Part:BBa_K2379006
pH sensitive system with RNA pHmeter (wild type)
The RNA pHmeter with natural scar site BBa_K2379002(BBa_K2379002) is under the regulation of the pLAC promoter(BBa_R0011). Following the RNA pHmeter, the construct contains GFP (BBa_E0040) as the reporter which is followed by the double terminator (BBa_B0014).Expression of GFP is regulated by the RNA pHmeter and hence will be produced only at alkaline pH
Usage and Biology
ABOUT THIS PART:
We used this part to regulate gene expression at higher pH (above 7.0). This part functions as a pH-sensitive riboswitch which contains the SraF gene that serves as a 5’UTR part for the alx locus in E. coli . This variant of the part is one with the natural 4bp scar site. This part works as expected and when GFP is placed downstream of this gene, maximum fluorescence was observed at pH 8.5.
CHARACTERISATION STUDIES FOR THIS PART:
1. The above graph indicates the fluorescence expression at three different time periods (each recorded at an interval of 60 minutes)
2. The cell cultures were grown at two different pH : 7.0 and 8.5. Fluorescence values were recorded at excitation and emission peaks of 460nm and 515nm respectively.
3. Normalised fluorescence was calculated by dividing observed fluorescence by cell density at OD600.
4. Duplets were used for characterisation and the error bars indicate the Standard Deviation of the mean values.
CONCLUSION:
Expected results were obtained for the RNA pHmeter (Wild scar) while using GFP as the reporter gene. There was only an average 20.5% increase in the expression of GFP from pH 7 to pH 8.5 at all the three time points . Though this was not the case in the native operon where the expression was influenced largely by the SraF gene, the discrepancy in the values are the due to the influence of both the pLac promoter and the SraF gene on the expression of GFP. Yet the SraF gene holds its control over gene expression which is evident from the maximum fluorescence peak observed at pH 8.5. This serves as a proof of concept for working of RNA pHmeter with wild scar.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 173
Illegal BsaI.rc site found at 906
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