Can I directly clone, propagate and express in BL21 without using TOP10?

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Answer

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.

Answer Id: E3845

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63006cff9bec9dc6e8b0c7ce150a6aa6_FAQ

Can I co-express two proteins in E. coli?

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Answer

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.

Answer Id: E9694

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1508af4a76ec602d3e7058e20dca7ed0_FAQ

What is the optimal spacing between the ribosome-binding site (RBS) and the ATG when cloning into a bacterial expression vector?

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Answer

The sequence between the RBS and ATG should be between 8-12 bp and not contain any palindromic sequence.

Answer Id: E9696

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f1e4bdf843dce7298f7b20bc710eee56_FAQ

How much IPTG can I use to induce expression from a T7 promoter containing bacterial expression vector?

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Answer

This can vary somewhat, but we typically suggest a starting range of 0.1-5 mM IPTG.

Answer Id: E9697

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e4e6a95cab4cfe766a5e387e67a2bfb7_FAQ

I am using a bacterial expression vector containing the T7 promoter. At what OD should I induce my cells?

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Answer

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.

Answer Id: E9698

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62ffa992aa9dfb1868d48cc17fc16884_FAQ

I am using a bacterial expression vector containing the T7 promoter. At what temperature should I perform my induction?

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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.

Answer Id: E9699

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b1b83754b943cdb05783aa5122059768_FAQ

What are the advantages and mechanism of the T7 expression system?

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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 T7 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.

Answer Id: E9700

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a3495589334cbd266d4ec67eb4595c22_FAQ

What factors can affect expression in an inducible T7 system?

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Answer

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

Answer Id: E9701

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7e9294f6bf8daa8e3373a2fd1b9addae_FAQ

Can I use one of your mammalian expression vectors with a T7 promoter for expression in E. coli?

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Answer

Transcripts can be made, but there is no ribosome binding site or Shine Dalgarno sequence to initiate translation; therefore, little protein will be produced.

Answer Id: E9702

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fe0e0f9d3c8943b4416951bad45f7417_FAQ

What is the yield I should expect from my T7 promoter-based expression system?

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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.

Answer Id: E9703

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631abc33ce9db352a331181b21c34f89_FAQ

Can I use carbenicillin in place of ampicillin in my transformation/T7 expression experiments?

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Answer

Yes; in fact, carbenicillin is generally more stable than ampicillin and may help to increase expression levels by preventing loss of the pET plasmids.

Answer Id: E9704

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b7a4699cd0e58fe749edc71039fb440a_FAQ

What is the difference between the Champion™ pET expression systems and the T7 expression systems?

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Answer

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.

Answer Id: E9705

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0b6ec296535440a4109452530ba5248e_FAQ

What are BL21 cells derived from?

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Answer

All BL21 cells are derived from strain B834, and are therefore B strains.

Answer Id: E9719

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b9d3b3dafd484a0fdeb85afcf3be61a8_FAQ

What is the maximum size plasmid transformed for bacterial expression?

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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.

Answer Id: E9720

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334832896b5bdea613587af6e2ecc4c0_FAQ

What competent cell strain should I use for protein expression from a T7 promoter-based vector and why?

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Answer

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. The BL21-AI™ cell line can also be used to avoid basal expression with toxic proteins (see below for more details). 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.

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.

Answer Id: E9721

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a6090d45492f78c8112b82b4085db276_FAQ