Champion™ pET102 Directional TOPO™ Expression Kit with BL21 Star™ (DE3) One Shot™ Chemically Competent E. coli - FAQs

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

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 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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?

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

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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?

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.

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

- 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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?

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).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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 fraction; check cell lysates and not just the supernatant.
- 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.

- 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. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. Try using carbenicillin instead of ampicillin in the medium; wash and resuspend the overnight culture with LB containing fresh amp/carb before inoculation.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

- 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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.

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

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.

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What is the maximum size plasmid transformed for bacterial expression?

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.

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What is the difference between the Champion pET expression systems and the T7 expression systems?

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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

Yes; in fact, carbenicillin is generally more stable than ampicillin and may help to increase expression levels by preventing loss of the pET plasmids.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What is the yield I should expect from my T7 promoter-based expression system?

The T7 promoter-based expression systems usually give fairly high yield and can be scaled up easily. Yields will vary depending on the protein being expressed, but in general yields range from 100 µg to 10 mg per liter of culture.

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

Transcripts can be made, but there is no ribosome binding site or Shine Dalgarno sequence to initiate translation; therefore, little protein will be produced.

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What factors can affect expression in an inducible T7 system?

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


Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

What are the advantages and mechanism of the T7 expression system?

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 lacUV5 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.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

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

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.

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

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.

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

This can vary somewhat, but we typically suggest a starting range of 0.5-1 mM IPTG.

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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?

The sequence between the RBS and ATG should be between 8-12 bp and should not contain any palindromic sequence.

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Can I co-express two proteins in E. coli?

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.

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Do I need to include a ribosomal binding site (RBS/Shine Dalgarno sequence) or Kozak sequence when I clone my gene of interest?

ATG is often sufficient for efficient translation initiation although it depends upon the gene of interest. The best advice is to keep the native start site found in the cDNA unless one knows that it is not functionally ideal. If concerned about expression, it is advisable to test two constructs, one with the native start site and the other with a Shine Dalgarno sequence/RBS or consensus Kozak sequence (ACCAUGG), as the case may be. In general, all expression vectors that have an N-terminal fusion will already have a RBS or initiation site for translation.

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What is the best molar ratio of PCR product:vector to use for TOPO TA cloning? Is there an equation to calculate the quantity to use?

We suggest starting with a molar ratio of 1:1 (insert:vector), with a range of 0.5:1 to 2:1. The quantity used in a TOPO cloning reaction is typically 5-10 ng of a 2 kb PCR product.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 (insert:vector ratio)

What is the best ratio of insert:vector to use for cloning? Is there an equation to calculate this?

The optimal ratio is 1:1 insert to vector. Optimization can be done using a ratio of 0.5-2 molecules of insert for every molecule of the vector.

Equation:

length of insert (bp)/length of vector (bp) x ng of vector = ng of insert needed for 1:1 insert:vector ratio

Can I use Taq polymerase to generate my gene of interest for directional TOPO cloning?

No, your gene of interest must be amplified with a proofreading polymerase such as Platinum SuperFi DNA Polymerase or AccuPrime Pfx DNA Polymerase that leaves blunt ends for directional TOPO cloning.

What are the requirements for primer design when using directional TOPO cloning?

Please consider the following when designing your primers:

- The 3' pcr primer cannot contain homology to the 5' flap sequence GTGG.
- The enzyme you use must create a blunt-ended PCR product for cloning.
- Primers cannot contain 5' phosphates, which will block the 5' OH nucleophile reactive group.
- The reading frame must be considered when you are designing your primers.

Which PCR polymerases do you recommend for TA/Blunt/D-TOPO cloning and why?

TA Cloning:
- This cloning method was designed for use with pure Taq polymerases (native, recombinant, hot start); however, High Fidelity or Taq blends generally work well with TA cloning. A 10:1 or 15:1 ratio of Taq to proofreader polymerase will still generate enough 3' A overhangs for TA cloning.
- Recommended polymerases include Platinum Taq, Accuprime Taq, Platinum or Accuprime Taq High Fidelity, AmpliTaq, AmpliTaq Gold, or AmpliTaq Gold 360.

Blunt cloning:
- Use a proofreading enzyme such as Platinum SuperFi DNA Polymerase.

Directional TOPO cloning:
- Platinum SuperFi DNA Polymerase works well.

Where is the transcriptional start site of the T7 promoter featured in many of Invitrogen's vectors?

