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

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Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

77.0K 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

TransFLP &#x2014; A Method to Genetically Modify Vibrio cholerae Based on Natural Transformation and FLP-recombination

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids (e.g. harboring pir-dependent or temperature-sensitive replicons1,2). These plasmids are integrated into bacterial chromosomes based on homology-mediated recombination. Such insertional mutants are often directly used in experimental settings. Alternatively, selection for plasmid excision followed by its loss can be performed, which for Gram-negative bacteria often relies on the counter-selectable levan sucrase enzyme encoded by the sacB gene4. The excision can either restore the pre-insertion genotype or result in an exchange between the chromosome and the plasmid-encoded copy of the modified gene. A disadvantage of this technique is that it is time-consuming. The plasmid has to be cloned first; it requires horizontal transfer into V. cholerae (most notably by mating with an E. coli donor strain) or artificial transformation of the latter; and the excision of the plasmid is random and can either restore the initial genotype or create the desired modification if no positive selection is exerted. Here, we present a method for rapid manipulation of the V. cholerae chromosome(s)5 (Figure 1). This TransFLP method is based on the recently discovered chitin-mediated induction of natural competence in this organism6 and other representative of the genus Vibrio such as V. fischeri7. Natural competence allows the uptake of free DNA including PCR-generated DNA fragments. Once taken up, the DNA recombines with the chromosome given the presence of a minimum of 250-500 bp of flanking homologous region8. Including a selection marker in-between these flanking regions allows easy detection of frequently occurring transformants.
This method can be used for different genetic manipulations of V. cholerae and potentially also other naturally competent bacteria. We provide three novel examples on what can be accomplished by this method in addition to our previously published study on single gene deletions and the addition of affinity-tag sequences5. Several optimization steps concerning the initial protocol of chitin-induced natural transformation6 are incorporated in this TransFLP protocol. These include among others the replacement of crab shell fragments by commercially available chitin flakes8, the donation of PCR-derived DNA as transforming material9, and the addition of FLP-recombination target sites (FRT)5. FRT sites allow site-directed excision of the selection marker mediated by the Flp recombinase10.

A quick method to modify the genome of V. cholerae is described. These modifications include the deletion of single genes, gene clusters and genomic islands as well as the integration of short sequences (e.g. promoter elements or affinity-tag sequences). The method is based on the natural transformation and FLP-recombination.

TransFLP - A基因修改方法<em&gt;霍乱弧菌</em基于自然转化和FLP重组

Several methods are available to manipulate bacterial chromosomes1-3. Most of these protocols rely on the insertion of conditionally replicative plasmids ...

科研

免疫与感染

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity of cells within a test tube. By obviating the need to maintain the viability of the cell, and by eliminating the cellular barrier, CFPS has been foundational to emerging applications in biomanufacturing of traditionally challenging proteins, as well as applications in rapid prototyping for metabolic engineering, and functional genomics. Our methods for implementing an E. coli-based CFPS platform allow new users to access many of these applications. Here, we describe methods to prepare extract through the use of enriched media, baffled flasks, and a reproducible method of tunable sonication-based cell lysis. This extract can then be used for protein expression capable of producing 900 &#181;g/mL or more of super folder green fluorescent protein (sfGFP) in just 5 h from experimental setup to data analysis, given that appropriate reagent stocks have been prepared beforehand. The estimated startup cost of obtaining reagents is $4,500 which will sustain thousands of reactions at an estimated cost of $0.021 per &#181;g of protein produced or $0.019 per &#181;L of reaction. Additionally, the protein expression methods mirror the ease of the reaction setup seen in commercially available systems due to optimization of reagent pre-mixes, at a fraction of the cost. In order to enable the user to leverage the flexible nature of the CFPS platform for broad applications, we have identified a variety of aspects of the platform that can be tuned and optimized depending on the resources available and the protein expression outcomes desired.

Escherichia coli-Based Cell-Free Protein Synthesis: Protocols for a robust, flexible, and accessible platform technology

This protocol details the steps, costs, and equipment necessary to generate E. coli-based cell extracts and implement in vitro protein synthesis reactions within 4 days or less. To leverage the flexible nature of this platform for broad applications, we discuss reaction conditions that can be adapted and optimized.

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

Over the last 50 years, Cell-Free Protein Synthesis (CFPS) has emerged as a powerful technology to harness the transcriptional and translational capacity ...

