Having difficulties with your experiment?

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View the relevant questions below:

Transformation

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.
  • Lower the induction temperature to 30°C, 25°C, or 18°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°C for 3–4 hours, 25°C for 3–5 hours, or 18°C for overnight).
  • Grow at a higher temperature (30°C or 37°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.

Bacterial Protein Expression

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.

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).

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.

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.

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.

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., DH5α™).
  • If using BL21 (DE3) cells, try growing cells at room temperature rather than 37°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.