作者感谢他们的同事和那些支持这项技术的发展，现在和过去的实验室工作人员。 SP的实验室支持由加拿大卫生研究院工作格兰特MOP-84473和艾伯塔创新 - 健康解决方案（由阿尔伯塔遗产基金会的医学研究基金资助）。 DP的被授予MD001091-01和GM068855-02支持由美国国立卫生研究院。

1。细菌培养，工具和试剂的制备（第1天，下午） <ol><li>在下午晚些时候，接种1-5毫升灭菌LB培养基在无菌（例如，100 x 13毫米），高硼硅玻璃试管用小等分细菌（ 大肠杆菌和霍乱弧菌 ）的。 </li><li>将接种试管在辊筒被安置在37°C的温度（温暖的房间或孵化器），打开辊筒高速，孵化的O / N。 </li><li>制备细胞吊具在从玻璃棒加热棒的中间在本生灯的火焰“曲棍球棒”的形状，直至玻璃软化，然后直接用镊子温和或钳子弯曲成135°角。加热棒再次在端部和第一弯曲部之间的中间点，并在向内成45°角（在第一弯曲部的相反方向）弯曲。 </li><li>准备LB琼脂无抗生素，倒入培养皿中制备的含有适当抗生素（根据上被电穿孔的向量中的抗性标记），并储存于4℃。electrocompetence协议，和LB-琼脂平板上</li><li>准备消毒2毫米氯化钙为V。2溶液的过滤器霍乱弧菌 ，或高压灭菌双蒸2 O为E。大肠杆菌 ，并储存于4℃。 </li></ol> 2。电感受态细菌的生长（第2天，上午） <ol><li>递送100微升的O / N的细菌培养物到每个LB琼脂平板上，冷冻甘油原种也可使用，并直接传递到印版上。 </li><li>浸曲棍球棒形状的细胞吊具在100％乙醇，简要地漏和燃烧掉剩余的乙醇消毒它在使用前和蔓延。均匀分布的100微升O / N细菌培养用无菌玻璃细胞吊具确保不破坏（断裂）的琼脂表面。 </li><li>将培养板在37℃下进行4-6小时，或直到薄细菌生长的草坪是来区分。细胞是最有能力的时候积极增长。 </li></ol> 3。电感受态细菌细胞的制备（第2天，下午） <ol><li>收获细菌用无菌接种环，注意不要刺破或破坏琼脂的表面。一个直径为2毫米的细菌质量是足够的单个转换。通常，这需要刮薄草坪的表面用接种环2或3次。一些样品（典型地为4-6）可从相同的板收集。 </li><li>悬浮细菌量1毫升冰冷的2毫米氯化钙2（ 霍乱弧菌 ）或无菌DDH 2 O（ 大肠杆菌 ），搅拌均匀，直到没有团块可见，保持在冰上。 </li><li>离心机各细菌悬浮液5分钟，在5000×g离心在冷冻离心设定为4℃，或在存储在冷室中设定为4℃的离心</li><li>弃去上清液，resusp最终将细菌沉淀在冰冷的2毫米氯化钙2或无菌DDH 2 O相同的体积，并重复离心步骤为已完成之前两次，总共3次洗涤。 </li><li>弃上清，重悬，然后在40微升冰冷的彻底放松细菌沉淀2毫米氯化钙2或无菌DDH 2 O和保持在冰上。 </li></ol> 4。电感受态细菌转化（第2天，下午） <ol><li>高达1微克质粒DNA（在最多1μl水或的Tris-EDTA缓冲液）添加到40μl的细菌悬浮液，并将该混合物转移到预先冷却的，无菌的0.2cm间隙杯中。该DNA样品中的盐浓度要低一个 ，因为它会在接下来的步骤向脉冲电弧。 </li><li>插入反应杯插入脉冲控制模块的电穿孔室中，并电穿孔在1.8千伏，25μF。时间常数应为〜5.0毫秒，并且没有产生电弧应该发生。 </li><li>很快就被重新悬浮在1ml LB培养基恢复细胞悬液，转入以前蒸压硼硅玻璃试管中。 </li><li>允许细胞在37℃下在充气的生长条件孵育（在辊筒）30分钟无抗生素选择来恢复。 </li><li>铠细菌到预先制备的LB琼脂平板上在适当的选择剂（抗生素）的存在下，孵育在37℃的CO / N。如果细菌的转化有高浓度的纯化的载体（0.1-1微克超螺旋质粒）的，传送10微升细菌培养到LB琼脂平板上的边缘和用无菌接种环连胜使用象限条纹法分离菌落。如果一个连接混合物转化，提供100μl的细菌培养物到每个琼脂平板上，均匀地用无菌玻璃细胞吊具传播它。 </li></ol> 一个如果时间常数是短的（是低3.5毫秒），或脉冲导致​​电弧放电，然后再沉淀的DNA样品，用70％乙醇洗两次，干燥前除去盐类和重新悬浮于TE缓冲液中。

