这项工作已经支持K08奖CA116429 - 04到MC，并授予一个从征服癌症（关注）的基础＃8501

一，生成一个随机突变体库波尔我介导启动ColE1质粒复制（检讨（1-3），我们诱变的方法是放置在一个ColE1质粒相邻的目标序列的来源或复制和传播细胞表达低高保真DNA聚合酶I的基础（LF聚合物我）。LF聚合物我是一个突变的DNA聚合酶I的编码三种突变，减少复制的保真度，即I709N（在图案中的一个），A759R（图案乙）和D424A （灭活校对）4,5 LF聚合物LF -波尔，我是在大肠杆菌菌株，JS200，我波尔（polA12）（6）的温度敏感等位基因表达，使我成为主要活动在37 ° C.复制数量目标序列。限制性条件下生成一个随机的突变体库的结果在polA12细胞的突变是更有效的在4饱和的文化，对于目前仍不清楚的原因，是不连续的突变，即基因突变频率不呈线性增加几代人的文化一旦达到饱和，即使细胞可以进一步扩大新媒体，因此，进一步增加了图书馆的突变负荷，需要反复的诱变和质粒恢复轮，在这里，我们提供LF聚合物我诱变的协议。注意：这里介绍的协议已经大大简化，以便为实现所需的突变负载（图1）迭代的过程相对于我们原来的描述4。 材料<ul><li>细胞：JS200 recA718 polA12（TS）uvrA155 trpE65 LON - 11苏拉<ul><li> JS200的WT - POL：JS200细胞表达野生型（WT）波尔我</li><li> JS200低频聚合物我：JS200表达低的保真度（星期三）波尔我</li><li>读数应变：JS200的WT - POL I或（互补）的应变，缺乏一个特定的活动</li></ul></li><li>目标质粒<ul><li>质粒的克隆目标基因的复制ColE1起源</li></ul></li></ul> 1。前突变：JS200电主管LF聚合物我细胞的制备<ol><li>从30℃（宽松的条件，请选择）长出了LB平板单一JS200 LF聚合物我的殖民地一夜之间LF聚合物我（0.03mg / mL氯霉素）的质粒轴承包含适当的抗生素选择和放置到测试的殖民地管内含有与氯霉素的LB 8毫升。文化增长30 ° C和摇晃一夜之间在250rpm。 </li><li>在早上，扩大与氯霉素含有400毫升LB培养基的烧瓶中倒入8毫升JS200 LF - 波尔我的文化。让文化的增长在30 ° C，而在250rpm晃动，直到达到0.4-0.7一个600（一般为3 - 4H）。 </li><li>一旦在0.4-0.7一个600，冷却15分钟的湿冰文化。 </li><li>将冷冻文化离心适当的容器。沉淀细胞在4 ° C离心倒掉上清液，然后加10毫升无菌和冰鲜（湿冰）10％的甘油，使用血清吸管容器和重新暂停细胞。 </li><li>重悬细胞转移到50 mL锥形管。填充锥形管，用10％甘油45毫升标记，然后离心15分钟，在4 ° C和4000RPM。 </li><li>倒掉上清液，加一次10毫升10％甘油的锥形管，并重新暂停使用血清或正常吸管的细胞。同样，填补了10％甘油和15分钟的离心锥管45毫升标记在4 ° C和4000RPM。重复这一过程中，两次以除去所有盐的痕迹。 </li><li>最终分拆后，重新暂停在相等的部分10％的甘油的颗粒细胞（即重新暂停2毫升细胞与2毫升10％甘油）。 </li><li>分装在100μL和500μL悬浮细胞成几个存储管。速冻干冰的细胞，然后存放在-80 ° C。 </li><li>在使用电主管转换的细胞，慢慢在冰上解冻的细胞。 </li></ol> 2。突变：质粒转化的目标<ol><li>吸取40微升电主管JS200 LF聚合物我细胞与成2mm的差距电比色皿的目标质粒DNA 30 - 250ng。 注1：一个ColE1质粒含有绿色荧光蛋白可以通过与作为对照的靶基因平行突变进行。读出一步完成后，GFP可镀的LB琼脂平板和诱变可视化。出现深色或暗淡的殖民地包含失活的突变。 </li><li>在electroporator 1800V脉冲的混合物。检查的时间常数（T C），以确保对每一个样品的均匀电条件; 理想 T C = 5-6秒。 </li><li>恢复细胞/ DNA混合物在37℃（40分钟1 mL LB培养基中trictive条件）在250转摇晃。 </li><li>板50μL恢复文化的一个100mm的LB琼脂培养皿上预先加热至37 ° C含有氯霉素和目标质粒的抗生素选择适当的浓度。 注：＃2：目标板在“不久的草坪”浓度的细胞。一个“附近的草坪”的浓度被定义为一个鲜明的殖民地，但不可数数（&gt; 1000菌落/百毫米盘）。镀稀释，如果有的话，将取决于如何电主管细胞。如果细胞不是很电主管和一个“附近的草坪”是不是可以实现电镀恢复文化“整齐”，然后离心2分钟，恢复文化在4000RPM，倒出上清液，在50个重新暂停细胞μLLB培养基和板块的文化。 </li><li>孵育的Petri洗涤在37（S）一夜之间° C。 </li></ol> 3。质粒突变：恢复<ol><li>第二天，洗移液2 mL LB培养基中的细胞在培养皿中。从LB琼脂培养皿中的细菌菌落转移到LB培养基“净化”他们用消毒的三角形状的玻璃棒板。首先加入1 mL LB培养基中，收集洗，重复第二毫升LB肉汤步骤。 </li><li>隔离的质粒DNA从洗板。 （质粒DNA构成的库）。 注3：从LB平板收集洗可能过于密集在其全部的小预习。如果是这样的情况下，小型预习最大配发金额由您的迷你准备试剂盒（通常情况下，这涉及稀释洗OD = 1和预备〜3毫升稀释后的文化），或扩展到最大准备</li></ol> 4。迭代<ol><li>孤立的质粒DNA用限制性内切酶线性化LF聚合物我质粒1μg限制，但不削减目标质粒（补充图1）。 </li><li>使用DNA纯化试剂盒，清理限制性消化。 注4：这一步中删除所有的限制性内切酶和它的缓冲区的痕迹。这一步是必须保持低盐浓度为后续电主管转换。 </li><li>重新改造限制的目标质粒到新鲜JS200 LF聚合物细胞库回30 - 250ng通过随后的几轮的诱变库。 注5：POL我质粒用限制性内切酶线性化，确保目标质粒得到转化。 </li><li>重复第2和第3。 </li></ol> 5。读数<ol><li>限制孤立的质粒DNA用限制性内切酶（S）的线性化目标质粒和POL我质粒。运行在1％琼脂糖凝胶的精华，以确保质粒的数量和质量。限制〜400ng的孤立的质粒DNA通常是足够进行分析。 </li><li>孤立的质粒DNA用限制性内切酶线性化LF聚合物我质粒1μg限制，但不会削减你的目标质粒。 </li><li>使用DNA纯化试剂盒，清理限制性消化。 </li><li>转换成读出的应变表征突变质粒文库的限制目标。 </li></ol>二。突变屏幕和性状分析，采用梯度生长板。 为了说明如何广阔的遗传多样性存在于我们的图书馆，可以再加上一个功能选择，在这里我们提供了药物在体内的功能在大肠杆菌中选择一个协议大肠杆菌 。这种方法是根据药物在固体琼脂梯度沿增长。这使得多个（最多12个）样品的浓度范围的同步特性，提供更广泛的动态范围比单一药物治疗。另一个优点是，这个实验中的非线性读数，缓冲区内2倍的范围内，适度的差异，在可行性。因此，这种细胞毒性电阻检测提供了一个强大的和迅速的方式，选择突变体库，并确定个别突变体的表型剖面。图2显示了每个使用的例子：面板显示的单个突变体的选择从人类的氧化去甲基ABH2库。以上WT门槛越来越殖民地被选中为增加引起的细胞毒作用的甲基化剂甲基磺酸（MMS），7保护。 B组显示，梯度用于个别克隆鉴定的一个例子。耐第三代头孢类抗生素头孢噻肟的水平，是野生型β-内酰胺酶的琼脂梯度和两个广谱的突变体，R164H，E104K R164S G267R 4所示。请注意，根据对所观察到的效果的力量，比单盘可能是必要的足够的定量的：0.4mg/ml梯度，同时可以控制克隆的直接比较，最有力的突变体的阻力水平只能建立使用较高的浓度tration头孢噻肟（4mg/mL）。 材料<ul><li>设备<ul><li> 100x100x15mm平方米培养皿（费舍尔科学＃0875711A） </li><li> 100毫米一轮培养皿</li><li> 25x75x1mm玻璃显微镜幻灯片（菲舍尔科学＃1255015） </li><li> 50毫升管毕业</li></ul></li><li>媒体<ul><li> LB琼脂融化水浴中平衡，并在56 ° C 注6：媒体的温度可能会影响稳定，因而正在研究的药物或复合活动。 </li><li>软琼脂融化，在水浴中平衡至42 ° C </li></ul></li></ul> 1。建设梯度<ol><li>马克十车道，跨平方米的培养皿底部的一个边缘均匀间隔。 </li><li>放置在一个倾斜等，显着的底部边缘升高7毫米的菜; 厚的草或其他平面物体上，可以被用来作为支持提升菜。倒入25毫升温水（〜56 ° C）以及LB琼脂混合成斜盘选择代理的适当集中。这是一个渐变的底层。确保LB琼脂均匀覆盖的培养皿中，这样的菜明显升高，最终包含LB琼脂〜1mm和降低部分包含〜LB琼脂8毫米。然后让琼脂为10-15分钟。 注7：对于疏水性选择剂，可添加0.1％的表面活性剂（消泡剂乙乳液）的LB琼脂，便于悬挂和均匀分布的药物。添加表面活性剂的热情无菌的LB琼脂剧烈摇晃前除了选择代理。表面活性剂应暂停作为一个小水滴的罚款阴霾的，大的飞沫媒体太热，并可能抑制试验药物的均匀分布。 </li><li>第25 mL的LB琼脂硬化后，菜是一个平坦的表面上移动。下一步，没有选择剂25毫升LB琼脂倒入覆盖第一的LB琼脂表面。这是一个渐变的顶层。确保LB琼脂覆盖整个表面的底层。盖上盖通风那副，并允许琼脂为10-15分钟。 注8：了解气溶胶的危害和对测试化合物的潜在挥发;倒在化学安全要求“规定的化学或生物安全罩的梯度。 注9：渐变的食物应在4h内用于维持的药物或测试化合物的浓度梯度。 </li></ol> 2。邮票的细菌转移<ol><li>软琼脂应平衡至42 ° C。转移到一个100mm的圆形培养皿底部的盖子或2毫升的液体软琼脂。吸取40μL的细菌培养到软琼脂，然后混合摇板。 注10：细菌培养的成长阶段，可能会影响其药物或测试化合物的反应。日志相或固定相过夜培养的文化，应采用统一的结果的一致性。细胞密度也可以倾斜的相对结果，因此所有的文化，应稀释至 600密度值匹配。稀释过夜培养应该有一个600的密度小于1.0 </li><li>外套的长边与软琼脂混合物的玻璃显微镜幻灯片。然后，对准梯度菜涂层边缘的底部标志的幻灯片（从低到高浓度），触摸琼脂表面的幻灯片。柔软的触感足以转移的梯度表面的软琼脂剪彩。幻灯片，然后预留的清洁和重用。 </li><li>重复这一过程中，其余的细菌样本。参考样品应包括在每个梯度菜，如果正在运行多个梯度。 </li><li>梯度菜自上而下一夜之间在37 ° C。时间和孵化温度，可能会有所不同不同的读出的菌株，但一夜之间在37 ° C时的典型足够明显增长。 </li></ol> 3。成像和分析增长<ol><li>成像：经过一夜的增长，梯度可以直接成像或固定和0.2mg的/ mL的吖啶橙与95％乙醇溶液，以提高对比度染色。板在室温下孵育5分钟，染色溶液中，，然后用95％乙醇，然后在一个紫外线灯箱成像。 注11：小心不刷新板的殖民地，而是岩石板的染色和洗涤溶液和删除的解决方案，从角落。 </li><li>个别突变质粒表型分析，对浓度梯度增长的距离是一个标准，每个梯度归。这些相对值，然后可以跨梯度相比。 注12：根据不同的细胞毒作用的性质，锋利的边缘或更弥漫的边缘，可观察（例如主机A和乙在图2比较）。在弥漫边缘的情况下，最好是衡量边缘Øf持续增长，作为单独的殖民地往往表现出增加的变异。 </li><li>对于库中选择，浓度比亲本的野生型控制的生长的单个菌落分离和测序，以确定更多的保护作出贡献的突变。 </li></ol>三。代表性的成果： <img src="/files/ftp_upload/2505/2505fig1.jpg" alt="图1" /> 图1。液体和直接电镀突变协议之间的比较 。直接电镀突变协议（底部）速度更快，需要比我们原来的液体诱变协议（顶部）步骤更少。当GFP作为一个记者，在荧光的变化指示库中的遗传多样性目前。通常情况下，一个周期的荧光水平明显下降12-18％的菌落突变结果。 <img src="/files/ftp_upload/2505/2505fig2.jpg" alt="图2" /> 图2。坡度阻力分析。阻力增大，甲基甲烷磺酸（MMS），A组的选择 。质粒人类的氧化去甲基ABH2库被选定为阻力增大到MMS。两个代表两个和四个迭代的诱变协议库是比较父母的野生型（WT）和空载体（Δ）所示。白线表示上述个别突变菌落作进一步的表型分析隔离阈值。B组用头孢噻肟的保护，超广谱β-内酰胺酶的活性的指标。R164H，E104K R164H G267R，先前发现的两种β-内酰胺酶突变体LF - POL我诱变耦合氨曲南选择4，显示在0.4μg/mL和4μg/mL头孢噻肟梯度。需要注意的是野生型β-内酰胺酶，赋予细胞表达一个空向量，Δ相对没有保护。因此，这些突变的一个新的生化活动8,9发展。 <img src="/files/ftp_upload/2505/2505fig3.jpg" alt="图3" /> 图3。作为ORI距离（D）功能的突变频率 。