
	<li>重复整个实验，并共同培育标有yfp和cfp的同源菌株 （例如BP40（rocG+ gudBCR amyE::P古德布- yfp）和 BP41 （罗克格+ 古德布CR 艾米: :P古德布- cfp））。通过跟踪细胞种群的组成，估计两种氟磷中的任何一种对细菌的适应力的负面影响。</li>
</ol>

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作者宣称他们没有相互竞争的经济利益。

已经开发了几种方法来分析细菌16的竞争性健康。在许多情况下，细菌被标记为不同的抗生素耐药性盒17。与我们的方法类似，用抗生素耐药盒标记细胞可以评估在规定的生长条件下共生过程中细菌的竞争性健康。此外，这种方法可用于确定17号染色体上特定轨迹中彼此不同的细胞的竞争性适应性。然而，使用抗生素耐药盒来监测竞技体能存在一些缺点。由于耐药基因的表达主要由强度不平等的促进者驱动，因此对抗生素产生抗药性的酶可能产生于不同的水平。因此，这种方法可能检测不到虚弱的健身效果。在我们的方法中，两个氟化基因都与相同的选择标记集成，它们的表达是由相同的促进者2驱动的。用抗生素耐药性盒式磁带监测竞争体能的另一个缺点可能是，这种方法更加费力，因为殖民地计数需要两种加板，辅以适当的抗生素。或者，细菌的健康状况可以通过简单地监测增长率和计算所谓的健身指数16来确定。显然，这是最精确的方法，因为细菌是单独培养的，而由具有某种基因型的菌株产生的有毒化合物不会影响竞争菌株的生长。此外，没有必要使用可能影响细胞生长的抗生素。然而，这两种方法都不是很说明问题，因为描述竞技健身的数字只能以相当中性的方式呈现。
使用氟酚编码基因来监测和量化物种内部竞争比其他方法具有若干优势。如果两个菌株通过双同源重组将氟磷编码基因整合到染色体中，则无需在任何培养步骤中使用抗生素。因此，在种植过程中从培养物中采集的样本可以在同一生长介质上进行分析，并且两种菌株都能够生长。这种方法允许以非常说明性的方式可视化物种内部竞争。此外，使用这种半量度方法可以同时测试几个生长条件，并且可以并行比较许多不同的菌株。最后，没有必要将样本固定在显微镜幻灯片上，因为从共生过程中从细菌培养物中采集的样本可以储存在18号冰柜中。因此，可以同时分析所有样品和复制品。
必须提及我们协议中的两个关键步骤。需要注意的是，在每个竞争对手菌株的相同生长阶段，低温物质应包含相同数量的细胞。在开始实验之前，低温库存中的菌株的初始不成比例，从而在摇动烧瓶中，将对竞争实验的结果产生重大影响。因此，在实验前检查冷冻机的组成是明智的。此外，适当的细胞量应均匀地在板上繁殖。否则，出现的殖民地彼此过于接近，对幸存细胞的精确确定将变得困难。
氟化物方法也有一些局限性和缺点。由于氟磷的激发和排放光谱彼此过于接近，因此不可能在多井板读卡器中进行共生，同时检测 CFP 和 YFP 信号。然而，这种技术问题可能使用不同的荧光蛋白来规避，例如那些以相同蛋白质脚手架为最佳基础的发光和红光。氟化物方法的另一个缺点可能是，一些突变只导致细菌的微弱生长缺陷。因此，如果氟化编码基因中任何一个可能引起的生长缺陷比某一突变引起的生长缺陷更强，则基于氟磷的方法不适合分析物种内部竞争。因此，在创建一整套菌株之前，建议首先将父菌株与cfp和yfp一起标记，并共同培育菌株。生长实验将揭示氟磷对细菌健康的影响程度（见图5A和5B）。
将来，如果使用流动细胞测量来计算存活细胞，那么测试基于氟的监测物种内部竞争的方法是否会更准确、更不费力将是很有趣的。最近，流细胞测量已被证明是一个强大的工具，分析 B.亚提利斯 生物膜19的组成。此外，通过在发酵器中连续培养荧光标记细菌来分析影响细菌竞争健身的弱健身效果可能更合适。与摇动烧瓶相比，这种方法能够保持生长条件不变，从而长期监测物种内部竞争。