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没有披露适用。

转化效率受多种因素考虑用来制备感受态细胞的方法。类似的变量应用于此方法;大，必须小心，一旦细胞从琼脂平板上，使得它们保持在恒定的低温（不高于4℃）收获。细菌草坪必须积极成长在领取时，细胞必须处于生长对数中期阶段。 DNA（ 即盐的污染）的质量会影响转化效率。具体地，使用该方法时，在琼脂不应该被打破如在比色杯中的琼脂片段将导致脉冲的电弧。 用这种方法遇到的大多数问题都与两个问题有关;从琼脂平板上的时间适当的窗口过程中收集的细菌时，他们正在积极成长。过早地加以收集将产生足够的细胞数量和将呈现刮掉的LB琼脂平板上令人沮丧的，因为“pellicule”它们形成难以抬离该板的表面上。收集细菌来不及将大大减少他们的能力。的时间窗口取决于镀在LB琼脂平板上接种物的密度，自然会有所不同。第二个关键步骤是从它们被抬离琼脂平板上，再悬浮于氯化钙或双蒸2 O，直到它们被转化后接种于LB琼脂时刻保持细菌的冷（4℃）。含有细菌，比色皿和缓冲液的离心管应预冷，并保存在冰中在任何时候，直到细菌镀。但是，可能会出现其他问题时，无菌DDH 2 O或氯化钙含有沉淀物或污染。出于这个原因，我们建议准备这个试剂新鲜。 故障检修改造是通过将巴时，包括控制方便cteria任何方法呈现的主管。阴性对照由一个批次接种于含有选择性标记的LB琼脂平板上的未转化的感受态细菌中的将迅速帮助确定在平板上抗生素是否是有效的。与已知的质粒制备质量好，数量怀有同样的选择标记作为实验样品阳性对照转型将有助于确定这种细菌是否有能力也有实验DNA转型是否足够数量和质量。 这种技术的优点是多个，该方法可以进行该转换计划在当天内。我们发现，冷冻细菌甘油可直接在LB琼脂无electrocompetence任何损失进行电镀;我们在“紧急”情况下做到了这一点（忘记前一天开始的过夜培养后）。虽然我们无法测试电子邮件很紧张众所周知，任何E.大肠杆菌或V。霍乱弧菌菌株，可以通过电转化一直是服从该协议在我们的手中。该技术具有快速，有效，而且收益率转化效率堪比那些从准备采用传统方法电感受态细菌获得。该方法消除了对于高速离心机和相关联的转子，定轨摇床培养箱和无菌离心桶（容器），以及大容量的高压灭菌缓冲的要求。虽然具体的试剂和仪器的表还是比较全面的，大多数实验室是进行电穿孔将有大多数，如果不是全部，已有的这些材料。也许最重要的是，该技术可以容易地应用到产生电感受实验室专用菌株突变体背景迅速。我们都没有意识到这种技术的任何限制，因为它已经取代了准备ELEC的传统方法在我们的实验室trocompetent细菌。 我们在这里显示的实验的结果进行与已存储的在-80℃下不超过1周的传统方法生产的新鲜电感受态细胞。然而，冷冻电感受态细胞股市失去竞争力和生存能力或在储存较长时间。我们的删节协议允许新鲜的电感受态细胞的制备当天的改造计划没有关于这可能导致减少的转化产率的股票年龄的担忧。我们并没有试图存储产生电感受态细胞用快速的方法在另一天使用，因为这还没有必要。 虽然这里没有显示，使用我们的快速的方法就足够了结扎混合物制得的电感受态细胞的效率。报告的非O1 V的变革霍乱弧菌在最近的一篇文章Unterweger 等。17进行了使用通过此处描述的（那些通过接合进行除外）的方法，电感受态细菌。这种技术能提供研究人员改造的菌株与突变的背景迅速和有效，而无需编写细胞的大批量。这种快速的电感受态菌的制备方法可能适合于已被证明是适合进行电穿孔作为制剂的原理和电穿孔的设置都将是相同的传统协议的其它原核生物。

