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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E6738

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E4435

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E4181

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

What is the source of the EM7 promoter?

Product FAQ

Answer

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

You may have to try different ratios ranging from 1:1 to 15:1 insert:vector.

Equation:

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

Answer Id: E7645

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

Answer

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.

Answer Id: E6651

Was this answer helpful?

Yes
No
Thank you for your response

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

Product FAQ

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

Was this answer helpful?

Yes
No
Thank you for your response