以下单周期的直接电镀诱变的突变频率是100 BP间隔相对ColE1起源的复制的RNA / DNA开关。 1600和2400之间的区域，是不是代表，因为β-内酰胺酶，并不代表一个中立的目标。超出β-内酰胺酶的点代表200 bp的突变在这方面的整体低频的间隔。趋势（二项式方程的R 2 = 0.41），显示为一条线。 <img src="/files/ftp_upload/2505/2505fig1sup.jpg" alt="图1补编失踪" /> 补充图1。我含低频POL POL我质粒。一个序列 （FASTA格式）。序列信息确定适当的限制性内切酶（S）使用线性POL我质粒时，迭代（随机突变库第4步代）或读数（步骤5）。B一般功能和限制的质粒地图。 pSC101原产地复制，氯霉素抗性标记（CAT）和LF聚合物I基因的位置。以及单一酶切位点的位置表示。 <table border="1"><tbody><tr><td> 图书馆 </td><td></td><td> 直接电镀 </td><td> 液体 （1天） </td><td> 液体 （3天） </td></tr><tr><td> 突变（＃） </td><td></td><td> 95 </td><td> 40 </td><td> 142 </td></tr><tr><td> 克隆测序 </td><td> 288 </td><td> 96 </td><td> 190 </td></tr><tr><td> 全覆盖（BP） </td><td> 182000 </td><td> 102000 </td><td> 213000 </td></tr><tr><td> 突变频率（X10 3 BP） </td><td> 0.52 </td><td> 0.39 </td><td> 0.67 </td></tr><tr><td> D &lt;1000频率（X10 3 BP） </td><td> 0.92 </td><td> 0.41 </td><td> 0.70 </td></tr></tbody></table> 表1。突变频率为D &lt;1000，即频率（＃突变/ BP）在1000 BP相邻的RNA / DNA开关，3个突变协议，：直接电镀，液体饱和度（1天），液体hypersaturation（ 3天）。 <table border="1"><tbody><tr><td></td><td></td><td> 直接电镀 </td><td> 液体 </td></tr><tr><td> 突变（＃） </td><td></td><td> 95 </td><td> 182 </td></tr><tr><td> 频谱（％） </td><td></td><td></td><td></td></tr><tr><td> A至G </td><td> A至G </td><td> 6.3 </td><td> 19.2 </td></tr><tr><td></td><td> T到彗星 </td><td> 4.2 </td><td> 3.8 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 彗星为T </td><td> G为一个 </td><td> 27.4 </td><td> 13.7 </td></tr><tr><td></td><td> 彗星为T </td><td> 35.8 </td><td> 35.2 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> A至T </td><td> A至T </td><td> 5.3 </td><td> 8.2 </td></tr><tr><td></td><td> T为一个 </td><td> 5.3 </td><td> 7.1 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> T到摹 </td><td> T到摹 </td><td> 1.1 </td><td> 1.1 </td></tr><tr><td></td><td> A至C </td><td> 0.0 </td><td> 2.2 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> 摹到T </td><td> 摹到T </td><td> 1.1 </td><td> 2.7 </td></tr><tr><td></td><td> C到A </td><td> 2.1 </td><td> 2.7 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> C至G </td><td> C至G </td><td> 2.1 </td><td> 1.1 </td></tr><tr><td></td><td> G到彗星 </td><td> 5.3 </td><td> 2.7 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td> INDELS </td><td> 宏 </td><td> 3.2 </td><td> 0.0 </td></tr><tr><td></td><td> 德尔 </td><td> 1.1 </td><td> 0.0 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td> A至N </td><td> 11.6 </td><td> 29.7 </td></tr><tr><td></td><td> G至N </td><td> 33.7 </td><td> 19.2 </td></tr><tr><td></td><td> T到ñ </td><td> 10.5 </td><td> 12.1 </td></tr><tr><td></td><td> 彗星为N </td><td> 40.0 </td><td> 39.0 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td> TS </td><td> 73.7 </td><td> 72.0 </td></tr><tr><td></td><td> 电视机 </td><td> 22.1 </td><td> 28.0 </td></tr><tr><td></td><td> INDELS </td><td> 4.2 </td><td> 0 </td></tr></tbody></table> 表2。直接电镀液的诱变指标的突变 。表列出了观察到的突变数（数量）和突变谱（％）以下的单周期突变。互补对频谱分解，核苷酸的变化，突变类型。