自从20世纪80年代初的1&#39;以来，电已成为不可或缺的分子生物学技术，可能用于转化/转染活细胞圆形或线性核酸的一个最常用的方法。最初是为真核细胞的1转发达，电穿孔后来改编为E的转型大肠杆菌 2，3。在细菌学，电穿孔已经成为实验室标准并已被修饰以改变一个广泛的细菌包括革兰氏阴性绿脓杆菌 4， 沙门氏菌 5， 弧菌6，Serratiae，和志贺氏菌 7;革兰氏阳性梭菌 8，9 杆菌 ， 乳酸杆菌 10和肠球菌 11。即使是一些古细菌已被证明易于转化通过电穿孔，包括Methanococci </EM&gt; 12和硫化种13。对于一个全面的检讨，请参阅欧尼和Aachmann 14。改造通过电穿孔效率是10-20据说比化学能力或热休克2倍以上。然而，就像准备细菌化学能力在20世纪80年代15优化道格拉斯的Hanahan，细胞还必须准备进行电感受。 电穿孔是一种快速和有效的细菌转化的方法，要求电感受态细菌，纯化的DNA，它提供的电脉冲的脉冲控制模块，以及收容该充当电极小的一次性比色皿的腔室。简要地，冷，感受态细菌与质粒DNA，进行高电压脉冲在比色杯中，再悬浮于生长培养基中，培养在30-37℃下进行30-45分钟混合，然后接种于半固体培养基（营养琼脂板）以APPRopriate选择性抗生素。 与此相反的脉冲的步骤中，电感受态大肠杆菌的制备大肠杆菌仍然是一个多部分的程序，是传统上开展大量。简要地，细菌的过夜培养物接种到锥形烧瓶中，用100ml至1升的Luria肉汤（LB），生长至生长中期对数生长期（的&lt;1.0 OD 600），并进行串行洗涤，用无菌非离子在4℃体积减小缓冲区或双蒸水（双蒸2 O）大必须小心，以保持细胞寒冷整个过程，防止污染。这需要使用高压灭菌离心桶和非离子型缓冲器或双蒸2 O为轨道摇床培养箱，大容量高速冷冻离心机和一组转子。细菌被最后重新悬浮于小体积的缓冲液中补充了10％甘油和等分入〜40μl的体积，WHICH被储存在-80℃直至使用。低温贮存通常导致降低转化效率由于生存能力的损失随着时间的推移。 电感受态细菌细胞也可从各种商业来源获得，但只对（通常重组缺陷型）E.有限数量的大肠杆菌菌株通常用作宿主传播范围广质粒。因此研究人员依靠内部的方法来编写自己株/突变体进行改造。 另一种快速，高效的协议，用于电感受态大肠杆菌的制备大肠杆菌已经由少数分子细菌学实验室使用了一段时间，现在已经扩展到其他革兰阴性菌，在我们的案例五霍乱弧菌 。该协议的发起者不能被识别，因为它已被优化其他研究者随着时间的推移。这里提出的方法已在我们的LABORATOR被用于IES了近二十年，这是我们的目标与我们的同行分享这个有用的协议。

