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

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

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

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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.

Answer Id: E9704

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I'm getting no expression from my bacterial expression vector, but my cells are growing normally. What should I do?

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Answer

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 section; check cell lysates and not just the supernatant.
- 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. Try using carbenicillin instead of ampicillin in the medium; wash and resuspend the overnight culture with LB containing fresh amp/carb before inoculation.

Answer Id: E9736

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

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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.

Answer Id: E9727

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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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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?

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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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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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What does DE3 mean?

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Answer

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.

Answer Id: E9728

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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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I'm getting low protein yield from my bacterial expression system. What can I do to improve this?

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Answer

- 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.

Answer Id: E9738

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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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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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What is the maximum size plasmid transformed for bacterial expression?

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

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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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