<ol>
	<li>Camps, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+ColE1-like+plasmid+replication+for+recombinant+gene+expression.">Modulation of ColE1-like plasmid replication for recombinant gene expression.</a> Recent Pat DNA Gene Seq. 4, 58-73 (2010).</li><li>Itoh, T., Tomizawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+replication+of+plasmid+ColE1+DNA+by+RNA+polymerase,+ribonuclease+H,+and+DNA+polymerase+I.">Initiation of replication of plasmid ColE1 DNA by RNA polymerase, ribonuclease H, and DNA polymerase I.</a> Cold Spring Harb Symp Quant Biol. 43 Pt 1, 409-417 (1979).</li><li>Polisky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ColE1+replication+control+circuitry:+sense+from+antisense.">ColE1 replication control circuitry: sense from antisense.</a> Cell. 55, 929-932 (1988).</li><li>Camps, M., Naukkarinen, J., Johnson, B. P., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+evolution+in+Escherichia+coli+using+a+highly+error-prone+DNA+polymerase+I.">Targeted gene evolution in Escherichia coli using a highly error-prone DNA polymerase I.</a> Proc Natl Acad Sci U S A. 100, 9727-9732 (2003).</li><li>Shinkai, A., Loeb, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+mutagenesis+by+Escherichia+coli+DNA+polymerase+I.+Ile(709)+in+motif+A+functions+in+base+selection.">In vivo mutagenesis by Escherichia coli DNA polymerase I. Ile(709) in motif A functions in base selection.</a> J Biol Chem. 276, 46759-46764 (2001).</li><li>Uyemura, D., Lehman, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+characterization+of+mutant+forms+of+DNA+polymerase+I+from+Escherichia+coli.+I.+The+polA12+mutation.">Biochemical characterization of mutant forms of DNA polymerase I from Escherichia coli. I. The polA12 mutation.</a> J Biol Chem. 251, 4078-4084 (1976).</li><li>Sedgwick, B., Robins, P., Lindahl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+removal+of+alkylation+damage+from+DNA+by+AlkB+and+related+DNA+dioxygenases.">Direct removal of alkylation damage from DNA by AlkB and related DNA dioxygenases.</a> Methods Enzymol. 408, 108-120 (2006).</li><li>Aharoni, A., Gaidukov, L., Khersonsky, O., Mc, Q. G. S., Roodveldt, C., Tawfik, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+'evolvability'+of+promiscuous+protein+functions.">The 'evolvability' of promiscuous protein functions.</a> Nat Genet. 37, 73-76 (2005).</li><li>Elena, S. F., Lenski, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+experiments+with+microorganisms:+the+dynamics+and+genetic+bases+of+adaptation.">Evolution experiments with microorganisms: the dynamics and genetic bases of adaptation.</a> Nat Rev Genet. 4, 457-469 (2003).</li><li>Itoh, T., Tomizawa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FoFormation+of+an+RNA+primer+for+initiation+of+replication+of+ColE1+DNA+by+ribonuclease+H.">FoFormation of an RNA primer for initiation of replication of ColE1 DNA by ribonuclease H.</a> Proc Natl Acad Sci U S A. 77, 2450-2454 (1980).</li><li>Bryan, S. K., Moses, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sufficiency+of+the+Klenow+fragment+for+survival+of+polC(Ts)+pcbA1+Escherichia+coli+at+43+degrees.">Sufficiency of the Klenow fragment for survival of polC(Ts) pcbA1 Escherichia coli at 43 degrees.</a> C. J Bacteriol. 170, 456-458 (1988).</li></ol>

