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View additional product information for pYES2.1 TOPO™ TA Yeast Expression Kit - FAQs (K415001)
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我们不建议将感受态细胞保存在液氮中，因为极端温度会损害细胞。另外，装感受态细胞的塑料管子可能承受不了如此低的温度，从而发生破裂。
我们推荐将感受态细胞存储在-80摄氏度。高于这个温度，即使存储时间很短，也会显著降低其转化效率。
以下是用于培养Pichia pastoris和S. cerevisia的营养丰富型和基本培养基：
营养丰富型培养基：
适用于S. cerevisiae和Pichia pastoris
•YPD（YEPD）：酵母提取物、蛋白胨和葡聚糖
•YPDS：酵母提取物、蛋白胨、葡聚糖和山梨醇
仅适用于Pichia pastoris
•BMGY：缓冲型甘油复合培养基
•BMMY：缓冲型甲醇复合培养基
基本培养基（又名缺陷型培养基）：
适用于S. cerevisiae
•SC（SD）：完全合成培养基（YNB、葡聚糖（或棉籽糖或半乳糖）以及氨基酸）
适用于Pichia pastoris
•MGY：基本甘油培养基
•MD：基本葡聚糖培养基
•MM：基本甲醇培养基
•BMGH：缓冲型基本甘油培养基
•BMMH：缓冲型基本甲醇培养基
当α因子基因中的P因子被替换为α时，S. pombe不能生成P因子。但是，当P因子基因中的α因子被替换为P时，S. pombe能够生成α因子。有反面证据表明，S. pombe能够加工其自身的交配因子切割位点，但并不是S. cerevisiae α因子的所有切割位点。最好使用一个更通用的信号序列（而不是前导信号序列，如α）。如果必须走前导路线，最好使用S. pombe的P因子前导而不是S. cerevisiae的α前导。
是的，我们提供pYES2.1/V5-His-TOPO载体，它是pYES2.1 TA表达试剂盒（货号K415001）的一部分，可将Taq聚合酶扩增的PCR产物直接克隆到Saccharomyces cerevisiae中并利用半乳糖调控其表达。
在诱导期间，一些研究人员选择使酵母生长在以2%半乳糖作为唯一碳源的培养基中。但是，在含2%半乳糖和2%棉籽糖的诱导培养基中，酵母通常生长更快。棉籽糖是酵母的良好碳源，与葡萄糖不同，棉籽糖不会抑制GAL启动子的转录。棉籽糖是由半乳糖、葡萄糖和果糖依次连接而形成的三糖。大部分酵母可切割葡萄糖-果糖键，但不会切割半乳糖-葡萄糖键。果糖随后被用作碳源。
我们提供INVSc1酵母株。这是一种二倍体酵母株，仅作表达用途。该酵母株不能形成良好的孢子，因此不适用于酵母遗传研究。INVSc1酵母株的基因型和表型如下：
基因型：MATa his3Δ1 leu2 trp1-289 ura3-52/MATα his3Δ1 leu2 trp1-289 ura3-52
表型：His-, Leu-, Trp-, Ura-
注意：INVSc1缺乏组氨酸、亮氨酸、色氨酸和尿嘧啶。该酵母株在缺乏组氨酸、亮氨酸、色氨酸和尿嘧啶的SC基本培养基中不能生长。
TE、醋酸锂和PEG的储液可保存。但是，制备待转化细胞的混合溶液，必须每次现用现配。如果溶液不是新配制的，则可能损失转化效率。
OD600值为0.1，约等于3 x 106细胞/毫升。
转化效率很大程度上取决于酵母株，范围为1000-100,000个转化株/微克 DNA。
以下是用于S. cerevisiae转化的不同方法：
•S. cerevisiae EasyComp转化试剂盒：易于使用的即用型试剂 ◦感受态细胞可冻存。转化效率高于103转化株/微克 DNA。与使用新鲜制备的细胞相比，使用冻存细胞可得到更高的转化效率。
•小规模酵母转化实验方案（见使用手册第13页）
•醋酸锂转化：简单、自己动手的实验方案 ◦感受态细胞必须是新鲜制备的
•电穿孔：简单、高效，自己动手的实验方案 ◦感受态细胞必须是新鲜制备的
•原生质球试剂盒：高效，工作量大，不适用于抗生素筛选
注意：选择适当的接种密度。菌落将在几天内出现。不要挑选最大的菌落，因为它们通常为抑制子。
我们建议将酵母置于15%甘油中保存在–80°C。甘油储液可长期保存（除非经过多次冻融）。在制备甘油储液时，我们建议使用过夜培养物并将其浓缩2-4倍。将细胞离心，并使用原始体积25–50%的甘油/培养基重悬。最好使用新鲜培养基加甘油冻存细胞，而不仅仅是将甘油加到过夜培养物中这么简单。
我们提供original Pichia pastoris表达系统、PichiaPink表达系统和Saccharomyces cerevisiae酿酒酵母表达系统，用于重组蛋白表达。已知P. pastoris和S. cerevisiae遗传性质已十分明确，均可进行多种翻译后修饰。
P. pastoris毕赤酵母表达系统结合了大肠杆菌表达（高水平表达、易于扩大规模和低成本）和真核系统表达（蛋白加工、折叠和翻译后修饰）的优势，从而可对有功能活性的重组蛋白进行高水平生产。作为一种酵母，毕赤酵母Pichia pastoris与酿酒酵母Saccharomyces cerevisiae具有相似的分子和基因操作优势，而且其外源蛋白表达水平比酿酒酵母Saccharomyces cerevisiae高10-100倍。这些特性使毕赤酵母Pichia pastoris 非常适合用作蛋白表达系统。Pichia表达载体含有强乙醇氧化酶（AOX1）启动子，用于高水平、严格调控的诱导型的表达；或者含有甘油醛-3-磷酸脱氢酶（GAP）启动子，用于高水平的组成型表达。诱导型和组成型表达构建体均整合到P. pastoris基因组，建立蛋白表达水平极高的稳定宿主，特别是在使用发酵器的情况下。我们可提供的Pichia pastoris表达系统包括：
•PichiaPink酵母表达系统：最新的Pichia pastoris表达系统包含低拷贝和高拷贝质粒骨架、8种分泌信号序列和4种酵母菌株，这些有助于优化得到最高的重组蛋白产率。所有PichiaPink载体都含有AOX1启动子，用于高水平诱导型表达；还含有ADE2标记物，利用ADE2互补作用筛选转化株（即腺嘌呤缺陷型的互补）而不是抗生素筛选。但是，它们通过不同长度的启动子表达ADE2基因产物，从而决定了整合质粒的拷贝数。在pPink-LC载体中，ADE2标记物的启动子为82 bp，可提供低拷贝表达；在pPink-HC载体，ADE2标记物的启动子为13 bp，可提供高拷贝表达。该系统也可诱导pPinkα-HC载体（含S. cerevisiae α-交配因子前导序列）产生高拷贝数分泌表达，并且还包含8种分泌信号序列可优化分泌表达。
•EasySelect Pichia表达试剂盒：一种传统Pichia 表达试剂盒，包含pPICZ和pPICZα载体，可分别用于目的基因的细胞内和分泌表达。这些载体含有AOX1启动子，可产生高水平的诱导型表达；还含有Zeocin抗生素抗性标记物，可直接进行多拷贝整合载体的筛选。它们有助于对表达的蛋白进行简单的亚克隆、纯化以及快速检测。
•Original Pichia表达试剂盒：该试剂盒包含pPIC9、pPIC3.5、pHIL-D2和pHIL-S1载体，每种载体都含有AOX1启动子，可产生高水平的诱导型表达；还含有HIS4基因，用于在缺乏组氨酸的培养基上筛选his4酵母株。pPIC9带有S. cerevisiae α-因子分泌信号，而pHIL-S1带有Pichia pastoris碱性磷酸酶信号序列（PHO），可指导蛋白质向培养基的转运。pHIL-D2和pPIC3.5专为细胞内表达而设计。
•多拷贝Pichia表达试剂盒：该试剂盒专为最大化表达而设计，包含pPIC3.5K、pPIC9K和pAO815载体，可产生和选择含多个目的基因的Pichia菌株。它们可通过体内方法（pPIC3.5K和pPIC9K）或体外方法（pAO815）分离和生成多拷贝插入片段。所有这些载体都含有AOX1启动子，可产生高水平的诱导型表达；还含有HIS4基因，用于在缺乏组氨酸的培养基上筛选his4菌株。pPIC9K载体可指导表达蛋白的分泌，而从pPIC3.5K和pAO815载体表达的蛋白仍留在细胞内。pPIC9K和pPIC3.5K载体带有卡那霉素抗性标记物，从而使Pichia对Geneticin试剂产生抗性。可通过Geneticin试剂抗性水平高低来鉴定自发的多次插入事件。在缺乏组氨酸的培养基上对Pichia转化株进行筛选，并筛选它们对Geneticin试剂的抗性水平。在高浓度Geneticin试剂中的生长能力，表示多拷贝的卡那霉素抗性基因以及目的基因被整合到了基因组。