土壤细菌通常具有灵活的调控网络和广泛的代谢能力。这两个特征使细胞能够调整其催化物和合成代谢途径，以与研究员和其他微生物竞争营养物质，这些营养物质在给定的生态利基1中可用。然而，如果细菌不能适应其环境，其他机制可能决定一个物种的生存。事实上，由于许多细菌迅速增殖，种群可以达到高细胞密度亚种群，可能自发地积累了有益的突变，为细胞提供了选择性的生长优势，从而增加了细胞的适应能力。此外，突变热点和压力引起的适应性突变可以促进一种不适应细菌2，3的进化。因此，在连续选择下突变的积累和生长是巨大的微生物多样性的起源，即使在同一属4，5。与自然界一样，由于在选择下不断培养，细菌基因组的形成也会在实验室中发生。Gram阳性细菌B.亚提利的驯化就是例证，该细菌在全世界用于基础研究和工业。在20世纪40年代，B.亚提利斯接受了DNA损伤X射线治疗，随后在特定的生长条件下进行了培养。细菌在驯化过程中积累的突变导致许多生长特征的丧失，即B.亚提利实验室菌株168丧失了形成复杂菌落的能力7，8。
如今，对于研究最好的大肠杆菌和B.亚提利菌模型，各种强大的工具可用于基因操纵其基因组，以解决特定的科学问题。有时，兴趣基因的失活会导致严重的生长缺陷，然后在标准生长介质9上清晰可见。相比之下，导致生长缺陷微弱，从而只轻微影响菌株适应的突变往往被忽视。然而，在这两种情况下，长期潜伏和传递的突变菌株几代人通常会导致抑制突变体的积累，已经恢复了表型的父菌株2，9。抑制突变体的特征和突变的识别，已经恢复了母突变株的生长缺陷是一个非常有用的方法，允许阐明重要和往往新颖的细胞过程10，11。
我们有兴趣控制谷氨酸平衡在B.亚提利斯12。与大肠杆菌类似，B. 亚提利通过抑制突变体的积累对谷氨酸平衡（即谷氨酸降解2中的块）的扰动做出反应。这些自发突变获得的抑制突变体的基因组变化被证明可以迅速恢复谷氨酸平衡9，13。因此，在细菌驯化过程中，B.亚提利适应特定生长状况，反映在酶合成和进化的酶活性中，这不足为奇，这些活动涉及谷氨酸代谢12。有人提出，在驯化过程中生长介质中缺乏外源性谷氨酸，是实验室菌株1682，14中神秘谷氨酸脱氢酶（GDH）gudB CR基因出现和固定的驱动力。这一假设得到我们观察的支持，即实验室菌株中GDH活性的减少为细菌提供了选择性生长优势，而外源性谷氨酸是稀缺的2。此外，在没有外源性谷氨酸的情况下，培育B.亚提利菌株，合成GDH GudB，导致抑制突变体的积累，这些突变体已经灭活了gadB基因2。显然，阴性活性GDH的存在对细胞不利，因为内源性产生的谷氨酸，否则可用于合成，会降解为铵和2-牛油酸盐（图1）。相比之下，当谷氨酸由介质提供时，配备高级 GDH 活性的B. 亚提利菌株比只合成一种功能性 GDH 的菌株具有选择性生长优势。有理由假设，高GDH活性允许细菌利用谷氨酸作为第二碳源，除了介质2提供的其他碳源（见图1）。因此，GDH 活性强烈地影响细菌的健身，具体取决于外源性谷氨酸的供应情况。
在这里，我们提出了一个非常说明性的方法来监测和可视化两个 B.亚蒂利斯 菌株之间的物种间竞争，在染色体上的单个轨迹不同（图2）。这两种菌株被标记为编码氟磷YFP和CFP的 yfp 和cfp的yfp和 cfp 基因，并在不同的营养条件下共同培养。通过随着时间的推移采样，并通过在阿加板块上电镀适当的稀释，每个培养物中的幸存者可以使用常见的立体荧光显微镜轻松监测。本文描述的程序是容易执行和适合可视化快速克隆扩张和消除有益和有害的突变，分别在细胞群随着时间的推移。