本文介绍了突变的协议，它允许生成大量的随机突变库，而不需要进行克隆或PCR。这种方法是基于对容易出错的复制质粒的编码序列的利益。从理论上讲，基因突变应在很大程度上制约100-300位于下游的RNA / DNA开关立即基点，领导人链中间的大小产生波尔我 2,10 。我们发现，在本议定书中提出的条件下，波尔我的突变发生各地质粒，虽然在频率递减质粒ORI增加（图3）的距​​离。这一发现意味着过渡或“开关”我从波尔在ColE1质粒复制波尔三是比以前的报道更逐步至少在我们的实验条件，并同意与早先的研究表明POL我和波尔之间的功能冗余，三11。 我们原来的协议描述（4）液体培养的突变。该协议的产量在1000 BP相邻的RNA / DNA开关（见表1）D &lt;1000，即0.41突变/ KB。离开3天的液体培养摇床，没有增加任何新媒体Hypersaturation引发基因突变频率0.70突变/ KB，但在十分恶劣的质粒产量的结果（小于1％相比，每日1次;，数据未显示）（表1）。在这里，我们提出了一个简化的协议基础上，直接电镀固体琼脂质粒改造的目标，在37 ° C（图1）。此过程产生的最大频率/周期诱变突变（0.92突变/ D &lt;1000 KB），极大地方便了迭代。固体介质改变培养条件，也影响的突变谱（见表2）。直接电镀诱变降低之间存在明显的互补碱基对在液体培养基中看到换人的不对称性（比较C→T与摹→A和A→G与T→彗星）。另一方面，直接电镀生产更多的INDELS（小于0.5％至4％）和A→G突变的数量下降了3倍，领先的G / C突变（74％）的比例过高。总体而言，我们认为，更简单和直接电镀这里介绍的协议效率的提高超过了稍微平衡液体诱变产生的突变谱的好处。 在目标质粒，我们的方法所产生的突变是不限制到所需的靶序列。然而，新颖的生化活动的演变应依赖于内的靶基因的突变，因为它代表了定性，而不是一个由量变。因此，结合梯度板的突变体的筛选质粒突变，我们利用我们的系统演化的新的生化特性（大型图书馆和选择可用性）的优势。优化现有的酶活性的或随机的基因的特定领域，如随机突变的其他应用程序需要克隆的随机突变。在这种情况下，质粒库，而不是一个PCR扩增产物提高克隆的效率，促进扩增和限制性。 总之，我们展示了一个简单的协议创造一个给定的靶基因，因其简单性和生成的库的多样性，突出的随机突变体库。我们表明这种方法可以高效演进的新的生化活动的功能选择。此外，我们在体内生成的库可以轻松克隆，允许特定地点的突变或优化现有的活动。

