T7

Part:BBa_K4829000

Designed by: Aditya Kamath Ammembal, Dr. Raghavan Varadarajan, Dr. Debajyoti Chakraborty   Group: iGEM23_IISc-Bengaluru   (2023-10-04)

A modified T7 promoter, meant for use in mRNA synthesis, and capping with CleanCap®AG

Modified T7 promoter

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]

Profile

Base Pairs: 33bp
Origin: Escherichia coli, synthetic
Properties: Modifications specific for IVT and compatible only with CleanCap®AG

Usage and Biology

  • The 'gold standard' of capping reagents of mRNA in the market at present could very well said to be the CleanCap®AG reagent. However, for use of this reagent, the last 2 bases of the T7 promoter must be AG and NOT GG. For this reason, we are also unsure if ARCA or other such capping reagents would work with the modified T7 promoter, as we have used only CleanCap®AG for our experiments.
  • We have included the entire eINCV01R promoter region in this promoter (as mentioned in the references below) to ensure that the T7 RNAP binds efficently to the T7 promoter.
  • To maximise transcription efficiency, further, we have included the highly sequnce of GGGAGA downstream of the T7. We would recommend, however, that any users of this part try out GGGATAAT instead, as this is said to provide higher activity.

The T7 expression system is widely used to express proteins (esp. recombinant proteins) in E. coli. Many E. coli strains, like BL21(DE3), possess a gene that is expressed to produce T7 RNA polymerase. This polymerase is responsible for initiating transcription at the T7 promoter of the transformed site. Expression of a recombinant protein under the control of the T7 promoter has been shown to be 8x faster than protein expression control of E. coli RNA polymerase.[1] In our experiments the T7 promoter sequence has been slightly modified to be compatible with in-vitro transcription/translation kits rather than in-vivo transcription/translation through bacterial transformation.

References

  • Iost, I; Guillerez, J; Dreyfus, M (1992). "Bacteriophage T7 RNA polymerase travels far ahead of ribosomes in vivo". Journal of Bacteriology. 174 (2): 619–622.
  • Conrad, T., Plumbom, I., Alcobendas, M. et al. Maximizing transcription of nucleic acids with efficient T7 promoters. Commun Biol 3, 439 (2020). https://doi.org/10.1038/s42003-020-01167-x
  • Malladi, S. K., Singh, R., Pandey, S., Singh, R., Nadig, G., & Varadarajan, R. (2021). Design of a highly thermotolerant, immunogenic SARS-CoV-2 spike fragment. Journal of Biological Chemistry, 296, 100025. https://doi.org/10.1074/jbc.RA120.016284
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