<table><tbody><tr><td>(NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>3746</td><td></td></tr><tr><td>Agar</td><td>Difco, USA</td><td>214010</td><td></td></tr><tr><td>Ammonium ferric citrate (CAF)</td><td>Sigma-Aldrich, Germany</td><td>9714</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&amp;nbsp;&lt;/sub&gt;</td><td>Roth, Germany</td><td>5239</td><td></td></tr><tr><td>Glucose</td><td>Applichem, Germany</td><td>A3617</td><td></td></tr><tr><td>Glycerol</td><td>Roth, Germany</td><td>4043</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Roth, Germany</td><td>6878</td><td></td></tr><tr><td>KCl</td><td>Applichem, Germany</td><td>A3582</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Roth, Germany</td><td>3904</td><td></td></tr><tr><td>KOH</td><td>Roth, Germany</td><td>6751</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Roth, Germany</td><td>P027</td><td></td></tr><tr><td>MnCl&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Roth, Germany</td><td>T881</td><td></td></tr><tr><td>MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Merck Millipore, Germany</td><td>102786</td><td></td></tr><tr><td>NaCl</td><td>Roth, Germany</td><td>9265</td><td></td></tr><tr><td>Nutrient broth</td><td>Roth, Germany</td><td>X929</td><td></td></tr><tr><td>Potassium glutamate</td><td>Applichem, Germany</td><td>A3712</td><td></td></tr><tr><td>Tryptone</td><td>Roth, Germany</td><td>8952</td><td></td></tr><tr><td>Tryptophan</td><td>Applichem, Germany</td><td>A3445</td><td></td></tr><tr><td>Yeast extract</td><td>Roth, Germany</td><td>2363</td><td></td></tr><tr><td>1.5 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,690,001</td><td></td></tr><tr><td>2.0 ml Reaction tubes</td><td>Sarstedt, Germany</td><td>72,691</td><td></td></tr><tr><td>15 ml Plastic tubes with screw cap</td><td>Sarstedt, Germany</td><td>62,554,001</td><td></td></tr><tr><td>Petri dishes</td><td>Sarstedt, Germany</td><td>82.1473</td><td></td></tr><tr><td>1.5 ml Polystyrene cuvettes</td><td>Sarstedt, Germany</td><td>67,742</td><td></td></tr><tr><td>15 ml Glass culture tubes&amp;nbsp;</td><td>Brand, Germany</td><td>7790 22</td><td></td></tr><tr><td>100 ml Shake flasks with aluminium caps</td><td>Brand, Germany</td><td>928 24</td><td></td></tr><tr><td>Sterile 10 ml glass pipettes</td><td>Brand, Germany</td><td>278 23</td><td></td></tr><tr><td>Incubator (28 and 37 &amp;deg;C)</td><td>New Brunswick</td><td>M1282-0012</td><td></td></tr><tr><td>Standard pipette set (2-20 &amp;mu;l, 10-100 &amp;mu;l, 100-1,000 &amp;mu;l)</td><td>Eppendorf, Germany</td><td>4910 000.034, 4910 000.042,</td><td></td></tr><tr><td>Table top centrifuge for 1.5 and 2 ml reaction tubes</td><td>Thermo Scientific, Heraeus Fresco 21, Germany</td><td>75002425</td><td></td></tr><tr><td>Table top centrifuge for 15 ml plastic tubes</td><td>Heraeus Biofuge Primo R, Germany</td><td>75005440</td><td></td></tr><tr><td>Standard spectrophotometer</td><td>Amersham Biosciences Ultrospec 2100 pro, Germany</td><td>80-2112-21</td><td></td></tr><tr><td>Stereofluorescence microscope&amp;nbsp;</td><td>Zeiss SteREO Lumar V12, Germany</td><td>495008-0009-000</td><td></td></tr><tr><td>Freezer (-20 and -80 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Fridge (4 &amp;deg;C)</td><td>-</td><td>-</td><td></td></tr><tr><td>Autoclave</td><td>Zirbus, LTA 2x3x4, Germany</td><td>-</td><td></td></tr><tr><td>pH meter</td><td>pH-meter 766, Calimatic, Knick, Germany</td><td>766</td><td></td></tr><tr><td>Vortex</td><td>Vortex 3, IKA, Germany</td><td>3340000</td><td></td></tr><tr><td>Balance</td><td>CP2202S, Sartorius, Germany</td><td>replaced by</td><td></td></tr><tr><td>Black pen (permanent marker)</td><td>Staedler, Germany</td><td>317-9</td><td></td></tr><tr><td>Powerpoint program</td><td>Microsoft, USA</td><td>-</td><td></td></tr><tr><td>Office Excel program</td><td>Microsoft, USA</td><td>-</td><td>Program for data processing</td></tr><tr><td>Adobe Photoshop CS5</td><td>Adobe, USA</td><td>replaced by CS6, download</td><td>Computer program for image processing</td></tr><tr><td>Computer</td><td>PC or