Is the LMG194 sold by Life Technologies the same as that in the ATCC bank?

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Answer

Yes, it is the same strain. ATCC Number: 47090

Answer Id: E3246

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3048aa7a49c7ed43ab01e7652003f954_FAQ

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

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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

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6d668e5b96b629314469c673ebbc33f1_FAQ

Are NP40 and DTT required for SUMO™ Protease activity? Will they interfere with downstream applications?

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Answer

Yes, they are both required, although it might be possible to substitute the NP40 with another detergent via dialysis if you have concern about interference with mass spectrometry. DTT is an absolute requirement.

Answer Id: E4965

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8d2eba1a82c3ec7ef6b335907855f514_FAQ

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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1508af4a76ec602d3e7058e20dca7ed0_FAQ

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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Answer

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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f1e4bdf843dce7298f7b20bc710eee56_FAQ

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

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Answer

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

Answer Id: E9697

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e4e6a95cab4cfe766a5e387e67a2bfb7_FAQ

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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62ffa992aa9dfb1868d48cc17fc16884_FAQ

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

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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

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b1b83754b943cdb05783aa5122059768_FAQ

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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a3495589334cbd266d4ec67eb4595c22_FAQ

What factors can affect expression in an inducible T7 system?

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Answer

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

Answer Id: E9701

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7e9294f6bf8daa8e3373a2fd1b9addae_FAQ

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

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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

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fe0e0f9d3c8943b4416951bad45f7417_FAQ

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

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Answer

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.

Answer Id: E9703

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631abc33ce9db352a331181b21c34f89_FAQ

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

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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

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b7a4699cd0e58fe749edc71039fb440a_FAQ

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

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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

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0b6ec296535440a4109452530ba5248e_FAQ

Do you offer a bacterial expression vector with a tag that can be cleaved from the recombinant protein that will not leave any residual amino acids?

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Answer

We recommend our Champion™ pET SUMO Protein Expression System (Cat. No. K300-01). Clone and express your gene of interest as a fusion to SUMO. Use SUMO Protease to cleave SUMO, resulting in the production of native protein with no extra amino acids added between the cleavage site and the start of your protein.

Answer Id: E9706

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f43d74522059f63d1f39defcc625711e_FAQ