Name: a new screening method for the directed evolution of thermostable bacteriolytic enzymes
Uploaded: 2012-11-07T13:00:00.000+00:00
Duration: 13 min 30 s
Description: Watch this Scientific Journal Video about A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes at JoVE.com

作者希望，感谢Emilija仁科和珍妮特于提供技术援助。 DCN的支持，补助金从美国国防部（DR080205，DM102823，OR09055，OR090059），，。 RDH的支持，马里兰州农业试验站，在细胞和分子生物学的NIH训练的资助。

1。确定加热条件首先，必须通过实验确定最佳的温育温度和时间，在测定中使用的加热步骤。对于我们的PlyC模型，重要的是要注意， 大肠杆菌大肠杆菌对单独的表达质粒共转化与plyCA和plyCB基因已被证明，以形成一个全功能的PlyC全酶6。适于从在文献中提供的实施例12-16的96孔微量滴定板的制备以及作为细胞生长条件和随后副本电镀技术。从以前的热灭活实验的结果决定活动急剧下降，在短期在超过生理温度（未发表的观察）的环境中孵化，孵化时间30分钟将适合此法。的最佳培养温度为PlyC阐明通过下面的步骤: <ol><li>陈德良sform表达构建pBAD24:plyCA（安培）到主管E.大肠杆菌菌株DH5αpBAD33:plyCB（CM）。 </li><li>板上的Luria-Bertani（LB）补充氨苄青霉素（100微克/毫升）和氯霉素（35微克/毫升）的琼脂平板上的转化体。一夜之间孵育板在37°C。 </li><li>用无菌牙签，单个菌落接种到每个排孔（ 图2a）中的无菌的，清晰的，平底96孔，带盖的微量滴定板，其中包含200微升补充有氨苄青霉素和氯霉素的LB。 </li><li>可以安全地放置在96孔微量滴定板在37℃下摇床和长出的细菌在300rpm过夜。 </li><li>找回的96孔微量滴定板的37℃下摇床和副本板到一个新的96孔微量滴定板，如下所示: <ol class="lalpha"><li>加入180μl辅以氨苄青霉素和氯霉素的LB副本板的各孔。 </li><li>从原版20μl的细菌转移到相应的井在副本板。这将导致在初始OD 600为〜0.5。 </li></ol></li><li>安全地将副本板在37度，Ç颤抖的孵化器和孵化1小时，在300转板，然后添加inductant。在PBAD表达系统的情况下，inductant是0.25％阿拉伯糖。其他表达系统可能需要不同的inductants。板放置在37℃下摇床和摇一个额外的4小时，以允许用于蛋白质表达在300rpm。表达水平通常介于10-20微克的PlyC的。 </li><li>一旦蛋白的表达已经结束，检索的副本板和颗粒的细菌在冷冻离心机，包含摆动斗转子，适合96孔酶标板96孔酶标板。在3,000 rpm下离心10分钟，在室温下。</li><li>慢慢地翻转酶标板取出介质上清，轻轻地调迁的媒体。 </li><li>向每孔加入50μl的第二B-PER细菌蛋白抽提试剂溶解细胞。板在室温下孵育20分钟。 </li><li>增大音量，在各孔中，通过加入120微升70微升磷酸盐缓冲盐水（PBS），PH值7.2。 </li><li>沉淀不溶的细胞碎片，通过纺丝板以3,000 rpm，在冷冻离心机在4℃下10分钟。 </li><li>水溶性粗提液110微升转移到一个96孔的热循环仪板相应的孔中。 </li><li>使用预定的孵化时间为30分钟，暴露的可溶性裂解目前居住在96个热循环仪板了广阔的梯度温度范围15-20℃。注意，目标是要确定在什么温度下一个特定的酶失去了&gt; 95％的催化活性。 </li><li>孵化，孵化后的96孔温度循环器板在4℃下5分钟，从96孔的热循环仪板100微升的可溶性裂解物转移到其相应的在最后的96孔微量滴定板的井位。 </li><li>随着一个12通道multipipettor，每孔加入100μl的GAS菌株D471。