Is T7 promoter active in mammalian cells?

Stable and high level synthesis of external proteins could be achieved by usingT7 RNA polymerase activity and T7 promoter vectors in mammalian cells [1-7]. Many studies have shown that using CMV promoter as a viral promoter in plasmids is not suitable for stably expression of genes in mammalian cells.

What is T7 expression?

The T7 expression system allows high-level expression from the strong bacteriophage T7 promoter. It’s ideal for expressing soluble, nontoxic recombinant proteins in E. coli. The T7 expression vectors are designed to facilitate cloning using Gateway® technology, and easy protein purification and detection.

What is the function of T7?

T7 RNA polymerase is a very active enzyme: it synthesizes RNA at a rate several times that of E. coli RNA polymerase and it terminates transcription less frequently; in fact, its transcription can circumnavigate a plasmid, resulting in RNA several times the plasmid length in size.

How does T7 expression work?

T7 expression hosts, such as DE3 strains or T7 Express strains, carry a chromosomal copy of the phage T7 RNA Polymerase gene, which is controlled by a lac promoter. When inducer is added, T7 RNA Polymerase is expressed and becomes dedicated to transcription of the gene of interest.

Is T7 promoter constitutive?

These promoters are recognized by the T7 RNA Polymerase and are constitutive meaning that their activity is dependent on the availability of RNA polymerase, but is not affected by any transcription factors.

Is EF1a a strong promoter?

In human T cells EF1a (1.2kb version) is a strong and stable promoter (Michael C. Milone, Molecular Therapy, 2009). If it is not too big for your expression plasmid, it might be a safer choice.

Is T7 promoter a protein?

These cells were transformed with vectors bearing a recombinant gene under the control of promoters recognized by these polymerases. Currently, one of the most widely used prokaryotic protein-producing systems uses the T7 phage polymerase that recognizes the T7 phage promoter [3].

What is T7 Terminator?

The bacteriophage T7 terminator (T7-T phi) encodes an RNA sequence that can form a stable stem-loop structure followed by a run of six uridylate residues; termination occurs at a 3′ G residue just downstream of the U run.

What is the origin of T7 polymerase?

Bacteriophage T7 DNA replication is initiated at a site 15% of the distance from the genetic left end of the chromosome. This primary origin contains two tandem T7 RNA polymerase promoters (phi 1.1A and phi 1.1B) followed by an A + T-rich region.

How does Iptg induce expression?

What is IPTG? IPTG or Isopropyl β-D-1-thiogalactopyranoside is a chemical reagent mimicking allolactose, which removes a repressor from the lac operon to induce gene expression. It acts as an inducer to initiate the transcription of genes in the lac operon.

Is T7 a strong promoter?

T7 RNA polymerase is very selective and efficient, resulting both in a high frequency of transcription initiation and effective elongation. These features result in an RNA elongation that is approximately five-fold faster than for E. coli RNA polymerase; hence, the T7 promoter is a much stronger promoter than the E.

What kind of cell is suitable for T7 expression?

High efficiency chemically competent E. coli cells suitable for T7 protein expression. Chemically competent E. coli cells suitable for high efficiency transformation and protein expression. A T7 expression plasmid and the same plasmid containing a gene encoding a toxic mammalian protein were transformed into each host.

How is T7 expression used in E coli?

T7 Expression. Within the realm of E. coli expression, the T7 system is the most popular approach for producing recombinant protein. In this system, the target gene is cloned into an expression vector downstream of the T7 promoter and this construct is introduced into a T7 expression host.

How are recombinant proteins produced in the T7 system?

Within the realm of E. coli expression, the T7 system is the most popular approach for producing recombinant protein. In this system, the target gene is cloned into an expression vector downstream of the T7 promoter and this construct is introduced into a T7 expression host.

Which is T7 expression host carries the target gene?

In this system, the target gene is cloned into an expression vector downstream of the T7 promoter and this construct is introduced into a T7 expression host. T7 expression hosts such as DE3 prophage strains or T7 Express strains carry a chromosomal copy of the phage T7 RNA polymerase gene.