<table border="1">
	<tbody>
		<tr>
			<td>Name of the Equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Gene Pulser Module</td>
			<td>Bio-Rad</td>
			<td>165-2668</td>
		</tr>
		<tr>
			<td>Gene Pulser Cuvette Chamber</td>
			<td>Bio-Rad</td>
			<td>165-2669</td>
		</tr>
		<tr>
			<td>Electrocuvettes (0.2 cm gap)</td>
			<td>Bio-Rad</td>
			<td>165-2082</td>
		</tr>
		<tr>
			<td>13 x 100 mm Pyrex Rimless Culture Tubes</td>
			<td>Corning</td>
			<td>70820-13</td>
		</tr>
		<tr>
			<td>13 mm Culture Tube Closures</td>
			<td>Cole Parmer</td>
			<td>EW-04500-00</td>
		</tr>
		<tr>
			<td>6 x 250 mm Glass Rod</td>
			<td>Lab Source</td>
			<td>11381D</td>
		</tr>
		<tr>
			<td>Disposable Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>08-757-12</td>
		</tr>
		<tr>
			<td>Roller Drum Motor Assembly</td>
			<td>New Brunswick Scientific</td>
			<td>M1053-4004</td>
		</tr>
		<tr>
			<td>Roller Drum for 13 x 100 mm Culture Tubes</td>
			<td>New Brunswick Scientific</td>
			<td>M1053-0306</td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Chemicals</td>
			<td>C5670</td>
		</tr>
		<tr>
			<td>Ethanol, anhydrous/denatured</td>
			<td>Sigma Chemicals</td>
			<td>227649</td>
		</tr>
		<tr>
			<td>Inoculating Loop</td>
			<td>Decon Labs</td>
			<td>MP182-5</td>
		</tr>
		<tr>
			<td>Refrigerated Microcentrifuge 5451 R</td>
			<td>Eppendorf</td>
			<td>22621425</td>
		</tr>
		<tr>
			<td>Bunsen Burner</td>
			<td>Fisher Scientific</td>
			<td>03-917Q</td>
		</tr>
		<tr>
			<td>LB Broth MILLER</td>
			<td>EMD</td>
			<td>1.10285.0500</td>
		</tr>
		<tr>
			<td>Bacteriological Agar</td>
			<td>Fisher Scientific</td>
			<td>S25127</td>
		</tr>
	</tbody>
</table>

rapid protocol for preparation of electrocompetent escherichia coli and vibrio cholerae

电已成为一种广泛使用的方法，快速有效地引入外源DNA插入多种细胞。电转化已成为首选的用于将DNA导入原核细胞是不天然胜任的方法。电穿孔是一种快速，高效，精简的改造方法，除了纯化的DNA和感受态细菌，需要市售基因脉冲控制器和比色皿。与此相反的脉冲的步骤中，制备电感受态细胞中是费时和劳动密集的，涉及在降低的无菌，冷水体积反复多轮离心和洗涤的，或大体积培养物的非离子型缓冲器生长至对数中期相增长。时间和精力，可以从商业渠道购买电感受态细胞被保存，但常用的大肠杆菌的选择是有限的大肠杆菌实验室菌株。我们特此传播迅速，用于制备电感受态大肠杆菌有效的方法大肠杆菌 ，已在通过细菌学实验室使用一段时间后，可以适用于五霍乱和其他原核生物。虽然我们不能确定谁功劳开发独特的技术，我们特此使其成为科学界使用。