请注意，D471的细胞原本是从过夜培养冻干并重新悬浮用PBS pH值7.2〜2.0，得到的OD 600。 </li><li>立即放置在96孔板上，在微孔板分光光度计和通过测量的OD 600为20分钟，每15秒监测的酶动力学。被定义为在非允许温度下，对应于井，缺乏光密度的变化（ΔOD≤0.1）的最低温度孵育。 </li></ol>一个宽的温度屏幕之后执行，以确定非允许温度下，可以重复上述步骤，在一个更​​窄的范围内的温度（5℃-10℃），以澄清感兴趣的酶的精确的非允许温度下。请注意，非允许温度下，在该测定中确定比通过其他方式阐明的熔融温度可能会有所不同，由于体积差异，浓度等 2。生成的突变体库以进行筛选的突变体库的创建涉及随机纳入由以最小的核苷酸偏压在plyCA基因，使用如下的GeneMorph II随机诱变试剂盒的一个容易出错的DNA聚合酶的突变: <ol><li>设计相似的熔融温度（Tm）的55-72℃下与给出的限制性位点的5&#39;和3&#39;末端的plyCA基因之间的核苷酸引物。 </li><li>由于所需的突变率是2-3核苷酸每plyCA的 （1.4kb的），使用PCR反应组分的浓度，以及低的突变f由生产商推荐的条件的热循环仪requencies（0-4.5每KB）。 </li><li>的的诱变plyCA基因克隆到表达载体pBAD24。 </li><li>变换的的构造成DH5αpBAD33:plyCB背景和板补充与氨苄青霉素和氯霉素的LB琼脂平板上的转化体。 </li><li>此外，变换和板DH5αpBAD33:plyCB pBAD24含有的WT plyCA基因的表达载体。从这个板块的殖民地将作为筛选过程的控制。 </li></ol> 3。 96孔微孔板的制备及细胞生长条件<ol><li>填充在96孔微量滴定板的各孔中，用200μl补充有氨苄青霉素和氯霉素的LB。 </li><li>微量滴定板的原理图（ 图2b）之后，小心地从过夜的琼脂平皿上选择一个单独的菌落用无菌牙签和接种的细菌，在指定的96孔微量滴定解放军德。这是必要的，以确保只有一个殖民地，每孔接种。 </li><li>摇动牢固地固定的96孔微量滴定板在37℃下在300 rpm的转速下过夜</li></ol> 4。副本电镀，蛋白表达的诱导和裂解液的制备<ol><li>按照步骤1.5-1.11一节"确定最佳的加热条件"的一个修改。存储的原始板，在4℃下后的副本电镀步骤已经结束。 </li><li>步后1.11，转移除了阳性对照孔A1-D1的96孔的热循环仪板的相应孔中的可溶性的粗裂解物到110微升。关于与阳性对照溶胞产物（ 即裂解物，不加热），转移到相应的井在一个新的96孔微量滴定板，将作为最终测定板的实验和存储在冰上的溶胞产物的100μl的。 </li></ol> 5。可溶性细胞裂解液热处理<ol><li>热阴性对照和突变体可溶性裂解液目前只限于在步骤1中确定在优化非许可孵化温度为30分钟，在热循环仪96孔热循环仪板。 </li><li>非允许温度下温育后，在4℃下孵育96以及热循环仪板5分钟。 </li><li>将100微升的可溶性裂解物从96孔的热循环仪的板，其在最后的96孔微量滴定测定板已经含有可溶性裂解物的阳性对照相同的井位。 </li><li>随着一个12通道multipipettor，每孔加入100μl的GAS菌株D471。在微孔板分光光度计立即将板。 </li><li>监视的酶动力学通过测量OD 600每15秒20分钟。 <ol class="lalpha"><li>甲PlyC与进展的热行为的变体被定义为一个结构中，可降低50％原来的光学密度在少于900秒，在该非允许温度。此外，阳性对照（ 即非加热，WT PlyC）需要以显示WT的催化活性（ 的t 1/2≤100秒）和阴性对照（ 即加热，WT PlyC）应该是没有活性的。 </li></ol></li><li>一旦PlyC变种与进展热行为被识别，检索储存在4℃和接种细菌从个人以及特定的突变成与氨苄青霉素和氯霉素的新鲜LB培养基中的兴趣的原始板。过夜生长的接种物，在37°C。 </li><li>从文化的提取和纯化质粒DNA，然后进行测序，以确定的核苷酸突变，推测增强的热性能PlyCA提交。此外，补充从过夜培养的小等分试样用10％甘油和存储在-80℃下以供进一步使用。 </li></ol>

