Team:ZJU-China
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OVERVIEW
Currently, T7 promoter is one of the most widely used promoters for expression of heterogenous protein in some E.coli strains such as BL21(DE3). Though the wild-type T7 promoter has proven quite effective, in some cases, we need modified T7 promoters with even higher efficiency of protein expression to meet specific demands. Hence, we tried to transform the wild-type T7 promoter to get modified T7 promoters with increased strength .
T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+ 1) and extends from -17 to +6. As reported in some papers, the sequence specificty of T7 promoter is so strong that some point mutations between positions -11 and -7 may make T7 promoter fail to work. Thus, with the help of previous research, we carefully chose the site which would be mutated by PCR. These sites mainly distribute in the range from -4 to +6. The sequences of these modified promoters are shown in the Table below.
| Part Number | Sequence(-17~+6) |
|---|---|
| BBa_R0085(wild type) | TAATACGACTCACTATAGGGAGA |
| BBa_K2721000 | TAATACGACTCACTATCGCGGAG |
| BBa_K2721001 | TAATACGACTCACTCCAGCAATC |
| BBa_K2721002 | TAATACGACTCACTTCAGCGACC |
| BBa_K2721003 | TAATACGACTCACACGAGCGAGA |
To test the function of mutant promoters, we chose the GFP as our reporter. By assessing the absolute fluorescence units(RFU) and OD600, we can conclude the relative strength of all promoters. When the E.coli BL21(DE3) is cultured at the stage of logarithmic phase, we added IPTG to induce the expression of GFP in strains BL21(DE3) for 4 hours. And the result is shown as Figure and Table below.
Fig.1 Relative Strength of wildtype T7 promoter and mutant promoters
| Part Number | Relative Strength |
|---|---|
| BBa_R0085(wild type) | 1 |
| BBa_K2721000 | 20.99 |
| BBa_K2721001 | 17.75 |
| BBa_K2721002 | 7.63 |
| BBa_K2721003 | 13.92 |
As we can see from the figure, our mutant promoters showed largely increased strength compared with wild type T7 promoter. Therefore, our mutant promoters offer users more opportunity to control the expression of protein using T7 promoter and permit higher levels of target protein expression to be obtained.
References[1] Ikeda R A, Ligman C M, Warshamana S, et al. T7 promoter contacts essential for promoter activity in vivo[J]. Nucleic Acids Research, 1992, 20(10): 2517-2524.
[2] Paul S, Stang A, Lennartz K, Tenbusch M, Uberla K. Selection of a T7 promoter mutant with enhanced in vitro activity by a novel multi-copy bead display approach for in vitro evolution[J]. Nucleic Acids Research, 2013, 41(1):e29.
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