Difference between revisions of "Part:BBa K4241012"

(Added usage notes)
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===Results===
 
===Results===
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To compare the relative expression of 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.
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To compare the relative expression of 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.
 
[[File:BBa K4241010_1.png|center]]
 
[[File:BBa K4241010_1.png|center]]
 
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).
 
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).

Revision as of 11:11, 9 October 2022


TIR-2 translation initiation region 2


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 a enhanced RBS/start codon region which was proven to yield higher protein expression in bacterial chassis. This will initiate the protein translation at the start codon including a Met residue. Like any other pET plasmid expression chassis, it would have this scar.
This 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 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.
Similarly to other RBS and start codon sequences, this sequence of nucleotides are to cloned in place of the original start codon sequence.


Results

To compare the relative expression of 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.

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).