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该协议中，在图1中所概述，提出了一个96孔微量滴定板的方法，它允许一个利用定向进化增加任何溶菌酶的热稳定性。通过一个容易出错的DNA聚合酶的使用，人们可以引入随机突变，增加整体的动力学稳定性的翻译的溶菌分子的利益，这通常是由于分子重组组成的增加静电，二硫桥和疏水性相互作用，产生改善分子齐聚状态提出了更高的17日至18日的的表面电荷网络或加固包装，增强了修改。核苷酸突变引入后，一个广泛的筛选程序，然后使用，以确定突变是热有益。代表结果是根据上一轮的随机突变。在现实中，它已被认为随机诱变的至少三个连续的轮，每个使用最强大的突变体作为引线的酶，然后通过DNA改组是适合于这些类型的定向进化的热行为的研究2。 重要的是要了解，而该协议的识别，增强动力稳定性的突变，这些变异体的不一定是增加了热稳定性。的分子被认为是热稳定的，它有能力在一个较高的温度下保持其结构时。然而，在测定中提出，没有突变的溶胞产物的残余活性测定在相同温度下，他们最初孵育在所述热处理步骤期间，从而可以选择的突变促进复性，而不是增强的热稳定性19。虽然我们推断热行为作为热稳定性的指标，真正的热稳定性，必须凭经验测定生物物理学ICAL方法，如圆二色性（CD），差示扫描量热法（DSC）和差示扫描荧​​光（DSF）。从CD和DSF实验的初步数据表明，从介绍的方法确定的几个候选人确实显示进展的热稳定性（数据未示出）。 虽然这里介绍的方法是具体实施热行为增加PlyC，这同样的方法可以额外地提高热其他溶菌酶的活性有一些小的改变，如缓冲剂，突变率，加热条件和表达系统的变量。的一个关键的变量与该测定涉及的容易出错的DNA聚合酶的碱基突变率。我们选择使用容易出错的Mutazyme II DNA聚合酶在我们的定向进化分析由于实验证据表明这显示特定的酶量最少的偏见与问候中，核苷酸是随机掺入的感兴趣的基因时相比，其他的随机诱变技术，如使用羟胺，赋值函数E.大肠杆菌和其他容易出错的DNA polyermases 20。在一般情况下，较低的突变率往往是更可取的，原因有二。首先，工程热稳定性的蛋白质通常包括相对较少的氨基酸取代。高突变率可能会导致特定分子结构的错误折叠和功能的差异，才能最后导致巨大的结构性改变。例如，甘氨酸和脯氨酸残基的掺入可以破坏α-螺旋的二级结构21。第二，高核苷酸的突变率增加成功的机会将提前终止密码子，从而导致生物活性的分子，在截断。在一般情况下，是优选的突变率较低，因为较低的错误率的结果，在储液器适应性突变的功能明显下降，而较高的突变率产生中性或有害的突变22。 每一轮的随机突变，另一个关键的变量，必须优化涉及到孵化温度在筛选过程中使用。选择的实验性非允许温度下是比较具有挑战性。我们最初使用的孵化温度为10°C比非许可温度PlyC的，但是在3000突变体的筛选，我们无法识别任何有利的突变。牢记定向进化方法包括依次进行识别具有加性效应的有益的氨基酸替换，它被确定不那么严格的温度下，应使用在筛选过程中，即使它产生假阳性的机会增加。因此，我们使用了一个孵化温度只有几摄氏度以上的非permiss香港专业教育学院的温度和再筛选选定的候选人，以排除假阳性。 我们决定利用的孵化温度，是不是太温带（≤1℃，高于最低的非允许温度下），反之不是太苛刻（≥5°C高于最低的非允许温度下）。理想的温度，我们决定在这个特殊的检测使用的是一个温和的2°C高于实验确定的非许可温度。选择太温和的温度下，会导致高得多的假阳性比〜20％，我们观察到（ 表I）的识别率时，使用一个适度的孵化温度。此外，使用过于苛刻的孵化温度，最终可能会导致无法识别突变体具有显着增强的热性能。例如，使用的孵化温度≥5℃下将有导致无法识别看好29C3突变体以及大多数其他候选人在第一轮的筛选选择。 在总结中，我们提供了一个协议，工程溶菌酵素，获得增强的动力学稳定性。此外，这种相同的测定，未经加热步骤，可以使用催化活性的突变，增加屏幕。增强的催化活性和热稳定性是一个重要的发展障碍，面对任何治疗酶。虽然此协议的细节是特定的细胞内溶素PlyC，该方法可以适于任何溶菌酶只有一些改动。我们提出了初步结果只从第一轮诱变验证该功能的测定，导致产生显著大小分子的热稳定性，增加的突变，在执行和识别。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> B-PER II细菌蛋白抽提试剂</td><td> Thermo Scientific的</td><td> 78260 </td></tr><tr><td> GeneMorph II随机突变试剂盒</td><td>安捷伦科技公司</td><td> 200500 </td></tr><tr><td>清除96孔，平底微孔板盖</td><td> BD真空采血管医学实验医学</td><td> 353072 </td></tr><tr><td> 96好Nonskir​​ted之PCR板</td><td> Fisher Scientific则</td><td> 14-230-232 </td></tr><tr><td>水平转子</td><td> Eppendorf公司</td><td> A-2-DWP </td></tr><tr><td>氨苄西林钠</td><td> Fisher Scientific则</td><td> BP1760 </td></tr><tr><td>氯霉素</td><td> Fisher Scientific则</td><td> BP904 </TD&gt; </tr><tr><td>阿拉伯糖</td><td> Fisher Scientific则</td><td> BP2504 </td></tr><tr><td> pBAD24表达载体的构建</td><td> ATCC </td><td> 87399 </td></tr><tr><td> pBAD33表达载体的构建</td><td> ATCC </td><td> 87402 </td></tr></tbody></table>

