Part:BBa_K2230005
PI-RBS-RFP-T/pSB1A3
PI promoter [BBa_K861170] is a modified version to Pcar with correction of -10 position in the promoter region, which gives the promoter stronger activity and a weaker CRP interaction. The PI promoter [BBa_K861170] was used as a major part in the work of team NCKU_Tainan in 2016. These two parts have been used in our work.
Cloning
PI-RBS-RFP was amplified from RFP Coding Device (BBa_J04450) using a primer with PI-RBS (B0034) sequence. The PCR product was ligated to pSB1A3 cut by XbaI and PstI.
Function
RFP may be dose-dependently expressed in an increasing concentrations of glucose. According to teams WHU-China 2012 & NCKU_Tainan 2016, the promoter, PI, is glucose responsive.
Demonstration: Glucose responsive promoter activity
The result shown in Fig. 1 indicated that PI promoter has significant activity in LB culture media. However, the activity of Pcar promoter is greater than negative control but much smaller than PI and positive control. It’s consistent with the properties of PI and Pcar promoters just mentioned previously in GENE CLONING section and described previously in Part Resgistry [Part: BBa_K861170] by team WHU-China in 2012 who designed the promoters. In our experiment as presented in Fig. 2, PI and Pcar promoters just responded to various concentrations of glucose with a very slight dose-dependent increase. This phenomenon didn’t correspond to the data provided by team WHU-China in 2012 and team NCKU_Tainan in 2016. Maybe our measurement was not in an optimized condition. Or the reporter of RFP activity was not sensitive enough to respond this difference.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 617
Illegal AgeI site found at 729 - 1000COMPATIBLE WITH RFC[1000]
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