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>carbenicillin</td>
<td>&nbsp;</td>
<td>CellGro</td>
<td>46100R6</td>
<td>0.1mg/ml final</td>
</tr>
<tr>
<td>tetracycline</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP7640</td>
<td>0.05mg/ml final</td>
</tr>
<tr>
<td>chloramphenicol</td>
<td>&nbsp;</td>
<td>Genlantis</td>
<td>M120100</td>
<td>0.03mg/ml final</td>
</tr>
<tr>
<td>LB Agar Miller</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP1425</td>
<td>&nbsp;</td>
<tr>
<td>LB Broth Miller</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>244620</td>
<td>&nbsp;</td>
<tr>
<td>Soft Agar</td>
<td>&nbsp;</td>
<td>Difco</td>
<td>214580</td>
<td>Made in house</td>
</tr>
<tr>
<td>Acridine Orange</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A38401-1</td>
<td>&nbsp;</td>
<tr>
<td>Antifoam B emulsion</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A5757</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>332030025</td>
<td>&nbsp;</td>
<tr>
<td>100x100x15mm sq dish</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>0875711A</td>
<td>&nbsp;</td>
<tr>
<td>100mm rnd dish </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>0875712A</td>
<td>&nbsp;</td>
<tr>
<td>Microscope slide 25x75x1mm</td>
<td>&nbsp;</td>
<td>Gold Seal</td>
<td>3048</td>
<td>&nbsp;</td>
<tr>
<td>Electroporator 2510</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Electroporator 2510</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>2mm gap tubes</td>
<td>&nbsp;</td>
<td>MolBioproducts</td>
<td>5520</td>
<td>&nbsp;</td>
<tr>
<td>Zippy mini prep kit</td>
<td>&nbsp;</td>
<td>Zymo </td>
<td>D4020</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