为实现在S. cerevisiae中的表达，我们提供pYES 载体系列。每个pYES载体都带有GAL1基因的启动子和增强子序列，可实现诱导型表达。GAL1启动子是使用最广泛的酵母启动子之一，因为它在半乳糖的诱导下具有很强的转录活性。pYES载体还具有2 µ复制起点，可以维持游离的高拷贝数（10-40拷贝/细胞）。
酵母是一种单细胞的真核生物，在成分确定培养基中可快速生长（在含葡萄糖培养基中的倍增时间通常为2.5小时），相比使用昆虫或哺乳细胞生产重组蛋白更简单、更便宜（见下表）。这些良好的特性使酵母适用于从多孔培养板、摇瓶和持续搅拌槽生物反应器到小型试验工厂和工业规模反应器等多种形式的蛋白制备。
实验室最常用的酵母种类是酿酒酵母（Saccharomyces cerevisiae，又名Baker或Brewer酵母）和一些毕赤酵母属（Pichia）的甲醇营养型酵母。S. cerevisiae和P. pastoris的遗传性质均已明确，并能够对蛋白进行翻译后修饰，包括二硫键形成和糖基化，这对于一些重组蛋白发挥正常功能具有重要作用。但是，应注意酵母的糖基化与哺乳细胞的有所不同：在S. cerevisiae中，O-连接的寡糖只有甘露糖残基，而更高等的真核蛋白有唾液酸化的O-连接糖链。此外，已知S. cerevisiae可过度糖基化N-端位点，从而导致蛋白质结合和活性发生改变，并可能在治疗应用中产生异常的免疫原应答。在P. pastoris中，寡糖链的长度短很多，并且已有一个P. pastoris株被报导可产生复杂的、末端唾液酸化的或“人源化”的糖蛋白。
ATG通常对于高效的翻译启始是足够的，尽管翻译效率要视目的基因而定。最佳的建议应是保持cDNA中天然起始位点，除非确定这一位点的功能性不理想。如果从表达的角度来考虑，推荐构建并测试两种载体，一个具有天然的起始位点，另一个具有保守的Kozak序列。通常情况下，所有N-端融合型表达载体都已包含了一个RBS或翻译起始位点。
您可能需要尝试不同的插入片段:载体比例，范围从1:1至15:1。
公式：
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
(插入片段长度 (bp) X 载体重量(ng) ) / 载体长度 (bp) = 插入片段:载体比例为1:1时所需的插入片段重量(ng)
是的，该酶混合物会使一部分PCR产物留下3′端 A尾。然而相对于单独采用Taq聚合酶，克隆效率会大幅度下降。建议在PCR产物添加3′端 A尾后再进行TA克隆。
仅使用载体进行转化不是推荐的阴性对照实验。拓扑异构酶改构过程不是一个效率100%的过程，因此，在您的混合物中将会存在“不含插入片段的载体”，因此会产生克隆。
磷酸化的产物可以进行TA克隆但不能进行TOPO克隆。这是因为所需的磷酸基团已经包含在载体DNA和拓扑异构酶形成的中间体复合物中。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体的线性载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。
你可能克隆了一段假阳性序列。TA和TOPO克隆对于存在于某些PCR反应中的小片段（< 100 bp）非常高效。使用硅基DNA纯化系统对插入片段进行凝胶纯化或者电洗脱。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
出现这一问题的原因可能有：
•插入片段没有破坏lacZ基因的读码框。如果你的插入序列较短（< 500 bp），则可能出现浅蓝色克隆。可以分析一些这类克隆，因为它们可能含有插入片段。
•使用了不能在3´ 末端加A的聚合酶。如果您使用的是具有校读活性的聚合酶，例如AccuPrime Pfx 或Platinum Pfx，您将需要进行一个后续Taq酶处理步骤以添加3’ A突出末端。
• PCR产物在连接之前进行了凝胶纯化。凝胶纯化可以去除单个的3’ A突出碱基。如果没有进行凝胶纯化，那么请优化您的PCR反应以便可以直接从PCR产物进入克隆步骤。
• PCR产物在进行连接反应前被存放了很长时间。请使用新鲜PCR产物。即使仅存放1天，连接效率也会下降。
•连接反应中加入了太多扩增反应的成分。PCR反应体系内的高盐成分会抑制连接反应。在连接反应中应使用不超过2-3 μl的PCR反应混合物。
•连接反应中的载体：插入片段摩尔比可能不正确。估计PCR产物的浓度。使连接反应中载体和插入片段的摩尔比大约为1:1或1:3。
通常平板上应该有少量不含插入片段的白色克隆。这些克隆通常比其它克隆大一些，是由于罕见的重组事件会导致质粒上部分序列缺失引起的（通常是从多克隆接头到F1起始点附近的一个位点）。为找到含有插入片段的克隆，最好挑选各种颜色和形态的克隆进行分析。同一插入片段经常会由于插入方向的不同而产生两种完全不同的克隆形态。
无克隆产生的原因可能是出现了下列问题：
•细菌不是感受态细胞。使用包含在One Shot模块内的pUC18载体检查感受态细胞的转化效率。
•平板的抗生素浓度不正确，或者平板过于陈旧。使用100 μg/mL的氨苄青霉素或50 µg/mL的卡那霉素。确保氨苄平板是新鲜的（储存时间小于1个月）。
•产物被磷酸化了（仅针对TOPO克隆而言）。磷酸化的产物可以进行TA-克隆但不能进行TOPO-克隆。这是因为连接所需的磷酸基团已经包含在载体DNA和拓扑异构酶-形成的中间体复合物中了。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIP）处理。
请考虑以下可能原因：
• pH > 9：检查PCR扩增反应的pH值，并使用1 M Tris-HCl, pH 8的缓冲液进行调节。
• PCR产物过多（或过度稀释）：减少PCR产物的用量（或增加其浓度)
• PCR反应延伸不完全：确保PCR反应最后包含一个7到30分钟的延伸步骤。长的PCR产物将需要更长的延伸时间。
•克隆长插入片段（>1 kb）：请尝试以下一个或全部建议：提高插入片段用量。延长 TOPO克隆反应孵育时间。使用硅基DNA纯化系统（如PureLink系统）或者电洗脱法对插入片段进行凝胶纯化。确保所有溶液均不含核酸酶（例如避免共用溴化乙锭染色器皿）。
•尽管你使用了Taq聚合酶但是PCR产物仍然没有足够的3´ A突出：提高最后的延伸时间以确保所有的3´末端被腺苷化。Taq聚合酶在模板链的A后面再添加一个A时的效率较低。Taq聚合酶在模板链的C后边添加一个A时的效率最高。您可能需要重新设计引物以使它们包含一个5´ G而非一个5´ T。
请尝试以下建议以提高克隆数量。
•向反应中加入6X盐溶液后，提高TOPO反应在室温孵育的时间。
•电转化能显著提高克隆数量；通常能提高10到20倍。但是，如果进行电转化，重要的一点是TOPO反应混合物要包含稀释的盐溶液，或者，为获得最佳结果，在转化前进行沉淀。为获得高电转化效率，建议如下操作： ◦向 6 µL TOPO反应体系中加入100 µL双蒸水并在37度再孵育10分钟。
◦加入10 µL 3 M 醋酸钠，2 µL 20 µg/µL糖原，300 µL 100%乙醇进行沉淀。置于干冰上或–80摄氏度20分钟，4摄氏度最高速离心15分钟。使用800 µL 80%乙醇洗涤沉淀团块，最高速离心10分钟，倒去乙醇，离心1分钟，然后移除剩余乙醇，避免碰到沉淀团块。干燥沉淀团块（空气干燥或真空吸干）。
◦将沉淀重悬于10 µL ddH2O中并根据常用电转步骤使用3.3 µL 重悬的DNA进行电转化。该电转化将产生比使用同样的TOPO-反应体系进行化学转化时多出高达20倍的克隆。加入100 µL ddH2O并孵育10分钟并不是绝对必要，但是它能充分的稀释反应并可能有助于拓扑异构酶的失活，以使得电转化更容易进行。
我们建议您使用我们的TOPO TA cloning kit for sequencing，它包含pCR4 TOPO载体，或者使用我们的Zero Blunt TOPO PCR cloning kit for sequencing,，它包含pCR4Blunt-TOPO载体。
因为您的PCR产物较小，我们建议您使用pCR2.1 TOPO载体。这一大小的片段将不能完全破坏pCR4-TOPO载体内的ccdB基因，因此你可能不能得到克隆，因为ccdB对E. Coli是致死的。
常规 TOPO TA克隆试剂盒对于2-3 kb左右的PCR产物能进行高效克隆。当PCR产物大于3kb时，克隆效率明显下降。TOPO XL PCR克隆试剂盒已优化可用于长(3- 10kb) PCR产物的TOPO克隆。如果使用常规TOPO试剂盒，这里是一些提高效率的建议：
1.使用结晶紫替代溴乙锭（EtBr）在凝胶分离过程中对PCR产物染色以避免DNA切口的出现。
