Difference between revisions of "Part:BBa K4241010"
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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). |
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<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K4241010 parameters</partinfo> | <partinfo>BBa_K4241010 parameters</partinfo> | ||
Revision as of 09:48, 9 October 2022
Consensus φ10 (T7) promoter (T7pCONS)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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.
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).