Difference between revisions of "Part:BBa K4241015"

Latest revision as of 16:56, 9 October 2022

T7pCONS-TIR-2

Overview

This is a composite part combining an enhanced T7 promoter region with an enhanced translation initiation region. The combined effect allows for up to a 40-fold increase in protein expression.
This was derived from the following publication: https://www.nature.com/articles/s42003-020-0939-8

Usage and Biology

The typical T7 consensus sequence used in the ubiquitous pET system, such as in popular plasmids such as pET28a has inherent flaws that inhibit the maximal expression and yield of proteins. Firstly, The nucleotide sequence of the T7 promoter which is derived from the consensus φ10 promoter in the T7 phage. The original consensus sequence is 23 nucleotides long spanning from -17 to +6 bases relative to the mRNA start site. However it is noted that in the pET system, the sequence is truncated at 19 nucleotides from -17 to +2 bases relative to the mRNA start site. By incorporating the full consensus sequence, the protein expression and yield is thus enhanced. Secondly, the Translation Initiation Region (TIR) is a stretch of nucleotudes that are recognised by the 30S ribosomal subunit during translation initiation. TIR-2 is an engineered translational initiation region. When combined with the enhanced T7pCONS promoter yields an increase of 121-fold over the standard pET28a in the synthesis of sfGFP.
This combined promoter, RBS and TIR region is to be used in place of typical promoters in bacterial chassis. It should be placed before any coding sequence of the protein of interest.

Results

To compare the relative expression of the enhanced T7 and the enhanced TIR: The pET system, pET system with cloned RBS and enhanced T7 were cloned into pET28(a)-FGF2 and the yield of the 21kDa product was compared. The constructs were then transformed into T7 Express lysY Competent E. coli.

Fig.1. The SDS PAGE result of expression of a 21kDa using standard pET system and TIR (control), pET system with cloned RBS (Normal T7+RBS) and enhanced T7 with TIR-2 (T7pCONS+TIR-2).

Parts that comprise this composite component

Consensus φ10 (T7) promoter (T7pCONS) - Part:BBa_K4241010
Scar for pMET-T7-RBS - Part:BBa_K4241011
TIR-2 translation initiation region 2 - Part:BBa_K4241012

References

Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., & Daley, D. O. (2020). Improved designs for pet expression plasmids increase protein production yield in escherichia coli. Communications Biology, 3(1). https://doi.org/10.1038/s42003-020-0939-8