2.提高TOPO反应孵育时间至30分钟。
3.保持较低的插入片段：载体摩尔比，最佳比值为1:1。
4.将反应稀释至20 µl，同时保持相同数量的载体和插入片段。将盐溶液体积增加到3.7 µl以补偿增加的体积。稀释反应可以降低对于载体末端的竞争。
将TOPO载体和插入片段反应混合物在4°C保存一星期被证明不会影响 TOPO反应的克隆效率，因为仍有超过95%的克隆含有插入片段。但是，总克隆数降低了10倍。将TOPO反应混合物放于4°C过夜后总克隆数和新鲜TOPO反应混合物比较没有或仅有轻微降低。
6X 终止溶液的成分是0.3 M NaCl，0.06 M MgCl2，而6X盐溶液的成分是1.2 M NaCl，0.06 M MgCl2。终止溶液仅包含在 TOPO XL 克隆试剂盒，而盐溶液目前包含在所有其它TOPO试剂盒中。这些溶液可以阻止拓扑异构酶重新结合并使质粒产生切口，后者将降低TOPO反应的克隆数量。
在进行一个TOPO克隆反应时，2 µl的PCR反应可包含最多10% DMSO或1.3 M甜菜碱而不影响TOPO反应。甲酰胺和高水平的甘油将抑制反应。这些试剂通常会被加入到PCR反应中以提高PCR产物的产量，例如，用来降低二级结构的影响或帮助扩增高GC含量的序列。我们还没有测试甘氨酸或乙酰胺对TOPO克隆反应的影响。
在使用任何TOPO载体进行克隆时PCR引物都不能有5´磷酸基团，因为5´磷酸基团抑制TOPO克隆反应。磷酸化的产物可以进行TA克隆但不能进行TOPO克隆。这是因为所需的磷酸基团已经包含在载体的拓扑异构酶-DNA中间体复合物中。TOPO载体含有一个与拓扑异构酶共价结合的3' 磷酸基团和一个5'磷酸基团。非TOPO线性载体（TA和Blunt）含有一个3' OH基团和一个5'磷酸基团。磷酸化产物在进行TOPO-克隆之前应该用磷酸酶（CIAP）处理。使用CIAP处理可将效率提高至25%。使用5'-生物素标记的引物（或任何5'-标记，包括 5’-Cy5标记）生成的PCR产物都因为空间位阻而不能连接到任何TOPO载体中。
如果凝胶电泳显示PCR产物是干净的，没有可见的非特异性条带或引物二聚体，那么凝胶纯化是不需要的。如果PCR产物大于1.5 kb或如果凝胶电泳显示可见的非特异性条带或引物二聚体，则建议进行凝胶纯化。小片段产物比大片段产物克隆进载体的效率高很多，所以在克隆之前应该将它们去除。在PCR产物凝胶纯化后会损失一些A突起，以及一些PCR产物的损失，这些因素会导致总克隆数的轻微减少。但是，带有插入片段的克隆的比例不会发生变化，该比例通常大于90%。
我们建议使用新鲜PCR产物用于TOPO克隆反应。
TA克隆使用T4连接酶连接插入片段和载体，而TOPO TA克隆使用拓扑异构酶的本身性质，其能够在室温孵育5分钟内连接插入片段和载体。TOPO TA克隆重组率超过95%，而TA克隆重组率大于80%。
我们提供一项TOPO克隆载体改造服务。我们的科学家可以将您的载体改造为适用于blunt TOPO 克隆, TOPO TA 克隆, 或PCR产物定向TOPO 克隆。
可以，我们的pCR2.1 TOPO TA (货号450641), pCR4-TOPO TA (货号450030), pCRBluntII-TOPO (货号450245)均可提供单独的不含感受态的克隆试剂盒。
我们不建议这么做，因为这些载体的末端结合有拓扑异构酶DNA蛋白复合物。
我们建议起始摩尔比为1:1(插入片段：载体)，范围为0.5：1到2：1(插入片段：载体)。TOPO克隆反应中，2kb PCR产物的ng值应在5- 10ng之间。
请在TOPO克隆前考虑以下问题：
• TOPO克隆不能连接带有5’磷酸基团的DNA。
• TOPO克隆效率会随着插入片段长度增加而降低，超过3 kb 建议使用TOPO XL克隆试剂盒。
• TOPO载体包含不同的抗生素抗性标记，这一点应该在购买之前考虑。
• TOPOTA载体可连接带有3’ A突出末端的片段，而Zero Blunt载体可连接带有平末端的片段。
TOPO及TOPO TA载体（非定向）加入了酚红染料。室温下溶液应该为粉红色（或者黄色）。如果在加入PCR产物后溶液变为蓝色，说明PCR缓冲液太偏碱性，转化的克隆数会变少。当溶液为黄色时，表明pH值为酸性。在pH 2.0条件下，TOPO载体仍能保持较高克隆效率。定向的TOPO及Zero Blunt TOPO载体加入了溴酚蓝。
亚克隆级别的DH5α可以使用，但是转化的效率会低一些（10e6 vs 10e9），因此得到的克隆数可能较少。 Top10F’细胞也可以使用，但是如果要进行蓝白斑筛选，需要同时加入IPTG和X-gal，因为带有F’游离体的细胞会表达lacIq抑制子。
确保您使用了正确的抗生素和合适的浓度。另外，确保抗生素没有过期。如果克隆的形态和预期不同，那么污染可能是一个原因。检测您的SOC培液和LB培液。
以下是一些建议：
•小片段/接头而不是您的插入片段被克隆进了您的载体。要解决这一问题，请在连接前对插入片段进行凝胶纯化。
•确保使用了正确的X-gal和/或IPTG（当载体带有lacIq标志物时）浓度。
•在平板上涂布X-gal和/或IPTG时，留出足够的时间让试剂充分扩散进入平板。
•孵育平板足够长的时间以确保颜色完全显现。
虽然没有特定菌株更适合进行大质粒转化，但是关注转化效率是非常重要的。对于大质粒，推荐使用效率最高的化学感受态细胞，例如OmniMAX 2，TOP10，等等。对于超过20 kb的质粒，建议使用电转感受态细胞以获得最高的转化效率。
无论使用何种菌株，您所要做的第一件事都是降低生长温度至30摄氏度或更低（室温）。慢速生长通常可以让E. coli更加耐受复杂序列。如果降低生长温度没有帮助，则您可能需要考虑使用其它感受态细胞比如Stbl2或Stbl4细胞，这类细胞已被证明在同等条件下对于上述复杂序列的耐受程度比其它菌株高。
我们建议尝试以下措施：
•进行pUC19转化对照；这可以告诉您所用感受态细胞的转化能力信息。
•检查平板的过期时间以及是否使用了正确的培液（LB/琼脂）。
•确认使用了正确的抗生素并且浓度也是对的。
TOP10F’中的F’游离体带有一个lacIq标志物，它可以过表达lac抑制子。必须将IPTG和X-gal一起加入平板中才能在该菌株内看到β-半乳糖苷酶的表达和蓝色的出现。而TOP10则不需要IPTG就能进行蓝白斑筛选。
以下这些因素会导致这一情况：涂板时所用的SOC培液或其它培液受到了污染，DNA被具有氨苄抗性的微生物所污染，平板时间过久氨苄降解失效，或者感受态细胞本身被污染了。
如果您使用的是一种mcr/mrr(+)感受态细胞株，其细胞内的酶可能会识别酵母基因组DNA上的真核细胞甲基化序列并将其删除或重排。请尝试用一种mcr/mr(–)菌株例如Top10, DH10B, 或OmniMAX 2进行转化。
XbaI切割位点是一个Dam甲基化敏感限制性酶切位点。TOP10是一种dam(+)菌株，它表达甲基化酶Dam。您可以试试重新转化一种dam(–)菌株，例如INV110。其它dam(–) (以及dcm(–))敏感的限制性酶切位点包括：
Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
•Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I
1.使用pUC或基于pUC的载体，这些载体含有lacZ基因的一部分，可以进行α补偿。
2.选择一种带有 lacZdeltaM15标记的E. Coli菌株。
3.将转化混合液涂布在含有X-gal的平板上。在一个100 mm平板上涂布50 µg的2% X-gal或者100微升2% bluo-gal （均可溶解于DMF或50:50的DMSO：水混合物中）并晾干。此外，也可在倒板前直接向冷却后的培液中加入终浓度50 µg/mL的X-gal或300 µg/mL的bluo-gal。平板在4摄氏度可稳定保存4周。
4.如果菌株带有lacIq标志，则需加入IPTG以诱导lac启动子。在100 mm平板上涂布30 µl的100 mM IPTG（溶于水中）。此外，也可以在倒板前直接向冷却后（大约59度）的培养液中加入IPTG至终浓度1 mM。平板在4摄氏度可稳定保存4周。
5.如果要使用 X-gal或bluo-gal进行蓝白斑筛选，那么不要将E. coli 涂布在含有葡萄糖的培养基上。因为葡萄糖会作为底物和X-gal或bluo-gal竞争从而阻止细胞变蓝。
对于长期存储，建议制作甘油存储液并保存在-70摄氏度。请按以下步骤操作：
1.挑取1个克隆到5 mL的LB培液或SOC培液中。37摄氏度培养过夜。
2.制备甘油溶液：6 mL的SOB培液加4 mL甘油。
3.细胞与甘油溶液等体积混合并混匀。
4.在乙醇/干冰混合物中冷冻然后存储在-70摄氏度。
是的，这是可以的。我们推荐使用饱和量的DNA（每种质粒10 ng）。确保不同质粒的复制起始点不同，以便它们可以同时存在于细胞中。如果复制起始点相同，则两种质粒会互相竞争，其中一种质粒即使稍有劣势也可能会丢失。另外，带有一个质粒的细胞可以通过电转导入第二个质粒，而不会破坏已有质粒。
在平板上，我们建议50 µg/mL X-gal和1 mM IPTG (0.24 mg/mL)。当直接涂布到琼脂平板上时，我们建议在琼脂平板表面使用40–50 µl溶于DMF的40 mg/mL 的X-gal (2% 储液)和30–40 µl的100 mM IPTG。让X-gal和IPTG向琼脂内进行扩散大约1小时。不要在含有葡萄糖的培养基上涂布，因为葡萄糖会和X-gal或bluo-gal竞争从而阻止细胞变蓝。
感受态细胞效率通过测定转化效率得到。转化效率等于每µg质粒DNA形成的转化子的数量，或者克隆数量(cfu/ µg)。
下面是一些能帮助您获得最高转化效率的建议：
•将感受态细胞在冰浴中解冻而不是在室温中解冻；不要对细胞进行涡旋震荡。
•感受态细胞解冻后，立刻在其中加入DNA。
•确保孵育时间是符合感受态细胞实验方案中针对该菌种所列出的要求的；改变孵育时间会降低效率。
•去除您的DNA样本中的盐分及其它污染；转化前可以使用离心柱或苯酚/氯仿抽提、乙醇沉淀的方法纯化DNA。
DH5α细胞是常规克隆的常用菌株，但它是mcr/mrr+的，因此不推荐用于基因组克隆。另一方面，TOP10感受态细胞包含突变的mcr/mrr，因此是常规克隆不错的选择而且可以用于甲基化的DNA的克隆，如真核基因组DNA。我们的Mach1菌株是生长最迅速的具有T1噬菌体抗性的菌株。
这些小的克隆很有可能是由于氨苄活性降低产生的。这些克隆是生长在氨苄活性降低的LB培养板上的未转化细胞。为了避免这种情况，你可以尝试：
1.降低细胞铺板密度。
2.使用新鲜的LB氨苄培养板或者用羧苄青霉素代替氨苄青霉素。
3.培养板不能在37摄氏度下孵育超过20小时。β-内酰胺酶是一种由氨苄抗性基因产生的酶，它由具氨苄抗性的转化菌分泌，能够使转化的菌落周围的抗生素失活。由于筛选试剂的失活而使得卫星菌落（不是真正的氨苄抗性）能够生长。如果使用羧苄青霉素也会产生同样情况。
其中一个原因是可能与插入片段的毒性有关。这种毒性不能影响在固体培养基上缓慢生长的细胞，但是在快速生长条件（如液体培养基中）下，其毒性更强。建议：
使用TOP10F’或其他带有LacIq阻抑物的菌种。
2.尝试使用任何其它的适用于克隆的菌株。
3.将培养温度降至27至30摄氏度并延长培养时间。
4.不生长的另一可能的原因是噬菌体污染。此种情况下，我们推荐使用具有T1噬菌体抗性的菌株，如DH5α-T1R。
这可能是由于插入DNA在TOP10大肠杆菌中不稳定造成的。此种情况下，Stbl2、Stbl4等大肠杆菌菌种对于有正向重复的DNA、反转录病毒序列、及高GC含量的DNA扩增的效果优于其它菌种。
一些可能的原因及补救措施如下：
•连接酶活性低。检查连接酶生产日期以及缓冲液的功能。
•感受态细胞没有转化能力。使用超螺旋载体，如puc19，检测感受态细胞效率。
•两个片段都已去磷酸化。
•限制性内切酶及残留的缓冲液抑制连接反应。不妨尝试转化未切割载体，使用苯酚去除内切酶，或者连接前进行PCR产物纯化／胶回收。
•抗生素使用错误。检查质粒及培养板并确保使用了正确浓度的抗生素。
如果以上均不适用，转化后克隆数低或没有克隆可能是由于DNA插入片段在感受态细胞中不稳定造成的。此种情况下，Stbl2、Stbl3、或Stbl4等大肠杆菌菌种对于有多片段重复的DNA、反转录病毒序列、及高GC含量的DNA扩增的效果优于其它菌种。
如果使用包含lac启动子和LacZ α片段（用于α互补）的载体，蓝/白筛选可作为筛选插入片段是否存在的工具。X-gal作为LacZ酶的底物需要加到反应板中，是蓝/白斑筛选必需的。完全破坏LacZ基因表达所需的最小插入片段大小为400 bp。如果存在Laclq抑制子（来自宿主细胞，如TOP10F'，或由质粒表达），它将抑制lac启动子介导的表达，从而阻碍蓝/白筛选。因此，当存在Laclq抑制子时，必须使用IPTG抑制Laclq。只有抑制了Laclq，才能实现蓝/白筛选中的lac启动子介导的表达。
含有LacZ-ccdB表达框的TOPO载体，可通过破坏对大肠杆菌致死的基因ccdB的表达而直接筛选重组体。PCR产物的连接，可破坏LacZ-ccdB融合基因的表达，从而仅允许转化所得的阳性重组体生长，而含有非重组载体的细胞则将死亡。因此，无需蓝/白筛选。对含有LacZ-ccdB表达框的TOPO载体的克隆进行蓝/白筛选时，可看见菌落呈现不同的蓝色阴影。根据我们的经验，浅蓝色和白色的菌落通常含有插入片段。浅蓝色克隆的形成是由于在有插入片段的情况下发生了部分LacZ基因的转录但ccdB的表达没有达到杀死细胞的水平，这是和插入片段有关的。为了完全破坏lacZ基因的表达，插入片段必须>400 bp；因此，一个300 bp的插入片段可产生浅蓝色菌落。出现不含插入片段的白色菌落，通常是因为ccdB基因的自发性突变。
为了确保破坏ccdB基因表达，防止细胞死亡，需要的插入片段最小为150 bp。（参考文献：Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.）
含F质粒的菌株，如TOP10F’，不推荐用于转化和筛选任何含ccdB基因的TOPO载体的重组克隆。F质粒可编码ccdA蛋白，一种ccdB旋转酶-毒素蛋白的抑制剂。ccdB基因也存在于F质粒的ccd（细胞死亡控制）位点。该位点含2个基因，ccdA和ccdB，可分别编码含72和101个氨基酸的蛋白。ccd位点通过杀死分裂后不含F质粒的细胞而维持F质粒的稳定性的。当ccdA蛋白不发挥抑制作用时，ccdB蛋白是一种强力的可以杀死细胞的蛋白。
如果使用包含lac启动子和LacZ α片段（用于α互补）的载体，蓝/白筛选可作为筛选插入片段是否存在的工具。X-gal作为LacZ酶的底物需要加到反应板中，是蓝/白斑筛选必需的。完全破坏LacZ基因表达所需的最小插入片段大小为400 bp。如果存在Laclq抑制子（来自宿主细胞，如TOP10F'，或由质粒表达），它将抑制lac启动子介导的表达，从而阻碍蓝/白筛选。因此，当存在Laclq抑制子时，必须使用IPTG抑制Laclq。只有抑制了Laclq，才能实现蓝/白筛选中的lac启动子介导的表达。X-gal（也称为5-溴-4氯-3-吲哚-β-D-半乳糖苷）可溶于DMSO或DMF，其溶液形式可在冰箱中储存长达6个月。溶液需避光保存。X-gal和IPTG在琼脂板中的终浓度：倾注平板前，在培养基中加入X-gal至终浓度20 mg/mL，IPTG 加至终浓度0.1 mM。若直接涂在反应板表面，则加入40 µl X-gal (20 mg/mL储液)和4 µl IPTG (200 mg/mL储液)。
不可以，这些载体不包含功能性启动子以表达您的目的基因。这些载体通常用于亚克隆或测序。
TA克隆
这种克隆方法最初是为配合纯Taq聚合酶（天然的、重组的、热启动）使用而设计的；然而，某些高保真Taq酶和Taq酶混合物通常也适合TA克隆。即使Taq与具有校正能力的聚合酶以10:1或15:1的比例，仍可以产生足够的3’ A突出端去做TA克隆。
推荐使用的我公司的聚合酶包括Platinum Taq、Accuprime Taq、Platinum或Accuprime Taq High Fidelity、AmpliTaq、AmpliTaq Gold或AmpliTaq Gold 360等。
平末端克隆
使用Platinum SuperFi DNA聚合酶等具有校对能力的酶。
定向TOPO克隆
Platinum SuperFi DNA聚合酶效果良好。
原核生物mRNA含有Shine-Dalgarno序列，也称为核糖体结合位点(RBS)，它是由AUG起始密码子5’端的多嘌呤序列AGGAGG组成。该序列与16S rRNA 3’端的互补，有助于mRNA有效结合到核糖体上。同理，真核生物（特别是哺乳动物）mRNA也含有完成有效翻译所需的重要序列信息。然而，Kozak序列不是真正的核糖体结合位点，而是一种翻译起始增强子。Kozak共有序列是ACCAUGG，其中AUG是起始密码子。-3位的嘌呤(A/G)具有重要作用；若-3位是一个嘧啶(C/T)，翻译过程会对-1、-2和+4位的改变更敏感。当-3位从嘌呤变为嘧啶时，可使表达水平降低多达95%。+4位对表达水平的影响相对较小，可以使表达水平降低约50%。
注：果蝇的最佳Kozak序列稍有不同，酵母完全不遵循这些规则。见下列参考文献：
•Foreign Gene Expression in Yeast: a Review. Yeast, vol. 8, p. 423-488 (1992).
