You searched for: 

K10001

Product FAQ

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?

Answer

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.

Answer Id: E9740

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

Answer

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., DH5?™).
- 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.

Answer Id: E9741

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Product FAQ

What is the maximum size plasmid transformed for bacterial expression?

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

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Product FAQ

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

Answer

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.

Answer Id: E3259

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Product FAQ

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?

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

Was this answer helpful?

Yes
No
Thank you for your response

Product FAQ

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

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

Product FAQ

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

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

Product FAQ

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

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

Product FAQ

What is the source of the EM7 promoter?

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

Product FAQ

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

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