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

What is the genotype of the BL21-AI™ competent cells?

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Answer

BL21-AI™: F- ompT hsdSB (rB-mB-) gal dcm araB::T7RNAP-tetA

The BL21-AI™ strain is an E. coli expression strain that is deficient in the lon protease and outer membrane protease, OmpT. The lack of these proteases reduces degradation of proteins expressed in this strain. The strain carries a chromosomal insertion of a cassette containing the T7 RNA polymerase and tetA genes in the araB locus, allowing expression of the two genes to be regulated by the araBAD promoter. The presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline.

Answer Id: E3884

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6504e5cf4854dfd6f7a93629c746c535_FAQ

Is the BL21-AI™ strain the best for expressing toxic proteins?

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Answer

The BL21-AI™ strain is suitable for high-level recombinant protein expression from any T7-based expression vector. Because T7 RNA polymerase levels can be tightly regulated, the BL21-AI strain is especially useful to express genes that may be toxic to other strains where basal expression of T7 RNA polymerase is leakier (e.g. BL21 Star™(DE3) or BL21(DE3)). The yield of recombinant protein obtained from BL21-AI is generally similar to that obtained from other BL21 strains.

Answer Id: E3885

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a292f6f76aa3229a4b6923ec19613b69_FAQ

What is the evidence that my protein is toxic to bacteria when using the BL21-AI™ cells?

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Answer

You can assume the protein may be toxic when any of the following occurs:
1) No colonies are obtained after transformation and growth on plates, or a combination of large and small, irregular colonies appears on the plate.
2) The initial liquid culture does not grow.
3) It takes longer than 5 hours after a 1:20 dilution of the initial culture for the expression culture to reach an OD600=0.4.
4) The cells lyse after induction with L-arabinose (or L-arabinose and IPTG).

Answer Id: E3886

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52beffc9d148df68df759154576f8e98_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 temperature should I perform my induction?

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Answer

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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Answer

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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Answer

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