•Caveneer, Nucleic Acids Research, vol. 15, no. 4, p. 1353-1361 (1987).
We do not recommend storing competent E. coli strains in liquid nitrogen as the extreme temperature can be harmful to the cells. Also, the plastic storage vials are not intended to withstand the extreme temperature and may crack or break.
We recommend storing our competent E. coli strains at -80°C. Storage at warmer temperatures, even for a brief period of time, will significantly decrease transformation efficiency.
Following are the rich and minimal media used for culturing Pichia pastoris and S. cerevisiae:
Rich Media:
S. cerevisiae and Pichia pastoris
YPD (YEPD): yeast extract, peptone, and dextrose
YPDS: yeast extract, peptone, dextrose, and sorbitol
Pichia pastoris only
BMGY: buffered glycerol-complex medium
BMMY: buffered methanol-complex medium
Minimal Media (also known as drop-out media):
S. cerevisiae
SC (SD): Synthetic complete (YNB, dextrose (or raffinose or galactose), and amino acids)
Pichia pastoris
MGY: minimal glycerol medium
MD: minimal dextrose
MM: minimal methanol
BMGH: buffered minimal glycerol
BMMH: buffered minimal methanol
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S. pombe cannot generate P factor when P factor is replaced for alpha in the alpha factor gene. It can, however, produce alpha factor when alpha is replaced for P in the P factor gene. This is negative evidence that S. pombe can process its own mating factor cleavage sites, but not all the cleavage sites of the S. cerevisiae alpha factor. It is better to use a more generic signal sequence (rather than a pre- pro- signal sequence such as alpha). If it is necessary to go the pre- pro- route, it is better to use the S. pombe P factor leader rather than the S. cerevisiae alpha leader.
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Yes, we do offer the pYES2.1/V5-His-TOPO vector, which is part of the pYES2.1 TOPO TA Expression Kit (Cat. No. K415001), for the direct cloning of Taq polymerase-amplified PCR products and regulated expression in Saccharomyces cerevisiae using galactose.
Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose, and fructose linked in that order. Most yeast can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.
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We offer the INVSc1 yeast strain. It is a diploid strain for expression purposes only. It does not sporulate well and is therefore not suited for yeast genetic studies. The genotype and phenotype of the INVSc1 strain are as follows:
Genotype: MATa his3D1 leu2 trp1-289 ura3-52/MATalpha his3D1 leu2 trp1-289 ura3-52
Phenotype: His-, Leu-, Trp-, Ura-
Note that INVSc1 is auxotrophic for histidine, leucine, tryptophan, and uracil. The strain will not grow in SC minimal medium that is deficient in histidine, leucine, tryptophan, and uracil.
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Stock buffers of TE, lithium acetate, and PEG can be stored. However, the combined solution used to prepare the cells for transformation must be made fresh every time. There is a loss in transformation efficiency if the solutions are not freshly prepared.
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OD600 of 0.1 = approximately 3 x 10e6 cells/mL
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The efficiency is very strain-dependent, but 1000 to 100,000 transformants per µg DNA is the range.
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Here are the different methods available for S. cerevisiae transformation:
- S. cerevisiae EasyComp Transformation Kit (K505001): easy-to-use, ready-made reagents
Competent cells can be stored frozen. Transformation efficiency is >10e3 transformants per µg DNA. Higher transformation efficiencies are often obtained with frozen versus freshly prepared cells.
- Small-scale yeast transformation protocol (page 13 of the manual)
- Lithium acetate transformation: easy, do-it-yourself protocol
Competent cells must be made fresh
- Electroporation: easy and high efficiency, do-it-yourself protocol
Competent cells must be made fresh
- Spheroplast Kit for Yeast (K172001): high efficiency, a lot of work, not suitable for antibiotic selection
Note: Plate an appropriate density. Colonies will appear over several days. Don't pick the largest colonies, as these are often suppressors.
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We recommend storing yeast frozen at -80 degrees C in 15% glycerol. Glycerol stocks are good indefinitely (unless there are numerous freeze-thaws). When making a glycerol stock, we recommend using an overnight culture and concentrating it 2-4 fold. Spin down cells and suspend in 25-50% of the original volume with glycerol/medium. It is better to store frozen cells in fresh medium plus glycerol, rather than simply adding glycerol into the overnight culture.
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We offer the original Pichia pastoris expression systems, PichiaPink expression system, and Saccharomyces cerevisiae yeast expression system for expression of recombinant proteins. Both P. pastoris and S. cerevisiae have been genetically well-characterized and are known to perform many posttranslational modifications.
The P. pastoris expression system combines the benefits of expression in E. coli (high-level expression, easy scale-up, and inexpensive growth) and the advantages of expression in a eukaryotic system (protein processing, folding, and posttranslational modifications), thus allowing high-level production of functionally active recombinant protein. As a yeast, Pichia pastoris shares the advantages of molecular and genetic manipulations with Saccharomyces cerevisiae, and it has the added advantage of 10- to 100-fold higher heterologous protein expression levels. These features make Pichia pastoris very useful as a protein expression system. The Pichia expression vectors contain either the powerful alcohol oxidase (AOX1) promoter for high-level, tightly controlled expression, or the glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter for high-level, constitutive expression. Both inducible and constitutive expression constructs integrate into the P. pastoris genome, creating a stable host that generates extremely high protein expression levels, particularly when used in a fermentor. The Pichia pastoris expression systems we offer include:
- PichiaPink Yeast Expression System: Newer Pichia pastoris expression system that contains both low- and high-copy plasmid backbones, 8 secretion signal sequences, and 4 yeast strains to help optimize for the highest yield possible of the recombinant protein. All PichiaPink vectors contain the AOX1 promoter for high-level, inducible expression and the ADE2 marker for selecting transformants using ADE2 complementation (i.e., by complementation of adenine auxotrophy) rather than antibiotic selection. However, they express the ADE2 gene product from promoters of different lengths, which dictate the copy number of the integrated plasmids. The pPink-LC vector has an 82 bp promoter for the ADE2marker and offers low-copy expression, and the pPink-HC vector has a 13 bp promoter for the ADE2marker and offers high-copy expression. The system also includes the pPinkalpha-HC vector (containing S. cerevisiae alpha-mating factor pre-sequence) for high copy number secreted expression, and provides eight secretion signal sequences for optimization of secreted expression.
- EasySelect Pichia Expression Kit: One of the original Pichia expression kits that contains the pPICZ and pPICZalpha vectors, for intracellular and secreted expression, respectively, of the gene of interest. These vectors contain the AOX1 promoter for high-level, inducible expression and the Zeocin antibiotic resistance marker for direct selection of multi-copy integrants. They facilitate simple subcloning, simple purification, and rapid detection of expressed proteins.
- Original Pichia Expression Kit: The kit includes the pPIC9, pPIC3.5, pHIL-D2, and pHIL-S1 vectors, each of which carries the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. pPIC9 carries the S. cerevisiae alpha-factor secretion signal while pHIL-S1 carries the Pichia pastoris alkaline phosphatase signal sequence (PHO) to direct transport of the protein to the medium. pHIL-D2 and pPIC3.5 are designed for intracellular expression.
- Multi-Copy Pichia Expression Kit: This kit is designed to maximize expression and contains the pPIC3.5K, pPIC9K, and pAO815 vectors, which allow production and selection of Pichia strains that contain more than one copy of the gene of interest. They allow isolation and generation of multicopy inserts by in vivo methods (pPIC3.5K and pPIC9K) or in vitro methods (pAO815). All of these vectors contain the AOX1 promoter for high-level, inducible expression and the HIS4 gene for selection in his4 strains, on histidine-deficient medium. The pPIC9K vector directs secretion of expressed proteins while proteins expressed from pPIC3.5K and pAO815 remain intracellular. The pPIC9K and pPIC3.5K vectors carry the kanamycin resistance marker that confers resistance to Geneticin Reagent in Pichia. Spontaneous generation of multiple insertion events can be identified by resistance to increased levels of Geneticin Reagent. Pichia transformants are selected on histidine-deficient medium and screened for their level of resistance to Geneticin Reagent. The ability to grow in high concentrations of Geneticin indicates that multiple copies of the kanamycin resistance gene and the gene of interest are integrated into the genome.
- For expression in S. cerevisiae, we offer the pYES Vector Collection. Each pYES vector carries the promoter and enhancer sequences from the GAL1 gene for inducible expression. The GAL1 promoter is one of the most widely used yeast promoters because of its strong transcriptional activity upon induction with galactose. pYES vectors also carry the 2m origin and are episomally maintained in high copy numbers (10-40 copies per cell).
Yeast is a single-celled, eukaryotic organism that can grow quickly in defined media (doubling times are typically 2.5 hr in glucose-containing media) and is easier and less expensive to use for recombinant protein production than insect or mammalian cells (see table below). These positive attributes make yeast suitable for use in formats ranging from multi-well plates, shake flasks, and continuously stirred tank bioreactors to pilot plant and industrial-scale reactors.