Mac</td><td>-</td><td></td></tr><tr><td>ZEN pro 2011 software for the stereofluorescence microscope</td><td>Zeiss, Germany</td><td>410135 1002 110</td><td>AxioCam MRc Rev. Obtained through Zeiss</td></tr><tr><td colspan="4">&lt;strong&gt;Specific solution recipes&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;SP Medium&lt;/strong&gt;</td></tr><tr><td>8 g Nutrient broth</td><td></td><td></td><td></td></tr><tr><td>0.25 mg MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>1 g KCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid SP medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>1 ml CaCl&lt;sub&gt;2&lt;/sub&gt; (0.5 M), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>1 ml MnCl&lt;sub&gt;2&lt;/sub&gt; (10 mM), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>2 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;LB Medium&lt;/strong&gt;</td></tr><tr><td>10 g Tryptone</td><td></td><td></td><td></td></tr><tr><td>5 g Yeast extract</td><td></td><td></td><td></td></tr><tr><td>10 g NaCl</td><td></td><td></td><td></td></tr><tr><td>15 g agar for solid LB medium</td><td></td><td></td><td>if required</td></tr><tr><td>add 1 L with H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;C-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%)</td><td></td><td></td><td>autoclaved for 20 min at 121 &amp;deg;C</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td colspan="4">&lt;strong&gt;CE-Glc Minimal Medium&lt;/strong&gt;</td></tr><tr><td>200 ml 5 x C salts</td><td></td><td></td><td></td></tr><tr><td>10 ml L-Tryptophan (5 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml ammonium ferric citrate (CAF, 2.2 mg/ml), sterilized by filtration</td><td></td><td></td><td></td></tr><tr><td>10 ml III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>20 ml Glutamate (40%)</td><td></td><td></td><td></td></tr><tr><td>25 ml Glucose (20%), autoclaved for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>5 x C salts&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>20 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>80 g K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;3H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>16.5 g (NH&lt;sub&gt;4&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>III&amp;rsquo; salts</td><td></td><td></td><td></td></tr><tr><td>0.232 g MnSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;4H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>12.3 g MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;7H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td></td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O, autoclave for 20 min at 121 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>40% Glutamate solution</td><td></td><td></td><td></td></tr><tr><td>200 g L-Glutamic acid</td><td></td><td></td><td></td></tr><tr><td>adjust the pH to 7.0 by adding approximately 80 g KOH</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">&lt;strong&gt;0.9% Saline (NaCl) Solution&lt;/strong&gt;</td></tr><tr><td>add 1 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td>50% Glycerol solution</td><td></td><td></td><td></td></tr><tr><td>295 ml Glycerol (87%)</td><td></td><td></td><td></td></tr><tr><td>add 0.5 L with sterile H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>autoclave for 20 min at 121 &amp;deg;C</td></tr><tr><td colspan="4">Bacteria (All strains are based on the &lt;em&gt;Bacillus subtilis&lt;/em&gt; strain 168)</td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP40 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-yfp)&amp;nbsp;</td><td>Laboratory strain collection</td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP41 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;CR&lt;/sup&gt; amyE::PgudB-cfp)&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP52 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-cfp)</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;Bacillus subtilis&lt;/em&gt; BP156 (rocG&lt;sup&gt;+&lt;/sup&gt; gudB&lt;sup&gt;+&lt;/sup&gt; amyE::PgudB-yfp)</td><td></td><td></td><td></td></tr></tbody></table>