a new screening method for the directed evolution of thermostable bacteriolytic enzymes

定向进化被定义为利用自然选择的方法，以便工程师来获得特别是不相关联的属性中的蛋白质性质的蛋白质。文学提供了大量的关于实施定向进化的例子，成功地改变分子的特异性和催化1。利用定向进化，而 ​​不是更理性为基础的方法对分子工程的主要优点涉及到的数量和多样性的变种，可以筛选2。定向进化的一个可能的应用涉及溶菌酶，如内溶素的结构稳定性的改善。噬菌体编码和表达的一个重要的共价键（ 即细胞壁的肽聚糖）的细菌内溶水解，产生的子代病毒在宿主细胞裂解和解放。值得一提的是，这些酶具有的能力，外在诱导裂解的易感已验证IBLE细菌噬菌体并且在没有在体外和体内对他们的治疗潜力3-5。我们的定向进化研究的主题涉及PlyC内溶素，它是由PlyCA和PlyCB亚基6。纯化和添加非本质当，PlyC全酶溶解在几秒钟内的A组链球菌（GAS）以及其他链球菌群体的而且已经通过验证在活体内对气体7。显着，监测后残余酶动力学，高温下孵化提供了明显的证据，PlyC失去催化活性突然在45°C，这表明一个短期治疗的保质期，这可能会限制进一步发展这种酶。进一步的研究揭示缺乏热稳定性只观察为PlyCA亚基，，而PlyCB亚基是稳定的高达〜90℃（未发表的观察）。除了PlyC，有s许多研究文献中的例子，描述了耐热性质的内。例如， 葡萄球菌溶素LysK和肺炎链球菌内溶CPL-1和好朋友自发失去活性，在42°C，43.5°C和50.2°C，8-10。根据Arrhenius方程，其中涉及的化学反应的速率在 ​​特定的系统中存在的温度，增加的热稳定性将与增加货架寿命11。为此，定向进化已被证明是一个有用的工具，改变自然界中各种分子的热活动的，但从来没有成功利用这个特殊的技术溶菌酶的研究。同样，成功的账目结构的稳定性，这个特定的类抗菌药物的进展完全是不存在的。在这段视频中，我们采用了一种新的方法，使用错误-PR一种DNA聚合酶，然后用96孔微量滴定板格式，以确定突变的PlyCA亚基的酶动力学稳定性的增加（ 图1）相关联的PlyC链球菌细胞内溶素的优化的筛选过程。结果只是随机诱变的一个轮后，建议的方法是产生保留的两倍以上时相比，野生型（WT）PlyC升高的温度下处理后的残余活性的PlyC变种。

