Part:BBa_K2213008
HighPromoter_PduD(1-20)_mCherry
This part is an improved version of https://parts.igem.org/Part:BBa_K562001, submitted by Dundee 2011.
Figure1. circuit diagram of BBa_K2213006.
Improvements
This part is an improved version of https://parts.igem.org/Part:BBa_K562001, originally submitted by team Dundee 2011. The original part contained an illegal XbaI site which has been removed to make it biobrick compatible.
This part has also been expressed under different strength promoters, by Manchester2017, to find the optimal level of induction.
- A high strength Anderson promoter (here)
- low strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213006)
- medium strength Anderson promoter (https://parts.igem.org/Part:BBa_K2213007)
Characterisation
The PduD tag was combined with the high strength Anderson promoter (https://parts.igem.org/Part:BBa_J23104) and mCherry.
Figure 2. Fluorescence microscopy images of Low, Medium and High strength Anderson promoter-PduD construct associated mCherry (OD600: 0.2) expressed in the absence of Eut.
A gradient of fluorescence is evident when compared to low and medium promoters.
Figure 3. Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.
The expression levels compared to low and medium are as shown. Fluorescence levels are similar to the medium promoter, this result was unexpected and should be taken into consideration when choosing either the medium or high promoter.
Figure 4. Optical Density (600nm) for Low, Medium and High strength Anderson promoter constructs with RFU values after 30 hours.
When expressed alone the distribution of the mCherry signal was homogeneous and fluorescence level was relatively high. In the presence of EutS the mCherry signal was less homogeneous but lacked obvious localisation, suggesting EutS isn’t enough to form proper BMCs. In the presence of EutSMN the mCherry signal clumped together, indicating localisation to the BMC.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 8
Illegal NheI site found at 31 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |