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View additional product information for One Shot™ BL21(DE3)pLysE Chemically Competent E. coli - FAQs (C656503)
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有多种措施可防止由毒性基因本底水平表达带来的问题。这些方法均基于T7或Champion表达质粒的设计和构建是正确的：
•在不含T7 RNA聚合酶（即，DH5α）的菌株中繁殖和维持表达质粒。
•如果使用BL21 (DE3)菌株，应在室温而非37°C下生长24-48小时。
•新鲜转化严格调控的大肠杆菌菌株，如BL21-AI菌株。
•转化实验后，将转化反应接种在含100 μg/mL氨苄青霉素和0.1%葡萄糖的LB板上。葡萄糖可抑制T7 RNA聚合酶的本底表达。
•完成BL21-AI菌株的转化后，挑选3或4个转化株直接接种到新制备的含100 μg/mL 氨苄青霉素或50 μg/mL 羧苄青霉素（以及0.1%葡萄糖，如果需要的话）的LB培养基中。当培养物的OD600值达到0.4时，可加入终浓度为0.2%的L-阿拉伯糖来诱导重组蛋白的表达。
•在表达实验中，在生长培养基中补充0.1%葡萄糖和0.2%阿拉伯糖。
•尝试使用调控型细菌表达系统，如我们的pBAD系统。
通常，如果您看见1-2条主带，则表示密码子使用偏好导致了翻译过早终止。发生降解时，通常可看到梯度条带。发生降解时，可尝试在裂解缓冲液中加入蛋白酶抑制剂有助于防止降解。如果是这种情况，可通过时间点实验来确定收细胞的最佳时间。
如果存在溶解性问题，可尝试在诱导时降低温度或减少IPTG用量。也可尝试使用不同的、更严格的菌株进行表达。此外，在表达期间，也可将细菌培养基的葡萄糖含量增加至1%。
使用新鲜的细菌培养物进行接种，因为新鲜的细菌菌落通常可得到较高的蛋白得率。
检查重组蛋白序列中的不常用密码子。利用常用密码子代替稀有密码子，可显著提高表达水平。例如，精氨酸密码子AGG和AGA是大肠杆菌的不常用的密码子，所以这些密码子的tRNA水平低。
进行蛋白纯化时，在缓冲液中加入蛋白酶抑制剂，如PMSF。使用新鲜配制的PMSF，因为PMSF被稀释到水溶液中后30分钟内会失效。
如果您在表达实验中使用氨苄青霉素进行筛选，则可能会因为筛选条件缺失而出现质粒不稳定。这是因为氨苄青霉素被β-内酰胺酶破坏，或者在由细菌代谢物造成的酸性培养基条件下发生水解。在转化和表达实验中，可使用羧苄青霉素替代氨苄青霉素。
重组蛋白可能对细菌细胞具有毒性作用。应选择更严格调控的系统用于表达，如BL21-AI。也可考虑尝试使用不同的表达系统，如pBAD系统。
当您的目的基因具有毒性时这种情况很常见。尝试使用更严格调控的系统，如BL21 (DE3) (pLysS)或BL21 (DE3) (pLysE)或BL21(AI)。
请查看以下可能的原因和解决方案：
•载体中存在移码或提前终止密码子；应检查序列。
•使用了错误的菌株进行表达。
•使用甘油菌可能会改变质粒的完整性，因为大多数用于表达的菌株基因型不是RecA-和EndA-。应使用新鲜转化的细胞。
•蛋白质存在于不溶的组分中；应检查细胞裂解物，而不仅是上清液。
•目的基因使用了稀有密码子——检查所用密码子。
（http://nihserver.mbi.ucla.edu/RACC）
•在培养过程中，细胞剔除了质粒——这在氨苄青霉素抗性质粒中较为常见。尝试在培养基中使用羧苄青霉素代替氨苄青霉素；接种前，使用含新鲜氨苄青霉素/羧苄青霉素的LB培养基清洗和重悬过夜培养物。
•降低诱导温度至30°C、25°C或18°C，有助于增加溶解性和减少包涵体形成。温度越低，诱导所需时间越长（即，30°C需3-4小时，25°C需3-5小时，18°C需过夜）。
•在较高温度下生长（30°C或37°C）并达到合适的OD，加入诱导剂，然后降低温度。
•尝试不同剂量的IPTG（1 mM-0.1 mM IPTG）。
•使用低拷贝数的质粒。
•使用营养不丰富的培养基，例如以M9基本培养基代替LB。
•如果蛋白质需要辅因子，如金属，则在培养基中加入辅因子。
•增加葡萄糖至1%。
•尝试使用BL21-AI菌株并使用不同剂量的阿拉伯糖。
请查看以下关于无菌落生成的可能原因和建议：
•检查所用的抗生素
•转化pUC19对照载体，检验感受态细胞。
•如果目的基因具有毒性，并且启动子为T7启动子，可尝试使用BL21 (DE3) (pLysS)或(pLysE)或BL21 (AI)感受态细胞。您也可尝试在培养基中加入葡萄糖。
DE3是指菌株含有λ DE3溶源菌，其染色体上带有由lacUV5启动子控制的T7 RNA聚合酶基因。lacUV5启动子受内源性大肠杆菌lacl蛋白的调控，可被IPTG诱导。诱导T7 RNA聚合酶的表达需要IPTG。DE3 λ衍生株也含有噬菌体21的免疫性区域。
我们建议您在获得正确的构建质粒后，将克隆以甘油储液的形式进行保存。请遵循以下步骤来制备甘油储液：
•在含有50-100 μg/mL氨苄青霉素的LB培养基中，使1-2 mL菌株生长至饱和（12-16小时；OD600 = 1-2）
•取0.85 mL培养物，与0.15 mL无菌甘油混合。
•通过涡旋，混合溶液。
•转移至合适的冻存管中，盖上管帽。
•在乙醇/干冰浴或液氮中冷冻，然后转移至–80°C长期保存。
我们建议使用TOP10或与其相似的细胞株，如DH5α，进行融合、繁殖和维持研究。即使是无诱导剂的情况下，基础水平的T7聚合酶，也会导致目的基因发生表达。如果目的基因对大肠杆菌宿主具有毒性作用，则可导致质粒不稳定和/或细胞死亡。此外，BL21细胞是endA和recA野生型的，这使其成为一种难以繁殖和维持的宿主细胞株。
BL21 (DE3) Star细胞中编码RNase E (rne131) 的基因存在突变，RNase E (rne131) 是与大肠杆菌中mRNA降解相关的主要酶之一。该突变可显著改善mRNA转录本的稳定性，从而增加具有普通T7启动子的细胞株（如BL21 (DE3)）的蛋白表达得率。由于T7 RNA聚合酶合成mRNA的速度比大肠杆菌RNA聚合酶更快，因此，T7启动子的转录与翻译步骤并不同步，导致大量无保护的mRNA转录本留在细胞中。这些无保护的mRNA对内源性RNase的酶降解较为敏感，可大幅降低蛋白得率。BL21 Star细胞株中编码RNase E (rne131) 的基因存在突变，RNase E (rne131) 是与大肠杆菌中mRNA降解相关的主要酶之一。该突变可显著改善mRNA转录本的稳定性，从而增加蛋白产量。
BL21 AI大肠杆菌菌株可对使用T7启动子表达毒性蛋白进行最严格的调控。BL21 AI细胞株与传统BL21 (DE3)细胞株具有完全不同的诱导机制。该细胞株利用克隆到T7 RNA聚合酶上游的araBAD启动子，取代了驱动T7 RNA聚合酶等基因的lacUV5启动子，消除了传统BL21 (DE3)表达系统的渗漏。这消除了对pLysS和pLysE质粒的需求。通常，该细胞株的表达得率与其他BL21细胞株相近。
渗漏表达是指存在一些基础水平表达。例如，在所有BL21（DE3）细胞株中， T7 RNA聚合酶总是存在一些基础水平表达。如果毒性基因被克隆到T7启动子的下游，则这种“渗漏表达”可能导致生长率降低、细胞死亡或质粒不稳定。
在所有BL21（DE3）细胞株中，T7 RNA聚合酶总是存在一些基础水平表达（请注意BL21 AI细胞株除外）。如果毒性基因被克隆到T7启动子的下游，则这个毒性基因的基础表达可能导致生长率降低、细胞死亡或质粒不稳定。利用含有可编码T7溶菌酶的基因以及普通DE3元件的细胞株，可避免出现上述问题。T7溶菌酶已被证明可与T7 RNA聚合酶结合并抑制转录。这种活性能够降低T7 RNA聚合酶的基础水平。在IPTG的诱导下，lac阻遏物不再与lac操纵子区域结合，从而可生成T7 RNA聚合酶。T7 RNA聚合酶生成水平增加并超出了少量T7溶菌酶蛋白有限的抑制T7 RNA聚合酶的能力，导致目的基因表达。使用BL21-AI细胞株，也可避免毒性蛋白的基础表达（详情见下文）。T7溶菌酶是一种双功能酶，它除了具有T7 RNA聚合酶结合活性，还可切割大肠杆菌细胞壁肽聚糖层的特异键。这种活性可增强纯化前冻融循环产生的细胞裂解作用。
当未携带pLysS质粒的宿主生长至稳定期（16 hr；过夜培养）并且目的基因具有毒性时，将基础表达降至最低，对pET载体的表达尤为重要。当没有来自pLysS质粒的T7溶菌酶时，稳定期培养物中的基础表达水平会升高。如果目的基因具有毒性，为了维持质粒的稳定性，可能有必要在液体培养基和琼脂板中加入0.5–1%葡萄糖。在生长至稳定期的培养物中，含有pLysS的宿主表达溶菌酶的水平升高，从而降低目标蛋白的诱导水平。这可能是因为氯霉素乙酰转移酶（CAT）基因启动子在缺乏葡萄糖的情况下对cAMP刺激也具有敏感性，并且氯霉素乙酰转移酶（CAT）基因启动子位于pLysS T7溶菌酶基因的上游。
我们尝试使用过的最大质粒为16kb。但是，从理论上来说，在效率开始下降之前，您可能可以转化高达30kb的质粒。
BL21细胞均来源于B834菌株，因此是B菌株。
T7和Champion pET表达载体都具有强力的噬菌体T7启动子。使用IPTG诱导后，T7 RNA聚合酶会与T7启动子结合，使目的基因发生转录和翻译。研究表明，即使在无诱导剂的情况下，也一直会存在一些由λDE3溶源菌中lacUV5启动子介导的T7 RNA聚合酶基础表达（Studier and Moffatt, 1986（http://www.ncbi.nlm.nih.gov/pubmed/3537305））。通常，这不会带来问题，但是，如果目的基因对大肠杆菌宿主具有毒性，则目的基因的基础表达可能会导致质粒不稳定和/或细胞死亡。为解决这个问题，经过设计的Champion pET 载体含有T7lac启动子，可驱动目的基因的表达。T7lac启动子包含T7 启动子及其下游的lac操纵子序列。lac操纵子作为lac阻遏物（由lacl基因编码）的结合位点，可进一步抑制BL21 Star（DE3）细胞中由T7 RNA 聚合酶诱导的目的基因的基础转录。
可以。实际上，羧苄青霉素通常比氨苄青霉素更稳定，可防止pET质粒的丢失从而可能会有助于增加蛋白表达水平。
T7启动子表达系统通常可产生相当高的得率，可轻松进行放大。得率取决于所表达的蛋白，范围通常为每升培养物可得到100 µg至10 mg蛋白。
尽管可生成转录本，但是没有可起始翻译的核糖体结合位点或Shine Dalgarno序列；因此，不会生成蛋白质。
有多种因素可影响表达，包括：
•诱导剂（IPTG）的加入量
•诱导的开始时间点（进行诱导的最佳OD600值为0.4-0.6）
•诱导持续时长
•诱导温度
•载体本身
T7表达系统使用强力的噬菌体T7启动子可进行高水平表达。该系统依赖于T7 RNA聚合酶。尽管该聚合酶不是细菌内源产生的，但一些大肠杆菌菌株（如BL21（DE3）和BL21（DE3）pLysS）经过基因工程改造已携带可编码该RNA聚合酶的基因，称之为DE3噬菌体λ溶原菌。该λ溶原菌含有lacl基因、受lacUV5启动子控制的T7 RNA聚合酶基因以及一小部分lacZ基因。这个lac基因被插入到int基因中，从而使其失活。破坏int基因可防止在缺乏辅助噬菌体的情况下发生噬菌体切割（即，溶菌）。lac阻遏物可抑制T7 RNA聚合酶的表达。因此，当这些细胞处于正常情况时，lac阻遏物（lacl产物）结合到 lacUV5启动子与编码T7 RNA聚合酶的基因之间的lac操纵子区域。这有效阻止了T7 RNA聚合酶基因的转录。当然，总会存在少量的基础表达水平的T7 RNA聚合酶。这是因为lac阻遏物与lac操纵子区域处于平衡状态，使操纵子位点大部分被占据，但并不是一直被占据。
加入可阻止lac阻遏物与lac操纵子结合的化合物后，可诱导蛋白质表达。该化合物为异丙基硫代半乳糖苷（IPTG）。IPTG与lac阻遏物结合，改变其构象，从而使其不能与lac操纵子结合。这会导致细胞大量生成T7 RNA聚合酶。由于T7 RNA聚合酶对T7启动子（仅存在于转化质粒中）具有特异性，质粒编码的蛋白将发生过表达。
诱导的温度范围很大，可以从37°C到30°C乃至室温。较低温度通常需要较长的生长时间。
诱导时间差异很大。在使用T7系统的实验中，当OD600为0.1–1.2时进行诱导，获得了成功的诱导结果。一般来说，在高OD值进行诱导，会产生较低的表达得率，因为细胞密度过高会使细胞立即停止生长。进行诱导的最佳OD600为0.4-0.6。
IPTG的用量差异很大，我们通常建议的起始范围为0.1-5 mM IPTG。
RBS与ATG之间的序列应该为8-12 bp，并且不含有任何回文序列。
若要在大肠杆菌中同时表达2种蛋白，我们建议使用双启动子载体或同时使用2种不同的可兼容的载体。例如，您可尝试同时使用1个pET载体和1个pRSET载体，它们含有不同的复制起点（ORI），分别为pBR322和pUC复制起点。唯一的问题是，pRSET载体是高拷贝数的，而pET不是；因此，如果您将它们转化到同一个宿主细胞中，从pRSET中得到的蛋白表达可能明显多于pET。
Several precautions may be taken to prevent problems resulting from basal level expression of a toxic gene of interest. These methods all assume that the T7-based or Champion-based expression plasmid has been correctly designed and created.
- Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase (i.e., DH5α).
- If using BL21 (DE3) cells, try growing cells at room temperature rather than 37 degrees C for 24-48 hr.
- Perform a fresh transformation using a tightly regulated E. coli strain, such as BL21-AI cells.
- After following the transformation protocol, plate the transformation reaction on LB plates containing 100 µg/mL ampicillin and 0.1% glucose. The presence of glucose represses basal expression of T7 RNA polymerase.
- Following transformation of BL21-AI cells, pick 3 or 4 transformants and inoculate directly into fresh LB medium containing 100 µg/mL ampicillin or 50 µg/mL carbenicillin (and 0.1% glucose, if desired). When the culture reaches an OD600 of 0.4, induce expression of the recombinant protein by adding L-arabinose to a final concentration of 0.2%.
- When performing expression experiments, supplement the growth medium with 0.1% glucose in addition to 0.2% arabinose.
- Try a regulated bacterial expression system such as our pBAD system.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Typically, if you see 1-2 dominant bands, translation stopped prematurely due to codon usage bias. With degradation, you usually see a ladder of bands. With degradation, you can try using a protease inhibitor and add it to the lysis buffer to help prevent degradation. If degradation is the issue, a time point experiment can be done to determine the best time to harvest the cells.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
If you are having a solubility issue, try to decrease the temperature or decrease the amount of IPTG used for induction. You can also try a different, more stringent cell strain for expression. Adding 1% glucose to the bacterial culture medium during expression can also help.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
- Inoculate from fresh bacterial cultures, since higher protein yields are generally obtained from a fresh bacterial colony.
- Check the codon usage in the recombinant protein sequence for infrequently used codons. Replacing the rare codons with more commonly used codons can significantly increase expression levels. For example, the arginine codons AGG and AGA are used infrequently by E. coli, so the level of tRNAs for these codons is low.
- Add protease inhibitors, such as PMSF, to buffers during protein purification. Use freshly made PMSF, since PMSF loses effectiveness within 30 min of dilution into an aqueous solution.
- If you are using ampicillin for selection in your expression experiments, you may be experiencing plasmid instability due to the absence of selective conditions. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. You may want to substitute carbenicillin for ampicillin in your transformation and expression experiments.
- The recombinant protein may be toxic to bacterial cells. Try a tighter regulation system for competent cell expression such as BL21-AI. You may also consider trying a different expression system such as the pBAD system.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
This typically occurs when your gene of interest is toxic. Try using a tighter regulation system, such as BL21 (DE3) (pLysS) or BL21 (DE3) (pLysE), or BL21(AI).
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please view the possible causes and solutions to try:
- Frame shifts or a premature stop codon is present in the construct; check the sequence.
- The wrong cell strain was used for expression.
- If using a glycerol stock, the integrity of the plasmid can change because most cell strains for expression are not RecA and EndA-. Use freshly transformed cells.
- The protein is in the insoluble fraction; check cell lysates and not just the supernatant.
- Check the codon usage in the recombinant protein sequence for infrequently used codons. Replacing the rare codons with more commonly used codons can significantly increase expression levels. For example, the arginine codons AGG and AGA are used infrequently by E. coli, so the level of tRNAs for these codons is low.
- Rare codons were used in the gene of interest: check the codon usage. (http://nihserver.mbi.ucla.edu/RACC)
- The cells may be kicking out the plasmid during culture: this is more common in plasmids that are ampicillin resistant. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. Try using carbenicillin instead of ampicillin in the medium; wash and resuspend the overnight culture with LB containing fresh amp/carb before inoculation.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
- Lower the induction temperature to 30 degrees C, 25 degrees C, or 18 degrees C to help increase solubility and reduce the formation of inclusion bodies. The lower the temperature, the more time needed to do the induction (i.e., 30 degrees C for 3-4 hours, 25 degrees C for 3-5 hours, or 18 degrees C for overnight).
- Grow at a higher temperature (30 degrees C or 37 degrees C) to reach the proper OD, add inducer, then shift to the lower temperature.
- Try different amounts of IPTG (1 mM-0.1 mM IPTG).
- Use a low copy number plasmid.
- Use a less rich medium, such as M9 minimal medium instead of LB.
- If the protein requires a cofactor, such as a metal, add the cofactor to the medium.
- Add glucose to 1%.
- Try the BL21-AI strain and use different amounts of arabinose.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
Please check the following possibilities and suggestions for getting no colonies:
- Check the antibiotic used.
- Check the competent cells with pUC19 control reaction.
- If your gene of interest is toxic, try using BL21 (DE3) (pLysS) or (pLysE) or BL21 (AI) cells if the promoter is the T7 promoter. You can also try adding glucose to the medium.
Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.
The DE3 designation means the strains contain the lambda DE3 lysogen that carries the gene for T7 RNA polymerase under control of the lacUV5 promoter. This promoter is regulated by the endogenous E. coli lacI protein and is induced with IPTG. IPTG is required to induce expression of the T7 RNA polymerase. The DE3 lambda derivative also contains the immunity region of phage 21.
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Once you have obtained your desired construct, we recommend that you store your clone as a glycerol stock. Please follow these steps to create a glycerol stock:
- Grow 1 to 2 mL of the strain to saturation (12-16 hours; OD600 = 1-2) in LB containing 50-100 µg/mL ampicillin
- Combine 0.85 mL of the culture with 0.15 mL of sterile glycerol
- Mix the solution by vortexing
- Transfer to an appropriate vial for freezing and cap
- Freeze in an ethanol/dry ice bath or liquid nitrogen and then transfer to –80 degrees C for long-term storage.
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We suggest using TOP10 or a similar strain, like DH5α, for characterization of the fusion, propagation, and maintenance. The presence of T7 polymerase, even at basal levels, can lead to expression of the desired gene even in the absence of inducer. If the gene is toxic to the E. coli host, plasmid instability and/or cell death can result. Additionally, BL21 cells are endA and recA wild type. This makes them a poor propagation and maintenance host cell line.
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BL21 (DE3) Star cells contain a mutation in the gene encoding RNase E (rne131), which is one of the primary enzymes involved with mRNA degradation in E. coli. This mutation significantly improves the stability of mRNA transcripts and thus increases protein expression yields over regular T7 promoter-based cell lines like BL21 (DE3). Since T7 RNA polymerase synthesizes mRNA faster than E. coli RNA polymerase; transcription from the T7 promoter is not coupled to translation, leaving a pool of unprotected mRNA transcripts in the cell. These unprotected mRNAs are susceptible to enzymatic degradation by endogenous RNases, greatly reducing protein yield. BL21 Star strains contain a mutation in the gene encoding RNase E (rne131), which is one of the primary enzymes involved with mRNA degradation in E. coli. This mutation significantly improves the stability of mRNA transcripts and thus increases protein production.
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The BL21 AI E. coli strain offers the tightest regulation of expression for production of toxic proteins using the T7 promoter. The BL21 AI line uses a completely different mechanism of induction from that of the traditional BL21 (DE3) lines. This cell line utilizes an araBAD promoter cloned upstream of T7 RNA polymerase. This replaces the lacUV5 promoter driving the T7 RNA polymerase gene and all but eliminates the leakiness of the traditional BL21 (DE3) expression systems. This eliminates the need for pLysS and pLysE plasmids. In general, the expression yields from this strain are similar to that of other BL21 strains.
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Leaky expression means there is some basal level expression seen. For example, in all BL21 (DE3) cell lines, there is always some basal level expression of T7 RNA polymerase. This “leaky expression” could lead to reduced growth rates, cell death, or plasmid instability if a toxic gene is cloned downstream of the T7 promoter.
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In all BL21 (DE3) cell lines, there is always some basal level expression of T7 RNA polymerase (note that this is not true for the BL21 AI cell line). If a toxic gene is cloned downstream of the T7 promoter, basal expression of this gene may lead to reduced growth rates, cell death, or plasmid instability. Utilizing a variant cell line that contains a gene encoding the T7 lysozyme as well as the usual DE3 components can circumvent this problem. T7 lysozyme has been shown to bind to T7 RNA polymerase and inhibit transcription. This activity is exploited to reduce basal levels of T7 RNA polymerase. Upon induction with IPTG, the lac repressors no longer bind to the lac operator region and T7 RNA polymerase is produced. This increased level of T7 RNA polymerase production exceeds the limited capacity of the few T7 lysozyme proteins present to inhibit T7 RNA polymerase, resulting in expression of the gene of interest. T7 lysozyme is a bifunctional enzyme. This means that in addition to its T7 RNA polymerase binding activity, it also cleaves a specific bond in the peptidoglycan layer of the E. coli cell wall. This activity increases the ease of cell lysis by freeze-thaw cycles prior to purification. The BL21-AI cell line can also be used to avoid basal expression with toxic proteins.
Minimizing basal expression is particularly important for pET vector expression when hosts that do not carry the pLysS plasmid are allowed to grow to stationary phase (16 hr; overnight cultures) and when the target gene is toxic. Without the T7 lysozyme from the pLysS plasmid, basal expression levels are elevated in cultures grown to stationary phase. If the gene is toxic, the addition of 0.5-1% glucose to both liquid medium and agar plates may be necessary to maintain plasmid stability. Hosts containing pLysS may express an elevated level of lysozyme in cultures grown to stationary phase such that induced levels of the target protein are lowered. This is likely due to the fact that the chloramphenicol acetyl transferase (CAT) gene promoter is also sensitive to stimulation by cAMP in the absence of glucose and is upstream of the T7 lysozyme gene in pLysS.
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The largest size plasmid we've tried is 16 kb, but you should theoretically be able to transform a plasmid as large as 30 kb before efficiency begins to drop.
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All BL21 cells are derived from strain B834, and are therefore B strains.