The most commonly employed species in the laboratory are Saccharomyces cerevisiae (also known as Baker's or Brewer's yeast) and some methylotrophic yeasts of the Pichia genus. Both S. cerevisiae and P. pastoris have been genetically characterized and shown to perform the posttranslational disulphide bond formation and glycosylation that is crucial for the proper functioning of some recombinant proteins. However, it is important to note that yeast glycosylation does differ from that in mammalian cells: in S. cerevisiae, O-linked oligosaccharides contain only mannose moieties, whereas higher eukaryotic proteins have sialylated O-linked chains. Furthermore S. cerevisiae is known to hyperglycosylate N-linked sites, which can result in altered protein binding, activity, and potentially yield an altered immunogenic response in therapeutic applications. In P. pastoris, oligosaccharides are of much shorter chain length and a strain has been reported that can produce complex, terminally sialylated or “humanized” glycoproteins.
ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.
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The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.
Equation:
length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio
Yes, the enzyme mix leaves 3' A-overhangs on a portion of the PCR products. However, the cloning efficiency is greatly decreased compared to that obtained with Taq polymerase alone. It is recommended to add 3' A-overhangs to the product for TA cloning.
Using the vector only for transformation is not a recommended negative control. The process of TOPO-adaptation is not a 100% process, therefore, there will be “vector only” present in your mix, and colonies will be obtained.
Phosphorylated products can be TA cloned but not TOPO cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO cloning.
You may be cloning in an artifact. TA and TOPO Cloning are very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product using either a silica-based DNA purification system or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
There could be a few possibilities for this:
- The insert does not interrupt the reading frame of the lacZ gene. If you have a small insert (< 500 bp), you may have light blue colonies. Analyze some of these blue colonies as they may contain insert.
- A polymerase that does not add 3' A-overhangs was used. If you used a proofreading enzyme, you will need to do a post-reaction treatment with Taq polymerase to add the 3' A-overhangs.
- PCR products were gel-purified before ligation. Gel purification can remove the single 3' A- overhangs. Otherwise, optimization of your PCR can be performed so that you can go directly from PCR to cloning.
- The PCR products were stored for a long period of time before ligation reaction. Use fresh PCR products. Efficiencies are reduced after as little as 1 day of storage.
- Too much of the amplification reaction was added to the ligation. The high salt content of PCR can inhibit ligation. Use no more than 2-3 µl of the PCR mixture in the ligation reaction.
- The molar ratio of vector:insert in the ligation reaction may be incorrect. Estimate the concentration of the PCR product. Set up the ligation reaction with a 1:1 or 1:3 vector:insert molar ratio.
On a typical plate there are a few white colonies which do not contain insert. These are usually larger than the other colonies and are due to a deletion of a portion of the plasmid sequence by a rare recombination event (usually from the polylinker to a site in the F1 origin). To find a colony with an insert it is best to pick clones of a variety of color and pattern for analysis. Often an insert will generate two distinct patterns according to its orientation.
No colonies may occur due to the following problems:
Bacteria were not competent. Use the pUC18 vector included with the One Shot module to check the transformation efficiency of the cells.
- Incorrect concentration of antibiotic on plates, or the plates are too old. Use 100 µg/mL of ampicillin or 50 µg/mL kanamycin. Be sure ampicillin plates are fresh (< 1 month old).
- The product was phosphorylated (TOPO cloning only). Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. The TOPO vector has a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. The non- TOPO vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be phosphatased (CIP) before TOPO-cloning.
Please consider the following possible causes:
- pH > 9: Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
- Excess (or overly dilute) PCR product: Reduce (or concentrate) the amount of PCR product.
- Incomplete extension during PCR: Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
- Cloning large inserts (>1 kb): Try one or all of the following suggestions: Increase amount of insert. Incubate the TOPO cloning reaction longer. Gel-purify the insert using either a silica-based DNA purification system (e.g., PureLink system) or electroelution. Be sure that all solutions are free of nucleases (avoid communal ethidium bromide baths, for example.)
- PCR product does not contain sufficient 3' A-overhangs even though you used Taq polymerase: Increase the final extension time to ensure all 3' ends are adenylated. Taq polymerase is less efficient at adding a nontemplate 3' A next to another A. Taq is most efficient at adding a nontemplate 3' A next to a C. You may have to redesign your primers so that they contain a 5' G instead of a 5´ T.
Please try the suggestions below to increase the number of colonies.
- Longer incubation of the TOPO cloning reaction at room temperature, provided that the 6X Salt solution is added to the reaction.
- Electroporation can give significant increases in colony numbers; often 10-20 fold higher. However, if doing electroporation, it is important that the TOPO reaction mix contains diluted Salt solution or, for best results, precipitated prior to transformation. For high primary transformants by electroporation it is recommended to:
- Add 100 µL double diH2O to the 6 µL TOPO reaction and incubate 10 more minutes at 37 degrees C.
- Precipitate by adding 10 µL 3 M Na-Acetate, 2 µL 20 µg/µL glycogen, 300 µL 100% ethanol. Place on dry ice or –80 degrees C for 20 min, spin at top speed in a microcentrifuge at 4 degrees C for 15 min. Wash pellet with 800 µL 80% ethanol, spin at top speed for 10 min, pour off ethanol, spin 1 min, and remove remaining ethanol without disturbing pellet. Dry pellet (air-dry or speed-vac).
- Resuspend pellet in 10 µL ddH2O and electroporate 3.3 µL of resuspended DNA according to a normal electroporation protocol. This electroporation protocol can yield up to 20 fold more colonies than chemical transformation of an equivalent TOPO-reaction. The addition of the 100 µL ddH2O followed by 10 mins incubation is not absolutely necessary, but it sufficiently dilutes the reaction and may help inactivate topoisomerase so that it is more easily electroporated.
We would suggest using our TOPO TA cloning kit for sequencing, which contains the pCR 4 TOPO vector, or our Zero Blunt TOPO PCR cloning kit for sequencing, which contains the pCR4Blunt-TOPO vector.
Due to the small size of your product, we recommend using the pCR 2.1 TOPO vector for your cloning. This size fragment would not be able to fully interrupt the ccdB gene in the pCR4-TOPO vector, and therefore, you may not get colonies as ccdB is lethal to E. coli.
Regular TOPO TA Cloning kits are efficient for cloning PCR products up to approximately 2-3 kb. With PCR products larger than 3 kb, the efficiency of cloning drops significantly. The TOPO XL PCR Cloning Kit has been optimized for TOPO cloning of long (3-10 kb) PCR products.
If using the regular TOPO kits, here are some tips to improve efficiency:
1. Use crystal violet instead of ethidium bromide (EtBr) to visualize the PCR for gel isolation to avoid DNA nicks
2. Increase incubation time of the TOPO reaction to 30 mins
3. Keep insert:vector molar ratio low, optimally 1:1
4. Dilute reaction to 20 µL, while maintaining same amount of vector and insert. Increase the volume of the salt solution to 3.7 µL to compensate for the increase in volume. Diluting the reaction reduces the competition for the vector ends.
Storage of the TOPO vector plus insert reaction for 1 week at 4 degrees C has shown no detectable decrease in the cloning efficiency of the TOPO reaction, as >95% of the colonies have insert. However, the total number of colonies was decreased by 10-fold. Storage of the TOPO reaction mix overnight at 4 degrees C showed little to no decrease in the number of colonies when compared to fresh TOPO reaction mix.
The composition of the 6X Stop solution is 0.3 M NaCl, 0.06 M MgCl2, and the composition of the 6X Salt solution is 1.2 M NaCl, 0.06 M MgCl2. Stop solution is only included in the TOPO XL Cloning kit whereas Salt solution is currently included in all of the other TOPO cloning kits. These solutions prevent free topoisomerase from re-binding and nicking the plasmid, which would reduce the number of colonies from a TOPO reaction.
When doing a TOPO cloning reaction, 2 µl of a PCR reaction containing up to 10% DMSO or 1.3 M betaine will not interfere with the TOPO reaction. Formamide and high levels of glycerol will inhibit the reaction. These reagents are usually added to the PCR reaction to enhance the yield of the PCR product, e.g., to reduce the effect of secondary structure or assist in amplification of GC-rich sequences. The effects of tricine or acetamide have not been tested on the TOPO cloning reaction.
PCR primers should not have 5'-phosphates when cloning into any TOPO vector, as the presence of 5'-phosphates inhibit the TOPO cloning reaction. Phosphorylated products can be TA-cloned but not TOPO-cloned. This is because the necessary phosphate group is contained within the topoisomerase-DNA intermediate complex of the vector. TOPO vectors have a 3' phosphate to which topoisomerase is covalently bound and a 5' phosphate. Non-TOPO linear vectors (TA and Blunt) have a 3' OH and a 5' phosphate. Phosphorylated products should be treated with phosphatase (CIAP) prior to TOPO-cloning. Treatment with CIAP may raise efficiency to 25%. PCR products generated with 5'-biotinylated primers (or any other 5'-label including 5'-Cy5) will not ligate into any of the TOPO vectors due to steric hindrance.
Gel purification is not required if the gel indicates that the PCR product is clean with no visible non-specific bands or primer dimers. It is recommended if the PCR product is >1.5 kb or if non-specific bands and primer dimers are visible on the gel. Smaller products clone much more efficiently into the vector than larger products; therefore, they should be eliminated from the sample prior to cloning. There is some reduction in A-overhangs if the PCR product is gel purified, which along with PCR product loss during the procedure may slightly reduce total number of colonies. However, the percentage of colonies with insert does not change; it is typically >90% recombinant clones.
For optimal TOPO cloning, we recommend using fresh PCR products.
