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

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

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

Answer Id: E9697

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

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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 source of the EM7 promoter?

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The EM7 promoter is a synthetic promoter derived from the T7 promoter. The promoter is typically used to drive expression of antibiotic resistance genes for selection in E. coli.

Answer Id: E3270

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I'm trying to express my protein using a bacterial expression system and am getting inclusion bodies. What should I do?

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

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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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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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