我们进行了一组与大肠杆菌变换大肠杆菌和V。霍乱弧菌对我们快速的方法转化效率比较与（传统）方法最初由Dower 等 2所描述的电感受态细菌制备的一种适应。进行了传统的制备感受态细胞，加入500ml的LB在2升的三角烧瓶中接种大肠杆菌的500微升过夜培养大肠杆菌 DH5α或V。霍乱弧菌 O395，孵育在轨道摇床（215转，在37℃）和收获在0.5-1.0之间OD 600。将烧瓶在冰上冷却，并在培养物离心，在预冷的转子以4000×g离心10分钟，在4℃下将细胞沉淀物彻底在500毫升冰冷的2毫米氯化钙2为五重悬于霍乱和双蒸2 O为E。大肠杆菌 ，并再次在相同条件下离心。随后的几轮的再悬浮的ð离心分离，进行了减少的200卷和100毫升的体积使得在4℃下确定的培养维持冷藏最后，再悬浮和离心分离在一个10毫升的量，进行与10％甘油。最终的沉淀物再悬浮于2ml的10％甘油和40微升等分试样冷冻于-80℃，并存储不长于前一周电穿孔。 电穿孔条件包括脉冲设置和试管大小都相同的两个菌种中的所有电穿孔无论准备感受态细胞的方法。唯一的区别是，我们进行了清洗冷双蒸2 O为E。大肠杆菌和冷2毫米氯化钙2 V。霍乱弧菌 。 表1显示细菌通过电相同，但呈现的主管要么与快速的协议，或与传统方法转化的结果。我们采用应变O395和DH5α共同LY使用，代表五株霍乱弧菌和大肠杆菌大肠杆菌分别;类似的结果可以与相同物种的其他菌株获得的（虽然不是每个单一菌株已经过测试）以及可能适用于其它的革兰氏阴性细菌和可能超出。 为了确保平等的细胞数目存在于每个批次脉冲，产生利用快速的方法电感受态细菌汇集在收集，混合，保持均匀悬浮，并且不久之前，电分装在40微升卷。产生利用传统的方法电感受同样的细菌之前，在40微升等分冷冻治疗。分娩后的脉冲的，每批40微升电穿孔的细菌悬浮于400微升的LB和串行1:10稀释。每个稀释的一百微升接种在LB琼脂中的100微克/毫升氨苄青霉素的存在和传播使用无菌玻璃细胞吊具并在37℃培养过夜。量化细菌在每个变换采用的总数，每个批次准备的电感受态细菌的40微升体积中连续稀释，并接种在LB琼脂不含抗生素的相同并联。从每批同等对待的细菌之一等分试样电穿孔用1微升的Tris-EDTA（TE）缓冲液，但没有DNA（模拟变换），连续稀释并涂布在LB琼脂无抗生素来估计细胞的传递损失的数量电脉冲。最后，一​​个额外的变化来进行实验，以确定是否分30保温在37℃在1ml LB后电穿孔，但镀在LB-琼脂上之前，会影响转化细胞的恢复。菌落形成单位（CFU的）进行了列举翌日。 在这个实验中，我们采用pUC18中，一个常见的​​实验室质粒，并包括一个称职的细菌批pproximately 10 8 CFU的对细胞制备采用传统的方法和10 7 CFU的为准备利用快速的协议细胞。独自电（无DNA）减少可行的CFU了10-100倍，无论用于制备电感受态细胞的方法。如表1所示，转化产率是DNA的范围在三次重复实验（括号中的标准偏差）为E.在10 6 -10 7的CFUs /微克内大肠杆菌 （DH5α）和V。霍乱弧菌 （O395）分别为使用电感受细胞制备的传统，更长的方法。