化学

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the majority of Vibrio-associated infections. Thus, accurate differentiation among Vibrio spp. and detection of V. parahaemolyticus is critically important to ensure the safety of our food supply. Although molecular techniques are increasingly common, culture-depending methods are still routinely done and they are considered standard methods in certain circumstances. Hence, a novel chromogenic agar medium was tested with the goal of providing a better method for isolation and differentiation of clinically relevant Vibrio spp. The protocol compared the sensitivity, specificity and detection limit for the detection of V. parahaemolyticus between the new chromogenic medium and a conventional medium. Various V. parahaemolyticus strains (n=22) representing diverse serotypes and source of origins were used. They were previously identified by Food and Drug Administration (FDA) and Centers for Disease Control and Prevention (CDC), and further verified in our laboratory by tlh-PCR. In at least four separate trials, these strains were inoculated on the chromogenic agar and thiosulfate-citrate-bile salts-sucrose (TCBS) agar, which is the recommended medium for culturing this species, followed by incubation at 35-37 &#176;C for 24-96 hr. Three V. parahaemolyticus strains (13.6%) did not grow optimally on TCBS, nonetheless exhibited green colonies if there was growth. Two strains (9.1%) did not yield the expected cyan colonies on the chromogenic agar. Non-V. parahaemolyticus strains (n=32) were also tested to determine the specificity of the chromogenic agar. Among these strains, 31 did not grow or exhibited other colony morphologies. The mean recovery of V. parahaemolyticus on the chromogenic agar was ~96.4% relative to tryptic soy agar supplemented with 2% NaCl. In conclusion, the new chromogenic agar is an effective medium to detect V. parahaemolyticus and to differentiate it from other vibrios.

Development of a More Sensitive and Specific Chromogenic Agar Medium for the Detection of Vibrio parahaemolyticus and Other Vibrio Species

Detection and isolation of clinically relevant Vibrio species require selective and differential culture media. This study evaluated the ability of a new chromogenic medium to detect and identify V. parahaemolyticus and other related species. The new medium was found to have better sensitivity and specificity than the conventional medium.

对于检测到的敏感性和特异性显色琼脂培养基发展<em&gt;副溶血性弧菌</em&gt;及其他<em&gt;弧菌</em&gt;物种

Foodborne infections in the US caused by Vibrio species have shown an upward trend. In the genus Vibrio, V. parahaemolyticus is responsible for the ...

使用 大肠杆菌 dnaK756 细胞评估 Hsp70 伴侣活性

Escherichia coli -Based Complementation Assay to Study the Chaperone Function of Heat Shock Protein 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. While Hsp70 is not essential for E. coli cells growing under normal conditions, this chaperone becomes indispensable for growth at elevated temperatures. Since Hsp70 is highly conserved, one way to study the chaperone function of Hsp70&#160;genes from various species is to heterologously express them in E. coli strains that are either deficient in Hsp70 or express a native Hsp70 that is functionally compromised. E. coli dnaK756&#160;cells are unable to support &#955; bacteriophage DNA. Furthermore, their native Hsp70 (DnaK) exhibits elevated ATPase activity while demonstrating reduced affinity for GrpE (Hsp70 nucleotide exchange factor). As a result, E. coli dnaK756&#160;cells grow adequately at temperatures ranging from 30 &#176;C to 37 &#176;C, but they die at elevated temperatures (&#62;40 &#176;C). For this reason, these cells serve as a model for studying the chaperone activity of Hsp70. Here, we describe a detailed protocol for the application of these cells to conduct a complementation assay, enabling the study of the in cellulo chaperone function of Hsp70.

作者聚焦:探索疟疾和结核病感染中的热休克蛋白

This protocol demonstrates the chaperone activity of heat shock protein 70 (Hsp70). E. coli dnaK756 cells serve as a model for the assay as they harbor a native, functionally impaired Hsp70, making them susceptible to heat stress. The heterologous introduction of functional Hsp70 rescues the growth deficiency of the cells.

基于大肠杆菌的互补测定研究热休克蛋白的伴侣功能 70

Heat shock protein 70 (Hsp70) is a conserved protein that facilitates the folding of other proteins within the cell, making it a molecular chaperone. ...

生物化学

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global prevalence and severity of this disease, V. cholerae has been extensively studied in both environmental and laboratory settings, requiring proper maintenance and culturing techniques. Classical and El Tor are two main biotypes that compose the V. cholerae O1 serogroup, each displaying unique genotypic and phenotypic characteristics that provide reliable mechanisms for biotype characterization, and require distinct virulence inducing culturing conditions. Regardless of the biotype of the causative strain for any given infection or outbreak, the standard treatment for the disease involves rehydration therapy supplemented with a regimen of antibiotics. However, biotype classification may be necessary for laboratory studies and may have broader impacts in the biomedical field. In the early 2000&#39;s clinical isolates were identified which exhibit genotypic and phenotypic traits from both classical and El Tor biotypes. The newly identified hybrids, termed El Tor variants, have caused clinical and environmental isolate biotype identification to become more complex than previous traditional single assay identification protocols. In addition to describing V. cholerae general maintenance and culturing techniques, this manuscript describes a series of gene specific (ctxB and tcpA) PCR-based genetic screens and phenotypic assays (polymyxin B resistance, citrate metabolism, proteolytic activity, hemolytic activity, motility, and glucose metabolism via Voges-Proskauer assay) collectively used to characterize and/or distinguish between classical and El Tor biotypes. Together, these assays provide an efficient systematic approach to be used as an alternative, or in addition, to costly, labor-intensive experiments in the characterization of V. cholerae clinical (and environmental) isolates.