在结束了第一轮的随机突变，超过6000 PlyC的突变体的筛选，共35突变可能增加的热行为进行了鉴定，选择和测序。总结在表Ⅰ中 ，基因组分析，表明35候选人，7的构造包含的WT PlyCA序列翻译的电平，对应于由试验确定的误报。的余下的28名候选人，突变范围是1至6个碱基的突变与平均突变率的2.75的每plyCA基因，它是在2-3的碱基突变的目标的范围内，我们核苷酸。在翻译水平，这个特定的碱基突变范围和频率产生范围为1至5个氨基酸的氨基酸变异，与平均1.9每PlyCA多肽的氨基酸突变的突变率。 与至少一个氨基酸mutat的28个候选离子，这些突变构建四个随机选择验证的定向进化方法的广泛的筛选过程中确实正常作进一步鉴定。的突变PlyC酶进行纯化至&gt; 95％的均匀性，根据SDS-PAGE上分析，如先前描述的6-7。 WT PlyC和四个PlyC各突变体的酶动力学，其特征在于以相等的摩尔浓度纯化的酶的孵育后，在不同的温度升高。活动监测加入D471气体后，通过测量20分钟，每15秒，在600 nm的光密度。活性定义为热孵化后的酶的剩余的最高速度。随机挑选的四名候选人作进一步鉴定，突变体29C3表现出增强的热性能。 WT PlyC和29C3的热行为的影响在不同的温度下，同时温育并在PBS pH 7.2的活性测定在80 nM和40 nM的浓度，分别。 45-50℃的培养实验（ 图3）进行热循环仪中，在薄壁板在96孔热循环仪的总体积为120微升的样品孵育。 35℃，40℃和45℃下进行培养实验（ 图4和图5）中的热块在1.5ml微量离心管中的总体积为1.3毫升的样品孵育。所有实验均在一式三份。 WT PlyC和29C3没有表现出显着性差异，在动力学稳定性，在室温（25℃）作为描绘在图3中的第一组的酒吧。然而，从温度从45-50℃下30分钟的孵育后短期，29C3活性显着地大于在各温度点的WT构造特定的活性。例如，WT PlyC显示，44％的活性损失45.2℃，而29C3表明只有2％的损失，在相同温度下的活性。 孵育的长期研究，比较两个WT PlyC和29C3的残存活性，另外进行，在35℃和40℃的涉及在24和48小时的时间点测量的残余活性。 29C3显示在35°C，41％和176％活性高于WT PlyC在24和​​48小时的孵化时间点，分别为（ 图4a）。 29C3显示在40°C，28％和107％活性高于WT PlyC在24和​​48小时的孵化时间点，分别为（ 图4b）。 WT PlyC和29C3的残余活性也被监测，每20分钟共3小时，在45℃下，WT PlyC只能保留21％的活性，而29C3在此温度下保温3小时后能保留46％的活性。 WT PlyC和29C3的半衰期（的t 1/2）分别为67和147分钟，分别，建议<html> 29C3 ING，有2.2倍的增长在动力学稳定性，在45℃（ 图5）。 <table border="1"><tbody><tr><td>总计第1轮候选人</td><td> 35 </td></tr><tr><td>考生WT PlyCA序列</td><td> 7 </td></tr><tr><td>考生≥1氨基酸突变</td><td> 28 </td></tr><tr><td> -平均核苷酸突变率（NT / plyCA的 ） </td><td> 2.75 </td></tr><tr><td> - 核苷酸变异范围（NT） </td><td> 1-6 </td></tr><tr><td> - 平均氨基酸的突变率（AA / PlyCA） </td><td> 1.9 </td></tr><tr><td> - 氨基酸变异范围（AA） </td><td> 1-5 </td></tr></tbody></table> 表1。