Difference between revisions of "Part:BBa K4241010"
 
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===Usage and Biology===
 
===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. 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.
 
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. 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.
 +
<br/>
 +
Similarly to the T7 promoter, this enhanced T7 promoter is to be used in place of other promoters before the coding sequence for the protein of interest in bacterial chassis.
  
 
===Results===
 
===Results===
−
To compare the relative expression of the enhanced promoter 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.
+
To compare the relative expression of the enhanced promoter: 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.
 
[[File:BBa K4241010_1.png|center]]
 
[[File:BBa K4241010_1.png|center]]
−
Fig.1. The SDS PAGE result of expression of a 21kDa using pET system (control), pET system with cloned RBS (Normal T7+RBS) and enhanced T7 (T7pCONS+TIR-2).
+
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).
  
 +
===References===
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Shilling, P. J., Mirzadeh, K., Cumming, A. J., Widesheim, M., Köck, Z., &amp; 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
  
  
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<!-- Uncomment this to enable Functional Parameter display
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K4241010 parameters</partinfo>
 
<partinfo>BBa_K4241010 parameters</partinfo>

Latest revision as of 16:54, 9 October 2022


Consensus φ10 (T7) promoter (T7pCONS)


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]

Overview

This is an enhanced T7 promoter for high yield expression of protein compared to traditional T7 promoter.
This promoter is 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. 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.
Similarly to the T7 promoter, this enhanced T7 promoter is to be used in place of other promoters before the coding sequence for the protein of interest in bacterial chassis.

Results

To compare the relative expression of the enhanced promoter: 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.

BBa K4241010 1.png

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).

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