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Product FAQ

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

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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Product FAQ

How can I make a glycerol stock of my desired construct?

Answer

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.

Answer Id: E9727

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Product FAQ

My gene of interest is toxic to bacterial cells. Are there any precautions you can suggest?

Answer

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., DH5?™).
- 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.

Answer Id: E9741

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

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

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 ion 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: E9729

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Product FAQ

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

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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Product FAQ

What is the maximum size plasmid transformed for bacterial expression?

Answer

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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Product FAQ

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

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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Product FAQ

I am trying to express my protein using a T7 promoter-based vector. What does “leaky expression” mean?

Answer

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.

Answer Id: E9722

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Product FAQ

What factors can affect expression in an inducible T7 system?

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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Product FAQ

I'm getting no colonies with my T7 promoter-based bacterial expression system. What can I do?

Answer

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.

Answer Id: E9734

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Product FAQ

Can I use a competent cell that is meant for protein expression from a T7 promoter containing vector, for propagation and maintenance?

Answer

We suggest using TOP10 or a similar strain, like DH5?™, 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 not endA and recA wild type. This makes them a poor propagation and maintenance host cell line.

Answer Id: E9726

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Product FAQ

I'm trying to express my protein using a bacterial expression system. How do I know if I'm seeing degradation of my protein or if what I’m seeing is codon usage bias?

Answer

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 this is the case, a time point experiment can be done to determine the best time to harvest the cells.

Answer Id: E9740

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Product FAQ

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

Answer

You can assume the protein may be toxic when any of the following occurs:

- No colonies are obtained after transformation and growth on plates, or a combination of large and small, irregular colonies appears on the plate.
- The initial liquid culture does not grow.
- It takes longer than 5 hours after a 1:20 dilution of the initial culture for the expression culture to reach an AOD600 of 0.4.
- The cells lyse after induction with L-arabinose (or L-arabinose and IPTG).

Answer Id: E9730

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