TA cloning ligates the insert and vector using a T4 DNA ligase, while TOPO TA cloning uses the intrinsic properties of topoisomerase, which ligates the insert and vector during a 5 minute desktop reaction. TOPO TA cloning results in >95% recombinants, while TA cloning results in >80% recombinants.
We offer a custom service for TOPO cloning adaptation services. Our scientists can prepare your vector for either blunt TOPO cloning, TOPO TA cloning, or directional TOPO cloning of PCR products.
Yes, our pCR.1 TOPO TA (Cat. No. 450641), pCR4-TOPO TA (Cat. No. 450030), pCRBluntII-TOPO (Cat. No. 450245) are available separately.
No, we do not recommend this as these vectors contain the topoisomerase DNA protein complex conjugated to the end of the vector.
We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1 (insert:vector). The ng quantities should be between 5-10 ng of a 2 kb PCR product in a TOPO cloning reaction.
Please consider the following before TOPO cloning:
- TOPO cloning cannot ligate DNA with a 5' phosphate group.
- TOPO cloning will decrease in efficiency inversely with the size of the insert (above 3 kb) unless using the TOPO XL cloning kit.
- TOPO vectors contain different antibiotic resistance markers which should be considered before purchase.
- TOPO TA vectors accept fragments containing a 3' A overhang while Zero Blunt vectors accept fragments that are blunt-ended.
TOPO and TOPO TA vectors (non-directional) have phenol red dye added. The color should be pink (or yellow) at room temperature. If it turns blue when PCR product is added, the PCR product buffer is too basic and the number of transformed colonies will drop. When the solution is yellow, it signifies an acidic pH. At a pH 2.0, TOPO vectors still maintain high cloning efficiency. Directional TOPO and Zero Blunt TOPO vectors have bromophenol blue dye added.
Subcloning DH5? cells are a compatible strain, but you will get lower efficiency (10e6 vs 10e9) and therefore risk getting fewer clones. Top10F' is also compatible, but if blue/white screening is performed, IPTG along with X-gal will be needed for detection due to the expression of the lacIq repressor present in cells containing an F' episome.
Ensure that you are using the correct antibiotic at the appropriate concentration. Additionally, make sure the antibiotic is not expired. If colonies exhibit unexpected morphologies, contamination could be a factor. Check your S.O.C. medium and LB growth medium.
Here are a few suggestions:
- Small fragments/linkers are cloning in to your vector instead of your insert; to correct this, gel-purify the insert before ligation
- Ensure that the correct concentrations of X-gal and/or IPTG (if vector contains the lacIq marker) are used
- If spreading X-gal and/or IPTG on your plate, allow sufficient time for the reagents to diffuse into the plate
- Incubate your plate for a longer period to ensure full color development
While there is no specific strain that works better with large plasmids, it is important to focus on transformation efficiency. For larger plasmids, chemically competent cells with highest efficiency are suggested, such as OmniMAX 2, TOP10, etc. We would recommend using an electrocompetent cell strain with plasmids larger than 20 kb for best efficiency.
With any strain, the first thing to try would be to lower the growth temperature of the culture to 30 degrees C or even lower (room temperature). Slower growth will generally allow E. coli to tolerate difficult sequences better. If reducing the growth temperature doesn't help, you may want to consider using a competent cell strain such as Stbl2 or Stbl4 cells, which have been shown to accommodate this type of sequence better than other strains in the same conditions.
We recommend trying the following:
- Carry out the puc19 transformation control; this gives you information about the performance of the cells.
- Check plates for expiration and correct media used (LB/agar).
- Confirm that the correct antibiotic and concentration was used.
The F' episome in TOP10F' has a lacIq marker, which over-expresses the lac repressor. IPTG must be added to LB plates along with X-gal to see beta-galactosidase expression and blue color in this strain. TOP10, on the other hand, does not require IPTG for blue/white screening.
There are a few conditions that can lead to this: SOC medium or other media used when plating was contaminated, DNA was contaminated with amp-resistant microbes, you used old plates with degraded amp, or the competent cells themselves were contaminated.
If you are using a mcr/mrr(+) competent cell strain, cellular enzymes may be recognizing eukaryotic methylation patterns on the yeast genomic DNA and deleting or rearranging it. Try a mcr/mr(-) strain such as Top10, DH10B, or OmniMAX 2.
XbaI cutting site is a Dam-methylation sensitive restriction site. TOP10 is a dam(+) strain, which means it expresses the methylating enzyme, Dam. You can try re-transforming into a dam(-) strain, such as INV110. Other dam- (and dcm-) sensitive restriction sites include the following:
- Dam: Bcl I, Cla I, Hph I, Mbo I, Mbo II, Taq I, Xba I, BspH I, Nde II, Nru I
- Dcm: Ava II, EcoO 109 I, EcoR II, Sau96 I, ScrF, Stu I, Aat I, Apa I, Bal I, Kpn I, ISfi I
1. Use pUC or pUC-based vectors that contain the portion of the lacZ gene that allows for ? complementation.
2. Select an E. coli strain that carries the lacZdeltaM15 marker.
3. Plate transformations on plates containing X-gal. Spread 50 µg of 2% X-gal or 100 microliters of 2% bluo-gal (both can be dissolved in DMF or 50:50 mixture of DMSO:water) on the surface of a 100 mm plate and let dry. Alternatively, add directly to the cooled medium (~50 degrees C) before pouring the plates at a final concentration of 50 µg/mL for X-gal and 300 µg/mL for bluo-gal. Plates are stable for 4 weeks at 4 degrees C.
4. If the strain used carries the lacIq marker, add IPTG to induce the lac promoter. Spread 30 µl of 100 mM IPTG (in water) on 100 mm plates. Alternatively, add the IPTG directly to cooled medium (~50 degrees C) before pouring the plates to a final concentration of 1 mM. Plates are stable for 4 weeks at 4 degrees C.
5. Do not plate E. coli on medium containing glucose if using X-gal or bluo-gal for blue-white screening. Glucose competes as a substrate and prevents cells from turning blue.
For long-term storage, preparation of glycerol stocks stored at -70 degrees C is recommended. Follow the protocol below:
1. Pick one colony into 5 mL LB broth or S.O.C. medium. Grow overnight at 37 degrees C.
2. Prepare glycerol solution: 6 mL of S.O.B. medium and 4 mL of glycerol.
3. Take one volume of cells and add one volume of glycerol solution and mix.
4. Freeze in ethanol/dry ice. Store at -70 degrees C.
Yes, this is possible. We recommend using saturating amounts of DNA (10 ng of each plasmid). Make sure that the origin of replication is different in each plasmid so that they can both be maintained in the cell. If the ori is the same, the plasmids will compete for replication and the one with even a slight disadvantage will be lost. Alternatively, cells with a resident plasmid can be electroporated with a second plasmid without “electrocuring” taking place.
In plates, we recommend 50 µg/mL X-gal and 1 mM IPTG (0.24 mg/mL). When spreading directly onto agar plates, we recommend 40-50 µl of 40 mg/mL X-gal (2% stock) in dimethylformamide and 30-40 µl of 100 mM IPTG on top of the agar. Let the X-gal and IPTG diffuse into the agar for approximately 1 hour. Do not plate on media containing glucose, as it competes with X-gal or bluo-gal and prevents cells from turning blue.
Competent cell efficiency is measured by transformation efficiency. Transformation efficiency is equal to the number of transformants, or colony forming units, per microgram of plasmid DNA (cfu/microgram).
Some suggestions that will help you to obtain the highest transformation efficiency are:
- Thaw competent cells on ice instead of room temperature; do not vortex cells.
- Add DNA to competent cells once thawed.
- Ensure that the incubation times are followed as outlined in the competent cell protocol for the strain you are working with; changes in the length of time can decrease efficiency.
- Remove salts and other contaminants from your DNA sample; DNA can be purified before transformation using a spin column, or phenol/chloroform extraction and ethanol precipitation can be employed.
DH5? cells are commonly used for routine cloning, but are mcr/mrr+, and therefore not recommended for genomic cloning. The TOP10 competent cells, on the other hand, contain mutated mcr/mrr, and therefore are a good choice for routine cloning and can be used for cloning of methylated DNA, such as eukaryotic genomic DNA. Our Mach1 strain is the fastest growing cloning strain that is T1 phage resistant.
These small colonies are most likely caused by degradation of the Ampicillin. The colonies are just untransformed cells that grow on LB with degraded Amp. In order to circumvent this scenario, you can try to:
1. Plate cells at a lower density
2. Use fresh LB-Amp plates or replace Ampicillin with carbenicillin.
3. The plates should not be incubated for more than 20 hours at 37 degrees C. Beta-lactamase, the enzyme produced from the Ampicillin-resistance gene, is secreted from the Amp-resistant transformants and inactivates the antibiotic in the area surrounding the transformant colony. This inactivation of the selection agent allows satellite colonies (which are not truly Amp-resistant) to grow. This is also true if carbenicillin is being used.
One possible explanation could be toxicity associated with the insert. This toxicity does not affect slow growing cells on solid medium but is much stronger in faster growth conditions like liquid medium.
Suggestions:
1. Use TOP10F' or any other strain with the LacIq repressor
2. Try using any other strain appropriate for cloning.
3. Lower growth temperature to 27 - 30 degrees C and grow the culture longer
4. Another possibility to explain lack of growth is possible phage contamination. In this situation we recommend using an E. coli strain that is T1 phage resistant like DH5alpha-T1R.