产量转化为E.出现下降了10倍，DNA在三次重复实验（括号内标准差）10 5 -10 6的CFUs /微克大肠杆菌和V。使用快速的方法分别霍乱 。为此，我们通过将转化CFU的决定转化细菌的百分比通过CFU的数目由“模拟转换”（不带质粒）从回收的感受态细胞中其他相同批次。转化的细菌的百分比是一个日志（2.5-9.4％）内无论制备和转化（ 表1括号内的数字）物种的方法。这些结果表明，该快速方法的效率相媲美，制备感受态细胞和霍乱弧菌和大肠杆菌的代表菌株也同样适合于快速过程的传统的冗长程序。 我们发现，转化效率，对像的pUC18质粒携带β-内酰胺酶盒，不会受到在LB培养基中30分钟的恢复时间。转化子的数目相同，回收的细胞是否接种到LB琼脂在氨苄青霉素存在下直接电穿孔后，或者它们是否被允许一个30-45分钟的恢复时间在LB前电镀液中，在37℃。我们的研究结果，即氨苄青霉素抗性并不需要非选择性条件下生长表明，β-内酰胺酶的表达发生足够快时转换。然而，应该指出的是，我们进行了脉冲细菌的系列稀释液在LB肉汤中，这可能对恢复从电击贡献，并启动基因的表达。 <img alt="图1" src="/files/ftp_upload/50684/50684fig1.jpg" /> 图1。为电感受态细菌的快速制备方法的图形描绘。后4-6小时（取决于接种物的密度）足够数量的细菌可以被收集，进行四到六个转换从一个单一的LB琼脂平板上。以确保相等数量的感受态细胞中为每个最终体积被电穿孔，从LB琼脂平板上收集到的细菌最初被收集并洗涤，然后一起人iquoted在这取决于颗粒的大小将40μl体积（较大的颗粒稀释在更大的体积约40微升）。 <table border="1"><tbody><tr><td colspan="3"> 的氨苄青霉素抗性转化子/μgDNA的数目（氨苄青霉素抗性的转化体中回收[％]） </td></tr><tr><td> 应变 </td><td> 传统的方法 </td><td> 快速方法 </td></tr><tr><td> 大肠杆菌 （DH5α） </td><td> 2.3×10 6（±7.3×10 5）[9.4％] </td><td> 3×10 6（±7.1×10 5）[2.5％] </td></tr><tr><td> 霍乱弧菌 （O395） </td><td> 4×10 7（±1.7×10 7）[5％] </td><td> 6.7×10 5（±2.1×10 5）[3.3％] </td></tr></tbody></table> 表1中。电感受的B每DNA（pUC18中）微克的转化效率acteria由较快速的方法，传统的方法制备。 所有的转换进行了与500纳克的pUC18 DNA（〜80％的超螺旋DNA作为可视化通过1％琼脂糖凝胶电泳）和电穿孔的细胞进行系列稀释在LB肉汤10倍到电镀为CFU的LB-琼脂用抗生素之前。非电穿孔的细菌和细菌电穿孔的无质粒的对照，连续稀释并涂布在LB琼脂无抗生素进行了为每个组转换，以确定前和后脉冲的存活细菌的总数。电穿孔控制为代表的活菌成功转型的百分比底线。