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Both the T7 and Champion pET expression vectors contain a strong bacteriophage T7 promoter. After induction with IPTG, T7 RNA polymerase will bind the T7 promoter, leading to transcription and translation of your gene of interest. Studies have shown that there is always some basal expression of T7 RNA polymerase from the lacUV5 promoter in lambda DE3 lysogens, even in the absence of inducer (Studier and Moffatt, 1986 [http://www.ncbi.nlm.nih.gov/pubmed/3537305]). In general, this is not a problem, but if the gene of interest is toxic to the E. coli host, basal expression of the gene of interest may lead to plasmid instability and/or cell death. To address this problem, the Champion pET vectors have been designed to contain a T7lac promoter to drive expression of the gene of interest. The T7lac promoter consists of a lac operator sequence placed downstream of the T7 promoter. The lac operator serves as a binding site for the lac repressor (encoded by the lacI gene) and functions to further repress T7 RNA polymerase-induced basal transcription of the gene of interest in BL21 Star (DE3) cells.
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Yes; in fact, carbenicillin is generally more stable than ampicillin and may help to increase expression levels by preventing loss of the pET plasmids.
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The T7 promoter-based expression systems usually give fairly high yield and can be scaled up easily. Yields will vary depending on the protein being expressed, but in general yields range from 100 µg to 10 mg per liter of culture.
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Transcripts can be made, but there is no ribosome binding site or Shine Dalgarno sequence to initiate translation; therefore, little protein will be produced.
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There are several factors that can affect expression including:
- Amount of inducer (IPTG) added
- Time of induction (optimal OD600 for induction is 0.4 to 0.6)
- Duration of induction
- Induction temperature
- The construct itself
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The T7 expression system allows for high-level expression from the strong bacteriophage T7 promoter. The system relies upon the T7 RNA polymerase. While it is not endogenous to bacteria, some strains of E. coli (such as BL21 (DE3) and BL21 (DE3)pLysS) have been engineered to carry the gene encoding for this RNA polymerase in a piece of DNA called the DE3 bacteriophage lambda lysogen. This lambda lysogen contains the lacI gene, the T7 RNA polymerase gene under control of the lacUV5 promoter, and a small portion of the lacZ gene. This lac construct is inserted into the int gene, thus inactivating it. Disruption of the int gene prevents excision of the phage (i.e., lysis) in the absence of helper phage. The lac repressor represses expression of T7 RNA polymerase. Therefore, under normal circumstances in these cells, the lac repressor (the lacI product) binds to the lac operator region between the lacUV5 promoter and the gene encoding for T7 RNA polymerase. This effectively prevents transcription of the T7 RNA polymerase gene. Of course, there is always a small basal level of T7 RNA polymerase present. This is due to the fact that the lac repressor is in equilibrium with the lac operator region, causing the operator site to be occupied most, but not all of the time.
Adding a substance that prevents the lac repressor from binding to the lac operator then induces protein expression. This compound is isopropyl b-D-thiogalactoside (IPTG). IPTG binds to the lac repressor, changing its conformation in such a way that it is no longer able to bind the lac operator. This enables the cells to make T7 RNA polymerase in much more substantial amounts. As the T7 RNA polymerase is specific for the T7 promoter (which is only found in the transformed plasmid), the protein encoded by the plasmid will be overexpressed.
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Induction can be performed at a variety of temperatures, ranging from 37 degrees C to 30 degrees C to room temperature. A lower temperature typically requires a longer growth time.
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The time of induction can vary widely. Successful experiments using the T7 systems have induced at an OD600 of 0.1-1.2. Generally speaking, induction at high ODs will lead to lower expression yields, as the cells will stop growing rapidly after the density is too high. The optimal OD600 for induction is 0.4 to 0.6.
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This can vary somewhat, but we typically suggest a starting range of 0.5-1 mM IPTG.
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The sequence between the RBS and ATG should be between 8-12 bp and should not contain any palindromic sequence.
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To express two proteins at the same time in E. coli, we suggest using a dual promoter vector or using two different but compatible vectors at the same time. For example, you could try a pET vector with a pRSET vector, which contain different ORI (pBR322 origin and pUC origin, respectively). The only issue is that the pRSET vector is high copy number but pET is not; therefore, you may get significantly more protein expression from pRSET than from pET if you add them into one host cell.
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It is imperative that a cloning strain such as TOP10 be used for characterization of the plasmid, propagation, and maintenance. BL21 cells are wild-type for endA and recA, which could result in poor miniprep quality and a greater chance of plasmid rearrangements due to recombination. In addition, BL21 cells contain the T7 RNA polymerase gene which is expressed at low levels even in the absence of inducer. If the gene is toxic to E. coli, plasmid instability and/or cell death can result.