Although Invitrogen has not formally mapped the transcriptional start site of the T7 promoter, the following reference indicates that the start site occurs at the G following the CACTATA sequence found in the promoter: Nucleic Acids Research, Vol.20, No. 20, pp 4626-4634.

What PCR enzyme would you recommend for use with the Directional TOPO Cloning Kits?

For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

Does Thermo Fisher Scientific have a vector for co-expression of two proteins in E. coli?

A dual promoter vector is the best option for expressing two proteins at the same time. Unfortunately, we do not offer any prokaryotic expression vectors containing dual promoters. Another option is to use two different but compatible vectors at the same time. For example, you can try using a pET vector such as pET100/D-TOPO with a pRSET vector. Our pET vectors have a pBR322 origin and our pRSET vectors have a pUC origin, so they are able to replicate in E. coli at the same time. The only issue here is that pRSET is a high copy number vector and our pET vectors are not. Therefore, you may get significantly more protein expression from pRSET in comparison to pET if they are expressed in the same host.

Will carbenicillin in place of ampicillin help to increase expression levels from the pET TOPO vectors?

For most purposes, ampicillin works well for selection of transformants and expression experiments. However, degradation of ampicillin is enhanced during the late stages of bacterial growth, when the pH of the culture tends to decrease. This could result in non-selective conditions, resulting in the appearance of satellite colonies on plates, and if the transferred plasmid is unstable it may result in the loss of the plasmid and low expression levels. Carbenicillin is generally more stable under acidic conditions than ampicillin and studies have shown that using carbenicillin in place of ampicillin may help increase expression levels by preventing the loss of the pET TOPO plasmid. Use at a concentration of 50 ug/ml.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

Can you tell me the difference between a Shine-Dalgarno sequence and a Kozak sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. This sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA. Similarly, eukaryotic (and specifically mammalian) mRNA also contains sequence information important for efficient translation. However, this sequence, termed a Kozak sequence, is not a true ribosome binding site, but rather a translation initiation enhancer. The Kozak consensus sequence is ACCAUGG, where AUG is the initiation codon. A purine (A/G) in position -3 has a dominant effect; with a pyrimidine (C/T) in position -3, translation becomes more sensitive to changes in positions -1, -2, and +4. Expression levels can be reduced up to 95% when the -3 position is changed from a purine to pyrimidine. The +4 position has less influence on expression levels where approximately 50% reduction is seen. See the following references:

- Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283-292.
- Kozak, M. (1987) At least six nucleotides preceding the AUG initiator codon enhance translation in mammalian cells. J. Mol. Biol. 196, 947-950.
- Kozak, M. (1987) An analysis of 5´-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15, 8125-8148.
- Kozak, M. (1989) The scanning model for translation: An update. J. Cell Biol. 108, 229-241.
- Kozak, M. (1990) Evaluation of the fidelity of initiation of translation in reticulocyte lysates from commercial sources. Nucleic Acids Res. 18, 2828.

Note: The optimal Kozak sequence for Drosophila differs slightly, and yeast do not follow this rule at all. See the following references:

- Romanos, M.A., Scorer, C.A., Clare, J.J. (1992) Foreign gene expression in yeast: a review. Yeast 8, 423-488.
- Cavaneer, D.R. (1987) Comparison of the consensus sequence flanking translational start sites in Drosophila and vertebrates. Nucleic Acids Res. 15, 1353-1361.

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What is the source of the EM7 promoter?

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.

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What protein yields should I expect to get with the pRSET expression vectors?

We've expressed CAT and Beta-Gal in volumes from 2 ml to 50 ml and get about 20-50 µg protein/ml of culture. Protein yield is dependent on the type of protein being expressed and the culturing conditions.

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I sequenced one of your vectors after PCR amplification and observed a difference from what is provided online (or in the manual). Should I be concerned?

Our vectors have not been completely sequenced. Your sequence data may differ when compared to what is provided. Known mutations that do not affect the function of the vector are annotated in public databases.

Are your vectors routinely sequenced?

No, our vectors are not routinely sequenced. Quality control and release criteria utilize other methods.

How was the reference sequence for your vectors created?

Sequences provided for our vectors have been compiled from information in sequence databases, published sequences, and other sources.

What is a Shine-Dalgarno sequence?

Prokaryotic mRNAs contain a Shine-Dalgarno sequence, also known as a ribosome binding site (RBS), which is composed of the polypurine sequence AGGAGG located just 5’ of the AUG initiation codon. The Shine-Dalgarno sequence allows the message to bind efficiently to the ribosome due to its complementarity with the 3’-end of the 16S rRNA.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.