Watch this Scientific Journal Video about Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae at JoVE.com

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

电穿孔是用于将DNA导入细菌中称为转化的过程中通常采用的方法。为电感受态细胞的制备传统的协议是耗时且劳动密集型的。本文介绍了一种替代，快速，并为电感受态细胞通过一些实验室目前使用的高效制备方法。

对于电感受的制备快速协议<em&gt;大肠杆菌</em&gt;和<em&gt;霍乱弧菌</em

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

rapid-protocol-for-preparation-electrocompetent-escherichia-coli

Research

JoVE Journal

Biology

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

76.2K 次观看.  University of Texas Brownsville. 电穿孔是用于将DNA导入细菌中称为转化的过程中通常采用的方法。为电感受态细胞的制备传统的协议是耗时且劳动密集型的。本文介绍了一种替代，快速，并为电感受态细胞通过一些实验室目前使用的高效制备方法。 

视频: Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Vibrio cholerae: Model Organism to Study Bacterial Pathogenesis - Interview

霍乱弧菌：模式生物研究细菌的发病机制 - 访

科研

生物学

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

用于测量的活性测定大肠埃希氏菌诱导赖氨酸Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted into fuel alcohols1. The potential for enzymatic hydrolysis of cellulosic biomass to provide renewable energy has intensified efforts to engineer cellulases for economical fuel production2. Of particular interest are fungal cellulases3-8, which are already being used industrially for foods and textiles processing.
Identifying active variants among a library of mutant cellulases is critical to the engineering process; active mutants can be further tested for improved properties and/or subjected to additional mutagenesis. Efficient engineering of fungal cellulases has been hampered by a lack of genetic tools for native organisms and by difficulties in expressing the enzymes in heterologous hosts. Recently, Morikawa and coworkers developed a method for expressing in E. coli the catalytic domains of endoglucanases from H. jecorina3,9, an important industrial fungus with the capacity to secrete cellulases in large quantities. Functional E. coli expression has also been reported for cellulases from other fungi, including Macrophomina phaseolina10 and Phanerochaete chrysosporium11-12. 
We present a method for high throughput screening of fungal endoglucanase activity in E. coli. (Fig 1) This method uses the common microbial dye Congo Red (CR) to visualize enzymatic degradation of carboxymethyl cellulose (CMC) by cells growing on solid medium. The activity assay requires inexpensive reagents, minimal manipulation, and gives unambiguous results as zones of degradation (&#x201C;halos&#x201D;) at the colony site. Although a quantitative measure of enzymatic activity cannot be determined by this method, we have found that halo size correlates with total enzymatic activity in the cell. Further characterization of individual positive clones will determine , relative protein fitness. 
Traditional bacterial whole cell CMC/CR activity assays13 involve pouring agar containing CMC onto colonies, which is subject to cross-contamination, or incubating cultures in CMC agar wells, which is less amenable to large-scale experimentation. Here we report an improved protocol that modifies existing wash methods14 for cellulase activity: cells grown on CMC agar plates are removed prior to CR staining. Our protocol significantly reduces cross-contamination and is highly scalable, allowing the rapid screening of thousands of clones. In addition to H. jecorina enzymes, we have expressed and screened endoglucanase variants from the Thermoascus aurantiacus and Penicillium decumbens (shown in Figure 2), suggesting that this protocol is applicable to enzymes from a range of organisms.

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

We describe a low cost, high throughput method to screen for fungal endoglucanase activity in E. coli. The method relies on a simple visual readout of substrate degradation, does not require enzyme purification, and is highly scalable. This allows for the rapid screening of large libraries of enzyme variants.

真菌内切葡聚糖酶的活性高通量筛选大肠埃希氏菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

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

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 physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7, but GI information remains sparse for prokaryotes8, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10.
Here, we present the key steps required to perform quantitative E. coli Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format. Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g. the 'Keio' collection11) and essential gene hypomorphic mutations (i.e. alleles conferring reduced protein expression, stability, or activity9, 12, 13) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e. slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2 as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9.

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

Systematic, large-scale synthetic genetic (gene-gene or epistasis) interaction screens can be used to explore genetic redundancy and pathway cross-talk. Here, we describe a high-throughput quantitative synthetic genetic array screening technology, termed eSGA that we developed for elucidating epistatic relationships and exploring genetic interaction networks in Escherichia coli.