monitoring intraspecies competition in a bacterial cell population by cocultivation of fluorescently labelled strains

许多微生物，如细菌的增殖速度极快，种群可能达到高细胞密度。种群中很小一部分细胞总是积累的突变，这些突变对细胞有害或有益。如果突变的健身效应为亚人口提供了强大的选择性生长优势，这种亚种群的个体可能会迅速竞争，甚至完全消除其直接研究员。因此，小的基因变化和获得有益突变的细胞的选择驱动积累可能导致细胞种群的基因型完全改变。在这里，我们提出了一个程序，监测快速克隆扩张和消除有益和有害的突变，分别在细菌细胞群随着时间的推移，通过共生荧光标记个体的Gram阳性模型杆菌 亚蒂利斯。该方法易于执行，非常能说明细菌细胞群中个体之间的物种间竞争。

此处描述的方法成功地应用于可视化由 B. 亚蒂利斯 菌株组成的细胞群中的物种内部竞争，这些菌株分别标有编码荧光CFP和YFP的 cfp 和 yfp 基因。如 图3所示，该方法可用于以非常说明性的方式可视化物种内部竞争。通过在小区域发现样本，细胞群的克隆组成一目了然。虽然不适合定量分析，但这种方法有助于粗略估计不同生长参数（即氮源）对细胞群发育的影响，该细胞群最初包含两种菌株的量相等（图3）。此外，在小规模的方法中，可以使用单一的 agar 板测试在相同生长条件下培养的不同 B. 亚提利 菌株的适应性。对于定量分析，建议将样品传播到阿加板块的整个表面。这将防止殖民地的叠加，从而允许从单个细胞中出现的殖民地的明确识别和计数。通过在阿加板块上电镀适当的稀释，细胞种群的克隆组成可以精确地确定，只需计算黄色和蓝色荧光菌落（见 图4）。正如我们之前报道的，GDH活动强烈地影响 B.亚提利的 健身，这取决于外源性谷氨酸2的可用性。显然，在没有外源性谷氨酸高水平GDH活性的情况下，对细菌不利，因为酶RocG和GudB会降解合成代谢所需的谷氨酸（见 图1 和 图4A）。相比之下，如果提供给细菌，谷氨酸可以作为氨基组捐赠者在转氨反应。此外，谷氨酸可以输入碳代谢，并用作能量来源，由于存在催化活性GDH罗克和古德布（图1 和 图4B）。如 图4C 和 4D所示，染料开关实验也取得了类似的结果。同样，具有高GDH活性活性细菌的细胞在缺乏谷氨酸的生长介质中GDH活性降低，从而受到其竞争。相比之下，当介质补充谷氨酸时，仅从培养物中消除了只合成一种活性 GDH 的细菌。如 图5A 和5B所示，混合细胞种群的初始组成随着时间的流动几乎保持不变。因此，在竞赛实验中，消除两种含有不同量GDH活性菌株中的任何一种并不是由于氟磷引起的生长缺陷（见 图4）。 综合起来，氟磷的使用是监测细菌细胞群物种内部竞争的有力工具。
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51196/51196fig1highres.jpg" src="/files/ftp_upload/51196/51196fig1.jpg"> 图1。 碳和氮代谢在 B.亚提利物之间的联系。 当谷氨酸不是由介质提供时，合成所需的主要氨基供体通过谷氨酰胺合成酶 （GS） 和谷氨酸合成酶 （GOGAT） 的联合作用从铵和 2 氧化氧乙酸中合成。相比之下，在外源性谷氨酸的存在下，具有催化活性的GDH RocG和/或GudB可以将谷氨酸降解为铵和2-牛油酸盐，然后作为碳源。
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51196/51196fig2highres.jpg" src="/files/ftp_upload/51196/51196fig2.jpg"> 图2。实验工作流程。应变 1 （标记为yfp）和应变 2 （标记为cfp）在一个轨迹上彼此不同。在此处介绍的示例中，我们比较了外源性谷氨酸（效应）对细胞群的基因型转移的影响，该细胞群最初包含 50% 的rocG+ gudB+ （编码两个活性 GDH） 和 50% 的rocG+ gudBCR（编码一个活性 GDH） 细胞。<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51196/51196fig2highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 3" fo:content-width="5in" fo:src="/files/ftp_upload/51196/51196fig3highres.jpg" src="/files/ftp_upload/51196/51196fig3.jpg"> 图3。 以描述性的方式可视化物种内部竞争的半量化方法（见第 5 节）。 在共生（0小时）之前，在SP agar板上发现7小时和24小时生长稀释（10-4小时）细胞。在37°C的12小时孵化后形成菌落的存活细胞通过立体荧光显微镜鉴定。曝光时间，0.6秒:比例杆，1毫米。这个数字是由贡卡 等人修改的。20132.
<img alt="Figure 4" fo:content-width="5in" fo:src="/files/ftp_upload/51196/51196fig4highres.jpg" src="/files/ftp_upload/51196/51196fig4.jpg"> 图4。 物种内部竞争的量化。 在样品稀释和在SP介质上繁殖细胞（见步骤3.1-3.3）后，板在37°C处在一夜之间孵育。 黄色和蓝色殖民地的量化，如第4号和第6号议定书所述。黑色错误条表示至少四个独立重复实验的标准偏差。每个阿加板块包含至少100个可计数的殖民地。（A） 在没有外源性谷氨酸的情况下 ，B. 亚提斯 菌株 BP40（黄色）仅配备一个功能 GDH，可与 BP54（蓝色）菌株竞争，该菌株合成了谷氨酸降解酶 RocG 和 GudB。（B） 相比之下，当有外源性谷氨酸时，合成两种功能性GDH对细菌有利，因为除了葡萄糖外，谷氨酸还被用作碳源。如（C）和（D）所示，染料开关实验取得了类似的结果。这个数字是由贡卡 等人修改的。20132. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51196/51196fig4highres.jpg" target="_blank">单击此处查看更大的图像。</a>
<img alt="Figure 5" fo:content-width="5in" fo:src="/files/ftp_upload/51196/51196fig5highres.jpg" src="/files/ftp_upload/51196/51196fig5.jpg"> 图5。控制实验，以评估氟磷编码cfp和yfp基因对细菌的健身的影响。同源菌株 BP40 （罗克格+ 古德布Cr amye: yfp）和 BP41 （罗克格+ 古德布Cr 艾米: : cfp）或 BP52 （罗克格+古德布+艾米: cfp） 和 Bp156 （罗克格+古德布+艾米: yfp）是在外源性谷氨酸缺乏的情况下生长的 。幸存的细胞分别被计算在《议定书》1-4和6中。这些条形表示至少四个独立重复实验的标准偏差。这个数字是由贡卡等人修改的。20132.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51196/51196fig5highres.jpg" target="_blank">单击此处查看更大的图像。</a>

Watch this Scientific Journal Video about Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains at JoVE.com

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

细菌在有生之年可能会积累有害或有益的突变。在积累有益突变的细胞群中，个体可能迅速比其研究员竞争。在这里，我们介绍了一个简单的程序，以可视化物种内部竞争的细菌细胞种群随着时间的推移使用荧光标记的个人。