候选人基因组分析。 pload/4216/4216fig1highres.jpg"/&gt; 图1。定向进化分析方法。定向进化，一个人开始，与铅酶，将WT PlyC的第一轮。包含随机公正的plyCA基因的核苷酸突变的PlyC突变体库，然后构建一个容易出错的DNA聚合酶，克隆到表达载体pBAD24，转化到E.大肠杆菌菌株DH5α中已经含有pBAD33:plyCB个别的转化体接种到其自身特定的孔的96孔微量滴定板。通过广泛的筛选过程中，个别PlyC催化活性的突变体，在非容许的温度下温育后，被分类为动力学稳定性增强的突变体。 Theconstruct显示最进展的热性能，将因而成为铅的酶为下一轮的随机诱变。总体来说，有三个完整的轮拟人米突变，DNA改组在溶菌分子的与演化的热行为。 <a href="http://www.jove.com/files/ftp_upload/4216/4216fig1large.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" src="/files/ftp_upload/4216/4216fig2.jpg" /> 图2。定向进化测定96孔微量滴定板的模板的微量滴定板的最佳的加热条件下的测定期间的示意图（ 图2a）包括一个单一的父母WT PlyC克隆接种到微量滴定板的每一行的。的微量滴定板的示意图（ 图2b）的突变体的筛选期间，包括在第1列中的每个孔中接种，以及独特的父母WT PlyC克隆接种各孔列中的2-12具有鲜明PlyC突变克隆。被指定为家长积极控制，共同井A1-D1nsist WT PlyC结构不暴露在非允许的温度孵化。 E1-H1井被指定为负的家长控制功能，其中包括暴露在非允许的温度孵化的的WT PlyC结构。 <img alt="图3" src="/files/ftp_upload/4216/4216fig3.jpg" /> 图3。残余活性的动力学分析，比较WT PlyC和29C3酶被纯化至均一，并温育30分钟，在热循环仪中，以相等的摩尔浓度，在任一室温下或在范围从45-50℃的温度梯度酶的活性与特定温度下孵化后显示的最大速度。随着异常为25℃和47.7℃，在每个温度下的变化在WT和29C3之间的活动相关的p-值&lt;0.05。所有的数据被报告为三个独立实验的平均值±SEM。 </p&gt; <img alt="图4" src="/files/ftp_upload/4216/4216fig4.jpg" /> 图4。剩余活性动力学分析比较WT PlyC 29C3在35℃和40℃。平等摩尔浓度纯化的WT PlyC和29C3孵育的热块中，在35℃（ 图4a）或40℃（ 图4b）。在24和48小时的时间点，测定残留的酶活性。每种构建所显示的活性，被归一化到在时间点零显示的最大速度。活性在WT和29C3之间在每一个温度和时间点相关的变化的p-值&lt;0.05。所有的数据被报告为三个独立实验的平均值±SEM。 <img alt="图5" src="/files/ftp_upload/4216/4216fig5.jpg" /> 图5。剩余活性动力学分析比较WT PlyC 29C3在45°C的平等纯化WT PlyC和29C3的摩尔浓度的热块中孵育在45℃，测定残留的酶活性为3小时，每20分钟。每种构建所显示的活性，被归一化到在时间点零显示的最大速度。与异常的数据点在20分钟，在每个时间点的变化在WT和29C3之间的活性相关性的p-值&lt;0.05。所有的数据被报告为三个独立实验的平均值±SEM。