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

蛋白复合物的鉴定<em&gt;大肠杆菌</em使用顺序肽亲和纯化技术相结合串联质谱法

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for expressing proteins and genetic circuits in a controlled manner as well as for providing a prototyping environment for synthetic biology.2,3 To achieve the latter goal, cell-free expression systems that preserve endogenous Escherichia coli transcription-translation mechanisms are able to more accurately reflect in vivo cellular dynamics than those based on T7 RNA polymerase transcription. We describe the preparation and execution of an efficient endogenous E. coli based transcription-translation (TX-TL) cell-free expression system that can produce equivalent amounts of protein as T7-based systems at a 98% cost reduction to similar commercial systems.4,5 The preparation of buffers and crude cell extract are described, as well as the execution of a three tube TX-TL reaction. The entire protocol takes five days to prepare and yields enough material for up to 3000 single reactions in one preparation. Once prepared, each reaction takes under 8 hr from setup to data collection and analysis. Mechanisms of regulation and transcription exogenous to E. coli, such as lac/tet repressors and T7 RNA polymerase, can be supplemented.6 Endogenous properties, such as mRNA and DNA degradation rates, can also be adjusted.7 The TX-TL cell-free expression system has been demonstrated for large-scale circuit assembly, exploring biological phenomena, and expression of proteins under both T7- and endogenous promoters.6,8 Accompanying mathematical models are available.9,10 The resulting system has unique applications in synthetic biology as a prototyping environment, or "TX-TL biomolecular breadboard."

Protocols for Implementing an Escherichia coli Based TX-TL Cell-Free Expression System for Synthetic Biology

This five-day protocol outlines all steps, equipment, and supplemental software necessary for creating and running an efficient endogenous Escherichia coli based TX-TL cell-free expression system from scratch. With reagents, the protocol takes 8 hours or less to setup a reaction, collect, and process data.

协议用于实现<em&gt;大肠杆菌</em&gt;基于TX-TL无细胞表达系统合成生物学

Ideal cell-free expression systems can theoretically emulate an in vivo cellular environment in a controlled in vitro platform.1 This is useful for ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

检测现场的<em&gt;大肠杆菌</em&gt; O157：H7细胞的PMA-qPCR的

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble unimers below a characteristic transition temperature and aggregating into micron-scale coacervates above their transition temperature. The design of elastin-like polypeptides at the genetic level permits precise control of their sequence and length, which dictates their thermal properties. Elastin-like polypeptides are used in a variety of applications including biosensing, tissue engineering, and drug delivery, where the transition temperature and biopolymer architecture of the ELP can be tuned for the specific application of interest. Furthermore, the lower critical solution temperature phase transition behavior of elastin-like polypeptides allows their purification by their thermal response, such that their selective coacervation and resolubilization allows the removal of both soluble and insoluble contaminants following expression in Escherichia coli. This approach can be used for the purification of elastin-like polypeptides alone or as a purification tool for peptide or protein fusions where recombinant peptides or proteins genetically appended to elastin-like polypeptide tags can be purified without chromatography. This protocol describes the purification of elastin-like polypeptides and their peptide or protein fusions and discusses basic characterization techniques to assess the thermal behavior of pure elastin-like polypeptide products.

Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli

Elastin-like polypeptides are stimulus-responsive biopolymers with applications ranging from recombinant protein purification to drug delivery. This protocol describes the purification and characterization of elastin-like polypeptides and their peptide or protein fusions from Escherichia coli using their lower critical solution temperature phase transition behavior as a simple alternative to chromatography.

重组弹性蛋白样多肽及其融合物与从肽和蛋白质非色谱纯化<em&gt;大肠杆菌</em

Elastin-like polypeptides are repetitive biopolymers that exhibit a lower critical solution temperature phase transition behavior, existing as soluble ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

膜蛋白的绿色荧光蛋白为基础的表达筛选<em&gt;大肠杆菌</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III &#949; subunit (featuring 3&#8217;-5&#8217; exonuclease activity) is highly improved when the same protein is co-expressed with the &#945; and &#952; subunits (featuring DNA polymerase activity and stabilizing &#949;, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the &#945; subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the &#949; subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type &#949; subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-&#946;-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

Protein co-expression is a powerful alternative to the reconstitution in vitro of protein complexes, and is of help in performing biochemical and genetic tests in vivo. Here we report on the use of protein co-expression in Escherichia coli to obtain protein complexes, and to tune the mutation frequency of cells.

蛋白质的共表达了多方面的好处<em&gt;大肠杆菌</em

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in ...