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C601003

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 are BL21 cells derived from?

Answer

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

Answer Id: E9719

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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

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'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

I'm trying to express a toxic protein using a T7 promoter-based vector. What competent cell strain would you suggest?

Answer

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.

Answer Id: E9723

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Product FAQ

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

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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Product FAQ

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

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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Product FAQ

I'm having problems with protein solubility using a T7 promoter-based vector. What would you recommend to try?

Answer

- 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.

Answer Id: E9735

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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

My cells are growing very slowly, and I'm not getting any protein expression from my baterial expression system. What can I do to fix this?

Answer

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).

Answer Id: E9737

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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

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

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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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 and am getting inclusion bodies. What should I do?

Answer

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.

Answer Id: E9739

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