mutagenesis and functional selection protocols for directed evolution of proteins in e. coli

遗传多样性的有效代一个宝贵的工具，可用于标签的DNA合成，创造出独特的分子签名，​​或演变在实验室中的蛋白质分子。在这里，我们提出了一个协议，允许大（&gt; 10 月 11日）对于一个给定的目标序列的突变体库的生成。这种方法是基于一个ColE1质粒编码所需的顺序由低高保真DNA聚合酶变异的复制我（LF聚合物我）。目标质粒转化成五的mutator应变大肠杆菌和镀固体培养基上，在0.2和1个突变/ KB，根据靶基因的位置，之间产生。迭代这种突变的过程，取得了较高的突变频率。突变的替代方法相比，我们的协议代表了它的简单，没有克隆或PCR参与。因此，我们的方法是突变的质粒或其他POL我模板的标签，或探索在原来的目标，不存在活动的演变大部份的序列空间的理想选择。紧空间的控制，PCR或随机寡核苷酸为基础的方法提供也可以通过克隆库的特定部分的后续实现。在这里，我们提供的协议显示了如何创建一个随机突变体库，以及如何建立在大肠杆菌中的药物为基础的选择大肠杆菌对发现的突变体，表现出新的生化活动。

Watch this Scientific Journal Video about Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli at JoVE.com

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

在这里，我们展示了一个简单的协议来创建一个给定的目标序列的随机突变体库。我们表明，这种方法，这是在大肠杆菌体内进行，如何可以再加上功能选择进化出新的酶活动。

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 ...

mutagenesis-functional-selection-protocols-for-directed-evolution

Research

JoVE Journal

Biology

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

30.2K 次观看.  University of California Santa Cruz - UCSC. 在这里，我们展示了一个简单的协议来创建一个给定的目标序列的随机突变体库。我们表明，这种方法，这是在大肠杆菌体内进行，如何可以再加上功能选择进化出新的酶活动。

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

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

科研

生物学

Homemade Site Directed Mutagenesis of Whole Plasmids

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned target gene can be altered by substitution, deletion or insertion of a few bases directly into a plasmid. It works by simply amplifying the whole plasmid, in a non PCR-based thermocycling reaction. During the reaction mutagenic primers, carrying the desired mutation, are integrated into the newly synthesized plasmid. In this video tutorial we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid. The protocol works with standard reagents and is independent from commercial kits, which often are very expensive. Applying this protocol can reduce the total cost of a reaction to an eighth of what it costs using some of the commercial kits. In this video we also comment on critical steps during the process and give detailed instructions on how to design the mutagenic primers.

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. Here we demonstrate an easy and cost effective way to introduce base substitutions into a plasmid using standard reagents.

自制的网站定向诱变整个质粒

Site directed mutagenesis of whole plasmids is a simple way to create slightly different variations of an original plasmid. With this method the cloned ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

使用油脂介孔结构的测定结晶膜蛋白

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

异源表达在哺乳动物细胞离子通道的突变和功能分析

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

研究膜蛋白的构象动力学原位网站的荧光标记

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

使用机器人在油脂中间相高通量膜蛋白的结晶

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

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 ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

高通量定量表达筛选与纯化应用到重组二硫键丰富的蛇毒蛋白产于<em&gt; E。大肠杆菌</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

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 ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.