Part:BBa_K1913022
Synthetic plac-FixK2 hybrid promoter with RBS
Synthetic plac-FixK2 hybrid promoter is a promoter which can not only be activated by light sensors YF1-FixJ composite but also inhibited by the LacI repressor. The sequence of upper control element is from wild type FixK2 hybrid promoter on genomic sequence of Bradyrhizobium japonicum, which contains two typical FixJ boxes. The core element region is replaced by constitutive promoter BBa_J23106. Additional lac operators were included on the both sides of the promoter as second control elements that result in transcription repression.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 95
Illegal NheI site found at 118 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 48
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
In order to test the hybrid promoters, we constructed five composite parts with mRFP gene as reporter (BBa_K19103027, BBa_K19103028, BBa_K19103029, BBa_K19103030, BBa_K19103031). These composite parts were co-transformed with the light sensor part (BBa_K19103034) into E.coli strain BL21, cultured under dark for 24h and their fluorescence was tested. We included as controls cells that only contained the Fixk2 composite parts. The results (Figure 1) illustrated that hybrid promoter BBa_K1913022 has only very little different compared to control, which suggested that this promoter is not sensitive one for being induces by FixJ and it has strong leaky expression. Also, BBa_K19103022 did not show light sensitivity (Fig.2) under 24h light and dark condition culture.
Figure 1: Ratio of fluorescence value and absorbance of Fixk2 composite. Fixk2 composite and control was cultured under dark condition over 24h. Emission and excitation wavelength of mRFP are 607 and 584 nm respectively.
Figure 2. Ratio of fluorescence value and absorbance of Fixk2 composite. Fixk2 composite was cultured under dark and light condition over 24h. Emission and excitation wavelength of mRFP are 607 and 584 nm respectively.
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