Difference between revisions of "Part:BBa K4241016"

 
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K4241016 short</partinfo>
 
<partinfo>BBa_K4241016 short</partinfo>
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<partinfo>BBa_K4241016 SequenceAndFeatures</partinfo>
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===Overview===
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This is a composite part combining an enhanced T7 promoter retion, an enhanced translation initiation region along with a 6xHis-thrombin-T7 tag. This part should serve as a backbone to add a protein of interest. The combined effect of an enhanced T7 promoter retion, an enhanced translation initiation region results in an up to 40-fold increase in protein expression. Whereas the 6xHis-thrombin-T7 tag will serve to provide a simple and effective purification scheme.
  
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This part could be used to serve as a backbone to add target protein so it could achieve high yield expression, which could follow by a His-tag pulldown.
 
  
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<!-- Add more about the biology of this part here
 
 
===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. 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. The T7 tag is used to further increase yield of recombinant protein production.
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<br/>
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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.
 +
It is of note that this seqence does not contain a start codon, therefore to use this part it must be in frame with a promoter with a Met initiation site.
 +
<br/>
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For purification:
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1. His-pulldown complete construct
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2. Cleave thrombin
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3. His-pulldown once again to remove uncleaved product.
  
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<!-- -->
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===Results===
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<span class='h3bb'>Sequence and Features</span>
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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.
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<partinfo>BBa_K4241016 SequenceAndFeatures</partinfo>
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[[File:BBa K4241010_1.png|center]]
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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).
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===Parts that comprise this composite component===
 +
Consensus φ10 (T7) promoter (T7pCONS) - Part:BBa_K4241010 <br/>
 +
Scar for pMET-T7-RBS - Part:BBa_K4241011 <br/>
 +
TIR-2 translation initiation region 2 - Part:BBa_K4241012 <br/>
 +
6xHis-thrombin-T7Tag - Part:BBa_K4241013
  
  

Latest revision as of 11:22, 9 October 2022


Enhance-T7pro-RBS-Start-6xHis-T7tag


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 155
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Overview

This is a composite part combining an enhanced T7 promoter retion, an enhanced translation initiation region along with a 6xHis-thrombin-T7 tag. This part should serve as a backbone to add a protein of interest. The combined effect of an enhanced T7 promoter retion, an enhanced translation initiation region results in an up to 40-fold increase in protein expression. Whereas the 6xHis-thrombin-T7 tag will serve to provide a simple and effective purification scheme.


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. The T7 tag is used to further increase yield of recombinant protein production.
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. It is of note that this seqence does not contain a start codon, therefore to use this part it must be in frame with a promoter with a Met initiation site.
For purification: 1. His-pulldown complete construct 2. Cleave thrombin 3. His-pulldown once again to remove uncleaved product.

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.

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).

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
6xHis-thrombin-T7Tag - Part:BBa_K4241013