Watch this Scientific Journal Video about A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes at JoVE.com

耐热溶菌酶定向进化的新筛选方法- 中国优秀硕士学位论文全文数据库免疫学和感染|视频

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

热稳定性工程领域的一种新型的定向进化方法的具体开发，从而验证溶菌酵素。随机诱变只有一轮中，演进式溶菌酶，PlyC 29C3，显示后大于两倍的残留活性相比升高的温度下温育后的野生型蛋白。

耐高温溶菌酶的定向进化的一个新的筛选方法

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

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17.9K 次观看.  University of Maryland. 热稳定性工程领域的一种新型的定向进化方法的具体开发，从而验证溶菌酵素。随机诱变只有一轮中，演进式溶菌酶，PlyC 29C3，显示后大于两倍的残留活性相比升高的温度下温育后的野生型蛋白。

视频: A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Procedure for Adaptive Laboratory Evolution of Microorganisms Using a Chemostat

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) refers to the experimental situation in which evolution is observed using living organisms under controlled conditions and stressors; organisms are thereby artificially forced to make evolutionary changes. Microorganisms are subject to a variety of stressors in the environment and are capable of regulating certain stress-inducible proteins to increase their chances of survival. Naturally occurring spontaneous mutations bring about changes in a microorganism's genome that affect its chances of survival. Long-term exposure to chemostat culture provokes an accumulation of spontaneous mutations and renders the most adaptable strain dominant. Compared to the colony transfer and serial transfer methods, chemostat culture entails the highest number of cell divisions and, therefore, the highest number of diverse populations. Although chemostat culture for ALE requires more complicated culture devices, it is less labor intensive once the operation begins. Comparative genomic and transcriptome analyses of the adapted strain provide evolutionary clues as to how the stressors contribute to mutations that overcome the stress. The goal of the current paper is to bring about accelerated evolution of microorganisms under controlled laboratory conditions.

Here, we present a protocol to obtain adaptive laboratory evolution of microorganisms under conditions using chemostat culture. Also, genomic analysis of the evolved strain is discussed.

程序的微生物实验室自适应演化的恒化

Natural evolution involves genetic diversity such as environmental change and a selection between small populations. Adaptive laboratory evolution (ALE) ...

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遗传学

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

具有更严格特异性的限制内核内核酶的体外定向进化

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

X射线晶体学研究热木体马里蒂玛M42氨基肽TmPep1050的寡聚态过渡

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

生物化学

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

生物学

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident STIM1 regulates the activity of PM Orai1 channels in a process known as store operated calcium (Ca2+) entry which is the principal Ca2+ signaling process that drives the immune response. STIM1 undergoes post-translational N-glycosylation at two luminal Asn sites within the Ca2+ sensing domain of the molecule. However, the biochemical, biophysical, and structure biological effects of N-glycosylated STIM1 were poorly understood until recently due to an inability to readily obtain high levels of homogeneous N-glycosylated protein. Here, we describe the implementation of an in vitro chemical approach which attaches glucose moieties to specific protein sites applicable to understanding the underlying effects of N-glycosylation on protein structure and mechanism. Using solution nuclear magnetic resonance spectroscopy we assess both efficiency of the modification as well as the structural consequences of the glucose attachment with a single sample. This approach can readily be adapted to study the myriad glycosylated proteins found in nature.

Targeting Cysteine Thiols for in Vitro Site-specific Glycosylation of Recombinant Proteins

Biochemical and structural analyses of glycosylated proteins require relatively large amounts of homogeneous samples. Here, we present an efficient chemical method for site-specific glycosylation of recombinant proteins purified from bacteria by targeting reactive Cys thiols.

靶向半胱氨酸硫用于体外重组蛋白的特异糖基化

Stromal interaction molecule-1 (STIM1) is a type-I transmembrane protein located on the endoplasmic reticulum (ER) and plasma membranes (PM). ER-resident ...

免疫与感染

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 ...

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

细菌肽显示用于选择新型生物仿生酶

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

定向进化法<em&gt;酿酒酵母</em&gt;:突变体库的创建和筛选

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

多酶筛选使用高通量基因酶的筛选系统

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

<meta charset="utf8"></head><body>使用低成本 Thermomixer 进行嗜热体驯化的实验进化方案

Adaptation at the Extremes of Life: Experimental Evolution with the Extremophile Archaeon Sulfolobus acidocaldarius

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

<meta charset="utf8"></head><body>作者聚焦：使用创新技术了解微生物适应，探索嗜热性进化

Here, we present an experimental evolution protocol for adaptation in thermophiles utilizing low-cost, energy-efficient bench-top thermomixers as incubators. The technique is demonstrated through the characterization of temperature adaptation in Sulfolobus acidocaldarius, an archaeon with an optimal growth temperature of 75 &#176;C.

生命极端的适应：极端微生物 Sulfolobus acidocaldarius 的实验进化

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures ...

耐高温溶菌酶的定向进化的一个新的筛选方法

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