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

monitoring-intraspecies-competition-bacterial-cell-population

Nanyang Technological University

Research

JoVE Journal

Biology

8.5K 次观看.  Georg-August University. 细菌在有生之年可能会积累有害或有益的突变。在积累有益突变的细胞群中，个体可能迅速比其研究员竞争。在这里，我们介绍了一个简单的程序，以可视化物种内部竞争的细菌细胞种群随着时间的推移使用荧光标记的个人。

视频: Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Monitoring Spatial Segregation in Surface Colonizing Microbial Populations

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple genetic modification procedures of microbes facilitate the development of the sociomicrobiology field. To assess the fitness of certain microbial species, selected strains or their genetically modified derivatives within one population, can be fluorescently labelled and tracked using microscopy adapted with appropriate fluorescence filters. Expanding colonies of diverse microbial species on agar media can be used to monitor the spatial distribution of cells producing distinctive fluorescent proteins.
Here, we present a detailed protocol for the use of green- and red-fluorescent protein producing bacterial strains to follow spatial arrangement during surface colonization, including flagellum-driven community movement (swarming), exopolysaccharide- and hydrophobin-dependent growth mediated spreading (sliding), and complex colony biofilm formation. Non-domesticated isolates of the Gram-positive bacterium, Bacillus subtilis can be utilized to scrutinize certain surface spreading traits and their effect on two-dimensional distribution on the agar-solidified medium. By altering the number of cells used to initiate colony biofilms, the assortment levels can be varied on a continuous scale. Time-lapse fluorescent microscopy can be used to witness the interaction between different phenotypes and genotypes at a certain assortment level and to determine the relative success of either.

The role of assortment (spatial segregation) in evolutionary scenarios can be examined using simple microbial systems in the laboratory that allow controlled adjustment of spatial distribution. By modifying the founder cell density, various assortment levels can be visualized using fluorescently labelled bacterial strains in the colony biofilms of Bacillus subtilis.

在表面定植微生物种群监测空间分异

Microbes provide an intriguing system to study social interaction among individuals within a population. The short generation times and relatively simple ...

