Part:BBa_I712074

T7 promoter (strong promoter from T7 bacteriophage)

T7 promoter is very specific promoter which is transcribed only by specific T7 RNA polymerase. Usually this promoter is used in expression systems where T7 promoter is cotransfected with T7 RNA polymerase. That ensures strong transcription of desired genes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Contribution by NYU_Abu_Dhabi

Numerous studies have tried to stabilize the expression of the T7 promoter by T7 phage polymerase but there was no success in improving the gradual decline of target protein expression [1]. Moreover, despite the highly specific mechanism of how T7 polymerase binds the promoter during the transcription initiation, it is unknown to what extent sequences in the extended initiation bubble impact on transcription [2].

Novel approach to increase the gene expression level of the T7 promoter

Research published by Microbial Cell Factories highlights that the major contributing factor to decrease of gene expression by the described T7 system is a random chromosomal mutation [1]. Moreover, this mutation occurs in the sequence encoding the T7 phage RNA polymerase, which leads to non-overproducing cells to dominate the culture. Therefore, researchers tackled this issue by developing a method to eliminate non-overproducing cells from the culture [1]. In order to achieve low gene expression, a stable T7 system was created with dysfunctional T7 RNA polymerase. This goal was achieved by maintaining only target protein-expressing cells in the culture leading to a stable high level of recombinant protein expression [1].

Alternative approach

Another research tackled the same issue from yet another perspective. It has been shown that bacteriophage T7 lysozyme is a inhibitor of T7 RNA polymerase which naturally lowers gene activity [3]. In a paper published by Journal of Molecular Biology, it has been shown that if low levels of T7 lysozyme are supplied by plasmid they are sufficient to increase the target protein production upon the induction of T7 RNA polymerase [4]. This induces the activity of T7 RNA polymerase allowing further production of protein [4].

Reference

[1] Kesik-Brodacka, M., Romanik, A., Mikiewicz-Sygula, D., Plucienniczak, G., & Plucienniczak, A. (2012). A novel system for stable, high-level expression from the T7 promoter. Microbial cell factories, 11(1), 1-7. [2]Conrad, T., Plumbom, I., Alcobendas, M., Vidal, R., & Sauer, S. (2020). Maximizing transcription of nucleic acids with efficient T7 promoters. Communications biology, 3(1), 1-8. [3]Zhang, X., & Studier, F. W. (1997). Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme. Journal of molecular biology, 269(1), 10-27. [4] Studier, F. W. (1991). Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of molecular biology, 219(1), 37-44.