This may be caused by the instability of the insert DNA in TOP10 E. Coli. In this case, E.coli strains such as Stbl2, Stbl3, or Stbl4 have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.
Some possible causes and remedies are:
- Ligase function is poor. Check the age of the ligase and function of the buffer.
- Competent cells are not transforming. Test the efficiency of the cells with a control supercoiled vector, such as puc19.
- Both molecules were de-phosphorylated.
- Inhibition of ligation by restriction enzymes and residual buffer. Try transformation of uncut vector, clean up restriction with phenol, or carry out PCR cleanup/gel extraction before ligation.
- Incorrect antibiotic selection used. Check the plasmid and plates and make sure concentration of antibiotic used is correct.
If nothing above applies, low to no colonies may be due to instability of the insert DNA in your competent cells. In this case, E. coli strains such as Stbl2, Stbl3, or Stbl4 have been shown to support the propagation of DNA with multiple repeats, retroviral sequences, and DNA with high GC content better than other strains.
If working with a vector that contains the lac promoter and the LacZ ? fragment (for ? complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid), it will repress expression from the lac promoter thus preventing blue/white screening. Hence, in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening.
TOPO vectors containing the LacZ-ccdB cassette allow direct selection of recombinants via disruption of the lethal E. coli gene, ccdB. Ligation of a PCR product disrupts expression of the LacZ-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. When doing blue/white color screening of clones in TOPO vectors containing the LacZ-ccdB cassette, colonies showing different shades of blue may be observed. It is our experience that those colonies that are light blue as well as those that are white generally contain inserts. The light blue is most likely due to some transcription initiation in the presence of the insert for the production of the lacZ alpha without enough ccdB expressed to kill the cells and is insert dependent. To completely interrupt the lacZ gene, inserts must be >400 bp; therefore an insert of 300 bp can produce a light blue colony. A white colony that does not contain an insert is generally due to a spontaneous mutation in the ccdB gene.
A minimum insertion of 150 bp is needed in order to ensure disruption of the ccdB gene and prevent cell death. (Reference: Bernard et al., 1994. Positive-selection vectors using the F plasmid ccdB killer gene. Gene 148: 71-74.)
Strains that contain an F plasmid, such as TOP10F', are not recommended for transformation and selection of recombinant clones in any TOPO vector containing the ccdB gene. The F plasmid encodes the CcdA protein, which acts as an inhibitor of the CcdB gyrase-toxin protein. The ccdB gene is also found in the ccd (control of cell death) locus on the F plasmid. This locus contains two genes, ccdA and ccdB, which encode proteins of 72 and 101 amino acids respectively. The ccd locus participates in stable maintenance of F plasmid by post-segregational killing of cells that do not contain the F plasmid. The CcdB protein is a potent cell-killing protein when the CcdA protein does not inhibit its action.
If working with a vector that contains the lac promoter and the LacZ alpha fragment (for ? complementation), blue/white screening can be used as a tool to select for presence of the insert. X-gal is added to the plate as a substrate for the LacZ enzyme and must always be present for blue/white screening. The minimum insert size needed to completely disrupt the lacZ gene is >400 bp. If the LacIq repressor is present (either provided by the host cells, for example TOP10F', or expressed from the plasmid) it will repress expression from the lac promoter, thus preventing blue/white screening. Hence in the presence of the LacIq repressor, IPTG must be provided to inhibit the LacIq. Inhibition of LacIq permits expression from the lac promoter for blue/white screening. X-gal (also known as 5-bromo-4-chloro-3-indolyl β-D-glucopyranoside) is soluble in DMSO or DMF, and can be stored in solution in the freezer for up to 6 months. Protect the solution from light. Final concentration of X-gal and IPTG in agar plates: Prior to pouring plates, add X-gal to 20 mg/mL and IPTG to 0.1 mM to the medium. When adding directly on the surface of the plate, add 40 µl X-gal (20 mg/mL stock) and 4 µl IPTG (200 mg/mL stock).
No, these vectors do not contain a functional promoter to express your gene of interest. These vectors are typically for subcloning or sequencing.
TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum Taq, Accuprime Taq, Platinum or Accuprime Taq High Fidelity, AmpliTaq, AmpliTaq Gold, or AmpliTaq Gold 360.
Blunt cloning:
- Use a proofreading enzyme such as Platinum SuperFi DNA Polymerase.
Directional TOPO cloning:
- Platinum SuperFi DNA Polymerase works well.
S. cerevisiae can grow using either or both mechanisms of carbon metabolism. The balance between the two is different for glucose vs. galactose as a carbon source. Under ideal conditions, S. cerevisiae grows slower on galactose than on glucose, because production of glucose-6-P from galactose is rate limiting. (gal -> gal-1-P -> glu-1-P -> glu-6-P). Under non-ideal conditions (low oxygen, as in the center of a colony or a culture without really good oxygen feed), it becomes even worse because cells grown on galactose are using more respiration than fermentation relative to cells grown on glucose. Low oxygen makes fermentation more necessary, which cells growing on galactose are not good at.
Glucose will shut down completely expression from the GAL1 promoter. Glucose causes repression of the Gal1 promoter.
The doubling time of a pYES2 transformed S. cerevisiae strain grown on minimal media with glucose is approximately 2 hours. The doubling time on media with galactose is approximately 4 hours.
The 2 micron plasmid occurs naturally in some strains of S. cerevisae. When a plasmid contains the 2 micron origin of replication it is maintained at 10-40 copies per cell.
As with other sugars (e.g. glucose), D-raffinose is the biologically active carbon source for yeast. Pure L-raffinose will not work.
The suggested initial cell density for galactose induction is 1 to 5 X 10E6 cells/ml . The cells are allowed to divide one or two times and then induced with galactose. Galactose induction is best in log phase and the culture will probably approach static phase at 1 to 4 X 10E7 cells/ml. Induction of cells maintained in raffinose may begin in 15 to 30 minutes whereas induction of cells maintained in glucose may not first occur for an hour or more. Peak expression will often occur in 2 - 4 hours so time points should be taken every hour (or every other hour) for up to 10 hours. When using raffinose maintained cells, the induction is much faster than induction of glucose maintained cells. Maximal expression levels remain the same.
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Although there are no other genes on the plasmid that are induced, there certainly are a number of proteins in the cell that are turned on by galactose and any of those may be apparent. As with any experiment, one should always run the plasmid without insert, induced with galactose as a negative control. The additional bands could be due to post-translational modifications, such as glycosylation, that would increase the apparent molecular weight. Since the size of the sugar chains can be variable, glycosylated proteins often appear as less well defined bands compared to non-glycosylated proteins.
pYES2 does not have a CEN sequence. pYES2 has the 2µ ori which maintains a copy number of 30 to 50 copies per cell. The 2µ ori sequence is derived from the endogenous 2µ circle plasmid (it's the replication origin for 2µ circle).
'CEN' stands for 'CENtromere' sequence. This origin of replication keeps the copy number of that vector down to one or two per cell in yeast. CEN sequences are actual chromosome centromere sequences. CEN ori will keep copy number to 1 or 2 per cell (depending on whether the cell is haploid or diploid). This sequence allows the cell to recognize this plasmid as a chromosome; regulation of chromosome number is extremely rigid. Only functional CEN sequences from S. cerevisiae have been isolated and used on plasmids. CEN sequences are only a couple of hundred base pairs in size. Apparently functional centromere sequences from other eukaryotes, including Sc. pombe and Pichia, are too large to be isolated and utilized on a plasmid.
The 2µ sequence in pYES2 has the replication origin only--not sequences that code for the proteins required for self replication. Therefore, vectors such as pYES2 will replicate to high copy number (30 per cell) and are very stable in circle-plus (have endogenous 2µ) strains but are maintained only at low copy number (2 per cell) and are very unstable in circle-zero strains, with no endogenous 2µ.
The following relative expression levels were observed in beta-galactosidase expression assays:
0.1 units when repressed in the presence of glucose.
Greater than 2000 units when induced in the absence of glucose and the presence of galactose.
The following are commonly employed auxotrophic markers:
1) his3Δ1: Histidine requiring strain (from gene disruption) with a deletion in locus 1. The his3 denotes the disruption of the HIS3 gene. The Δ1 is a deletion that has been engineered to decrease the recombination between the incoming plasmid DNA and the chromosomal site.
2) leu2: Leucine requiring strain due to the disruption of the LEU2 gene.
3) trp1-289: Tryptophan requiring strain, developed from gene disruption and a further point mutation to decrease the recombination between the incoming plasmid DNA and the chromosomal site.
4) ura3-52: Uracil requiring.
For more detail on types and methods of gene disruption in yeast refer to METHODS IN ENZYMOLOGY Vol. 194.
Some researchers choose to grow yeast in medium containing 2% galactose as the sole carbon source during induction. However, yeast typically grow more quickly in induction medium containing 2% galactose plus 2% raffinose. Raffinose is a good carbon source for yeast, and unlike glucose, does not repress transcription from the GAL promoter. Raffinose is a trisaccharide of galactose, glucose and fructose linked in that order. Most yeasts can cleave the glucose-fructose bond, but not the galactose-glucose bond. Fructose is then used as a carbon source.
Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:
- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.
Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:
- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.
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Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.
No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.
Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.