科研

遗传学

Title Cell Encapsulation by Droplets

My name is Hang Moon. I'm working with the professor of condoms. He and me work with the XYJ stage to make a 3D structure of cell.Hi, I am PE and Lynn and I'm working with the cell printing project with cell encapsulation and media and gels. This is solenoid be for ejection of a cell and cell mixture of aros over cell media. The so valve is activated by two signal line.The signal line is came from a dry board here. Signal came from here. That line through the that line, that one is the function first generator.So first is generated by our programmed way. So if you want, you want to change the purse, you can change the program with the that switches line or you can control that process generator to computer. The cellular valve is open and closed by the electric signal, but the, the cell is supplied through that line.The cell and the media or agro gel is supplied through the syringe syringes. The syringes inside of the water, inside of syringe is the press light by the air. The pressurized air is supplied from a tank like here the line.So the little line and the ized air is fly through the air filter. The air filter is discriminate the dust and other kind of the dirt. That is pressure generator we call it, is generating the signal used for open the solar air bell or closed the solar air valve computer is the connected through P-I-B-U-S-V connector.So we can use the USB port by directly our computer, the laptop computer. So that one is connected to that side here. And then we can control through the control laptop here.So if we want to synchronize of the movement of the stage and opening the bell is can be controlled by the same controller as a laptop. So we can control the timing when it open, when it close, and when it moves and when it stops. And we can synchronize the motion and opening bell.That stage is the motorized stage. It is automatically automatically moved by through the death signal line and motor. So motorization is 0.1 micrometers.So it can ideally it can draw the line up to 100 millimeter line. So jet axis is moving, going up and down like that way. So it control the cap between the substrate and the solar belt.So it can control the height of ejection. That substrate is the connected to the XY stages here, X here and Y there. So it's moving around a plan XY plan.So it draws some feature demo right stages is three Xs, X, Y, and G.So the jet con jet axis or XY axis is controlled by signal here. So signal line is connected to the motion controller here. That motion controller doesn't have any manual switch at all because it cannot be controlled manually.It's controlled through the laptop computer. So laptop computer is connected. The backside of the controller, we have three xs for a printing cell XY jet.So it make three different kind of signal independently. And then motion controller is communicate through the rf 2, 3, 2 connectors is directly connected to the USV. And then USV is connected to laptop computer.So laptop controlled borrower. We use the AutoCAD program, we make a gen A path like that way bam. And then it change to the machine language like that.So machine language is transferred to the like that the simple character and numbers like that. There is each position related to the X, Y, Z stages here I choose the file, contain that kind of program and then open downloading program like that way. And then I choose from file like does exact the same, the text file.Just the text file is downloaded, choose text file and automatically download from laptop to com motion controller executive and then it going the BAM character as I mentioned before, BAM. In this flask are N IH 3G three mouse fiberblast cells. And I'm going to first aspirate the old media.This process is to passage the cells and the cells are adhered to the bottom of the flask. So after aspirating the media, I'm going to put in six RYS in which is an enzyme.Okay? So after the trypsin has been put into the flask, we need to wait three to five minutes for the cells to become detached from the bottom of the flask.Now we're ready to put in the media and passage the cells into a centrifuge tube. It's a one-to-one dilution. So I'm putting in six mil of this media right here and I'm gonna wash out the saws a little bit to make sure all of them are passaged.And now the cells are ready to be centrifuge in this tube. Now we're going to centrifuge these cells at a thousand RPM three to five minutes. Now that the cells have been spun down into a pellet, I'm going to aspirate the supernatant.Now I'm going to wash out the cells with PBS. And this is to, this is for staining the cells. The stains that we are using are esterase substrates.Okay, now we we're going to centrifuge again and spin down the cells into a palate. And I'm going to aspirate the PBS and I'm going to resin the cells. I'm in media to be counted.I'm mixing the cells so that when we take the a sample of it, the concentration will be even throughout the tube. Now I'm going to take a hundred microliters of the cells to measure the cell density. Now I'm putting in a hundred microliters of type and blue, which is a stain.We're going to wait three to five minutes in order for the stain to permeate dead cell membranes. When we count the cells, the blue ones we don't count. The cells are ready to be counted with the hemo cytometer, I'm going to transfer 50 microliters of the cell mixture to each side of the hemo cytometer.Okay, I'm going to cover it with a glass slide side. There's two sides to the hemo cytometer. I'm gonna count the first side and after counting both sides, I'm going to take the average and then multiply by two because we had a one-to-one dilution with the type in blue.So basically our cell density will be 135 times 10 to the four cells per mil. For our experiment, we will be using a cell solution with a concentration of half a million cells per mil. And so since we have 13.5 million cells, I will be resus suspending the cells in 27 mil of media.We are using two different types of stains. This is a live dead assay from molecular probes. Our first stain is called calcium am.Our second stain is called AUM homodimer. It stains dead cells. We use blue excitation to get green fluorescence and green excitation to get red fluorescence For these two stains, when we're imaging our dice solution will be prepared in PBS.So first I'm going to transfer five mil of PBS into a tube phosphate buffered saline. That's a saline solution. Now I'm going to add half a microliter of calcium am per mil.Now I'm adding 2.5 microliters of calcium am to the tube and two microliters of athe homodimer per mil of PBS. Here is the btex machine. It makes the cell and media well distributed through the whole volume.Now we load the cell 200 microliter onto the seven here. And then we put on the cap, this is a tight tightly sealed through like that way here I open the valve and then it's the pressure light air comes in and then I adjust the belt like that. And then it change the pressure.So I adjust. The pressure is five PSI. The inside of tubing pressure is five PSI.From here to here, it connected through the this inside the hole. So just we open the bell here and then the cell injected with the five PSI pressure and then starting open. And then Sona valve starting operate.And then we roll the substrate. It substrate is just a simple slide glass. And then we load the proper portion and I put some sketch tape to fix the substrate like that, right?And simply I just fix it. So we are setting whole thing the cell and ary valve and the substrate during the moving the ary working, start working. So we are set up whole thing and then open ary valve start.And then we move the stage like that, right? And then we make some pattern onto the substrate pattern onto the substrate and bam character. I'm going to immerse these droplets that we printed in this dye solution that we made earlier.And now the droplets will be incubated for 10 minutes and then imaged. First I'm going to adjust the image through the eyepiece. Our first image is just a bright field image.I'm gonna turn off the light and change the filter so that we have blue excitation, which will show green fluorescence. And the third image we'll be taking is the dead cells, which is the Thm Homodimer stain. And with green light, we will get red fluorescence.