Laboratory Techniques Used to Maintain and Differentiate Biotypes of Vibrio cholerae Clinical and Environmental Isolates

This manuscript describes proper Vibrio cholerae maintenance techniques in addition to a series of biochemical assays, collectively utilized for quick and reliable differentiation between clinical and environmental V. cholerae biotypes in a laboratory setting.

用于维持和区分生物型的实验室技术<em&gt;霍乱弧菌</em&gt;临床和环境隔离

The aquatic Gram-negative bacterium Vibrio cholerae is the etiological agent of the infectious gastrointestinal disease cholera. Due to the global ...

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ability to obtain single cells for fluorescence microscopy, which can be particularly challenging when imaging cells that exist within bacterial communities. For example, the human pathogen Vibrio parahaemolyticus exists as short rod-shaped swimmer cells in liquid conditions that upon surface contact differentiate into a subpopulation of highly elongated swarmer cells specialized for growth on solid surfaces. This paper presents a method to perform single cell fluorescence microscopy analysis of V. parahaemolyticus in its two differential states. This protocol very reproducibly induces differentiation of V. parahaemolyticus into a swarmer cell life-cycle and facilitates their proliferation over solid surfaces. The method produces flares of differentiated swarmer cells extending from the edge of the swarm-colony. Notably, at the very tip of the swarm-flares, swarmer cells exist in a single layer of cells, which allows for their easy transfer to a microscope slide and subsequent fluorescence microscopy imaging of single cells. Additionally, the workflow of image analysis for demographic representation of bacterial societies is presented. As a proof of principle, the analysis of the intracellular localization of chemotaxis signaling arrays in swimmer and swarmer cells of V. parahaemolyticus is described.

Induction of Cellular Differentiation and Single Cell Imaging of Vibrio parahaemolyticus Swimmer and Swarmer Cells

This protocol enables single cell microscopy of the differentially distinct Vibrio parahaemolyticus swimmer and swarmer cells. The method produces a population of swarmer cells easily available for single cell analysis and covers preparation of cell cultures, induction of swarmer differentiation, sample preparation, and image analysis.

诱导细胞分化和单细胞成像<em&gt;副溶血性弧菌</em游泳者和Swarmer细胞

The ability to study the intracellular localization of proteins is essential for the understanding of many cellular processes. In turn, this requires the ...

罗 伊氏利氏乳杆菌 DSM20016 中报告蛋白的测量

Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for use as a chassis for synthetic biological endeavors within the gastrointestinal tract. The wide phenotypic and genotypic variation observed within the genus led to a recent reclassification and the introduction of 23 novel genera.
Due to the breadth of variations within the old genera, protocols demonstrated in one member may not work as advertised with other members. A lack of centralized information on how exactly to manipulate specific strains has led to a range of ad hoc approaches, often adapted from other bacterial families. This can complicate matters for researchers starting in the field, who may not know which information does or does not apply to their chosen strain.
In this paper, we aim to centralize a set of protocols with demonstrated success, specifically in&#160;the Limosilactobacillus reuteri strain designation F275 (other collection numbers: DSM20016, ATCC23272, CIP109823), along with troubleshooting advice and common issues one may encounter. These protocols should enable a researcher with little to no experience working with L. reuteri DSM20016 to transform a plasmid, confirm transformation, and measure system feedback in a plate reader via a reporter protein.

作者聚焦:罗伊氏乳酸菌 DSM20016 中的电穿孔和转化确认方法  

Here, we present protocols for working with Limosilactobacillus reuteri DSM20016, detailing growth, plasmid transformation, colony PCR, fluorescent reporter protein measurement, and limited plasmid mini-prep, as well as common issues and troubleshooting. These protocols allow the measurement of reporter proteins in DSM20016, or confirmation via colony PCR if no reporter is involved.

罗伊氏乳酸杆菌电穿孔和转化确认的方法DSM20016

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for ...

对于电感受的制备快速协议

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此视频中的章节

TransFLP - A基因修改方法

大肠杆菌无细胞蛋白合成: 一种强大、灵活和可访问的平台技术的协议

对于检测到的敏感性和特异性显色琼脂培养基发展

基于大肠杆菌的互补测定研究热休克蛋白的伴侣功能 70

基于大肠杆菌的互补测定研究热休克蛋白的伴侣功能 70

基于大肠杆菌的互补测定研究热休克蛋白的伴侣功能 70

用于维持和区分生物型的实验室技术

诱导细胞分化和单细胞成像

罗伊氏乳酸杆菌电穿孔和转化确认的方法DSM20016

罗伊氏乳酸杆菌电穿孔和转化确认的方法DSM20016