通过飞沫标题细胞封装

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

的视觉检测，以监测T6SS介导的细菌竞争

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

免疫与感染

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, differentiation, and apoptosis1. During apoptosis, calcium accumulation in mitochondria promotes the release of pro-apoptotic factors from the mitochondria into the cytosol2. It is therefore of interest to directly measure mitochondrial calcium in living cells in situ during apoptosis. High-resolution fluorescent imaging of cells loaded with dual-excitation ratiometric and non-ratiometric synthetic calcium indicator dyes has been proven to be a reliable and versatile tool to study various aspects of intracellular calcium signaling. Measuring cytosolic calcium fluxes using these techniques is relatively straightforward. However, measuring intramitochondrial calcium levels in intact cells using synthetic calcium indicators such as rhod-2 and rhod-FF is more challenging. Synthetic indicators targeted to mitochondria have blunted responses to repetitive increases in mitochondrial calcium, and disrupt mitochondrial morphology3. Additionally, synthetic indicators tend to leak out of mitochondria over several hours which makes them unsuitable for long-term experiments. Thus, genetically encoded calcium indicators based upon green fluorescent protein (GFP)4 or aequorin5 targeted to mitochondria have greatly facilitated measurement of mitochondrial calcium dynamics. Here, we describe a simple method for real-time measurement of mitochondrial calcium fluxes in response to different stimuli. The method is based on fluorescence microscopy of 'ratiometric-pericam' which is selectively targeted to mitochondria. Ratiometric pericam is a calcium indicator based on a fusion of circularly permuted yellow fluorescent protein and calmodulin4. Binding of calcium to ratiometric pericam causes a shift of its excitation peak from 415 nm to 494 nm, while the emission spectrum, which peaks around 515 nm, remains unchanged. Ratiometric pericam binds a single calcium ion with a dissociation constant in vitro of ~1.7 &mu;M4. These properties of ratiometric pericam allow the quantification of rapid and long-term changes in mitochondrial calcium concentration. Furthermore, we describe adaptation of this methodology to a standard wide-field calcium imaging microscope with commonly available filter sets. Using two distinct agonists, the purinergic agonist ATP and apoptosis-inducing drug staurosporine, we demonstrate that this method is appropriate for monitoring changes in mitochondrial calcium concentration with a temporal resolution of seconds to hours. Furthermore, we also demonstrate that ratiometric pericam is also useful for measuring mitochondrial fission/fragmentation during apoptosis. Thus, ratiometric pericam is particularly well suited for continuous long-term measurement of mitochondrial calcium dynamics during apoptosis.

This protocol describes a method for real-time measurement of mitochondrial calcium fluxes by fluorescent imaging. The method takes advantage of a circularly permutated YFP-based dual-excitation ratiometric calcium sensor (ratiometric pericam-mt) selectively expressed in mitochondria.

使用一种基因编码的钙传感器来监测细胞凋亡过程中线粒体钙水平的动态变化

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. This method employs stable plasmids that allow each population to be differentially tagged with distinct antibiotic resistance capabilities and fluorescent proteins for selection and visual discrimination of each population, respectively. Here, we describe the preparation and coincubation of competing Vibrio fischeri strains, fluorescence microscopy imaging, and quantitative data analysis. This approach is simple, yields quick results, and can be used to determine whether one population kills or inhibits the growth of another population, and whether competition is mediated through a diffusible molecule or requires direct cell-cell contact. Because each bacterial population expresses a different fluorescent protein, the assay permits the spatial discrimination of competing populations within a mixed colony. Although the described methods are performed with the symbiotic bacterium V. fischeri using conditions optimized for this species, the protocol can be adapted for most culturable bacterial isolates.

Coincubation Assay for Quantifying Competitive Interactions between Vibrio fischeri Isolates

Bacteria encode diverse mechanisms for engaging in interbacterial competition. Here, we present a culture-based protocol for characterizing competitive interactions between bacterial isolates and how they impact the spatial structure of a mixed population.

用于量化Vibrio Fischeri分离物之间竞争交互的联合孵化测定

This manuscript describes a culture-based, coincubation assay for detecting and characterizing competitive interactions between two bacterial populations. ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

单细胞物种间细菌相互作用的动力学可视化

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

采用共培养，以检测化学介导的种间相互作用

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can be used to visualize and quantify competitive interactions between different bacterial strains at the single-cell level. The protocol described here provides methods for advanced approaches in slide preparation on both upright and inverted epifluorescence microscopes, live-cell and time-lapse imaging techniques, and quantitative image analysis using the open-source software FIJI. The approach in this manuscript outlines the quantification of competitive interactions between symbiotic Vibrio fischeri populations by measuring the change in area over time for two coincubated strains that are expressing different fluorescent proteins from stable plasmids. Alternative methods are described for optimizing this protocol in bacterial model systems that require different growth conditions. Although the assay described here uses conditions optimized for V. fischeri, this approach is highly reproducible and can easily be adapted to study competition among culturable isolates from diverse microbiomes.

This manuscript describes a method for using single-cell fluorescence microscopy to visualize and quantify bacterial competition in coculture.

使用单细胞荧光成像对菌际竞争进行量化

Interbacterial competition can directly impact the structure and function of microbiomes. This work describes a fluorescence microscopy approach that can ...

Bacterial Co-Incubation Assay: A Fluorescence Microscopy-Based Technique to Visualize Intraspecific Bacterial Competition at the Single-Cell Level

Source: Smith, S., et al., <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/62851">Quantification of Interbacterial Competition using Single-Cell Fluorescence Imaging.</a> J. Vis. Exp. (2021).
This video demonstrates a fluorescence microscopy-based method to study bacterial competition at the single-cell level. This method provides insights into the structure and function of bacterial communities.